CA2209247A1

CA2209247A1 - Nucleic acid probes for the detection and identification of fungi

Info

Publication number: CA2209247A1
Application number: CA002209247A
Authority: CA
Inventors: Bruce C. Kline; Gurpreet S. Sandhu
Original assignee: Individual
Current assignee: Bayer Corp
Priority date: 1995-01-13
Filing date: 1996-01-12
Publication date: 1996-07-18
Also published as: AU4313896A; PL321139A1; WO1996021741A1; MX9705292A; BR9607497A; JPH11500305A; US5763169A; KR19987001336A; US5958693A; AU693625B2; EP0804619A1

Abstract

Nucleic acid probes and primers are described for detecting fungi that cause disease in humans and animals, as well as spoilage of food and beverages.
These probes can detect rRNA, rDNA or polymerase chain reaction products from a majority of fungi in clinical, environmental or food samples. Nucleic acid hybridization assay probes specific for Acremonium sp., Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria sp., Bipolaris sp., Blastoschizomyces sp., Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var gattii serotype B, Cryptococcus neoformans serotype A, Cryptococcus laurentii, Cryptococcus terreus, Curvularia sp., Fusarium sp., Filobasidium capsuligenum, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus) neoformans var neoformans serotype D, Filobasidium uniguttulatum, Geotrichum sp., Histoplasma capsulatum, Malbranchea sp., Mucor sp., Paecilomyces sp., Penicillium species, Pseudallescheria boydii, Rhizopus sp., Sporothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae, and Trichosporon beigelii are also described.

Description

W ~96121741 PCT/l~-~r~0 6 NUCLE]~C ACID PROBES FOR THE DETECTION AND IDENTIFICATION
OF FUNGI

FIELD OF INVENTION

The inventions described and ~l~imed herein relate to ffle design and compositian of two nucleic acid probes capable of detectinf~ many dirr~ fimgal or~ni~m~ in clinical, food, ellvilol""~nt~l and other s~mples. The inventions 1~ described z~d c~l~ime(l herein also relate to the design and composition of probes capable of specific~lly detectin~ and idellli~yillg Acremonium sp., Aspergillus clavafus, Aspergillusflavus, Aspergillusfumigatus, Aspergillusglaucus, AspergiUus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria sp., Bipolaris sp., R~ tQsChi;7OmyCeSSp., Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida~u,~silosis, Candida tropicalis, Chrysosporium sp., Cladospor,ium sp., Coccidioides immitis, Cryptococcus neoformans var gattii sel~ly~e B, Cryptococcus neoformans seL~ulyl~e A, Cryptococcus laurentii, 25 Cryptococcus terreus, Curvularia sp., Fusarium sp., Filobasidium capsuligenum, Filob~ iella (C~yptococcus) neoformans var bacillispora se~oly~,e C, Filoba~i~iel~ (Cryptococcus) neoformans var neoformans sel~ ly~e D, Filobasidium uniguttu~(7tum, Geotrichum sp., Histoplasma capsulatum, Malbranchea sp., Mucor sp., Paecilomyces sp., Penicillium species, 30 P~eu~llescheria boydii, Rhizopus sp., Sporothrix schenkii, Scopulariopsis h~ WO 96/21741 PCT/lb5G~ C26 ~'-. .
brevicaulis sp., Scopulariopsis brumpti, Saccharomyces cerevisiae, and Trichosporon beigelii in clinical, food, environment~l and other samples.

, ~~~ Fungi are eukaryotic microorg~ni.eme that are ullivt;-~ally distributed. While -- 5 in nature fungi play a major role in the decomposition of plant materials, they are - also responsible for spoilage of food, beverage and ph~rm~ce~1tical preparations.
Out of an estim~te~l 100,000 species of fungi described by mycologists, ~yloxilllately 150 species are pathogenic to man and ~nim~le The incleas.llg incidence of AIDS and the development of newer tre~tmente for ht?m~to1ogic 10 m~ n~ncies and organ transplants has lead to an increase in the number of . , immunoconl~lulllised patients. These patients have a high risk of developing fungal infections, which if not rapidly diagnosed and treated are capable of causing death -in a matter of days. The number of antifungal drugs is limited and their toxic side effects on the patient are much higher than that of comparable antib~ct~ri~l therapy.
15 A rapid diagnosis of fungal infection and start of treatment is critical in these - patients. Books by Kwon-Chung and Bennett, along with Sarosi and Davies, ~ ~ provide an overview into the medical importance of fungi.
-- Fungal org~nieme are identified by morphology and nutritional 20 characteristics. Fungi may take anywhere from two days to several weeks to grow in culture and often the same organism can take radically diL~.e..l forms depending on the growth conditions. This makes timely identification diff1cult even for the ~: classically trained expert and impedes the treatment of patients where rapid identification of genus and species is of medical advantage.

The incidence and distribution of major pathogenic fungi varies by ~- geographic location. Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidiodes immitis, Cryptococcus neoformans, Histoplasma capsulatum, ~ -.

~ .... . .

W ~96121741 PCT/Lb~Gt~C26 Paracocc,'dioides brasiliensis, Psez~lescheria boydii and Sporothrix schenkii ,e~le3~lll some of the leading causes of mycotic infections.

Aspergillus fumigatus is among the top three causes of systemic fungal s infectio~ lreated in hospitals. It usually affects pz~ti~ntc; with organ tr~ncpl~ntc, acute lellk~mi~c and burns and can be rapidly fatal if not diagnosed quickly. With over 150 species of Aspergillus present in the soil, air and water, accurate detection of Aspergillus fumigatus becomes extremely important. Aspergillus clavatus, Aspergillvs flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus 10 nidulaMs, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis an,~Aspergillus ustus le~l~,s~llL a majority of Aspergillus species seen in clinical specimens and their presence can cause diagnostic difficulties. Aspergillus flavus, Asj7ergillus fumigatus and Aspergillus niger have been linked with disease in hllm~nc, vvith Aspergillus fumigatus being the pred~lllh~ pathogen in Nor~
15 ~meric~ ~ few immllnologic tests exist for Aspergillus fumigatus but these have limited sensitivity and specificity. There are also reports of development of polymerase chain reaction based tests for Aspergillus fumigatus based on the amplification of the Asp fl antigen gene and a ribosomal inl~l~,t;nic spacer (Spreadbury et. al.). The Spreadbury technique is based on the PCR amplification of 20 a 401 bp fragment ~ g the large subunit rRNA/intergenic spacer region. This relies on a pair of primers to specifically amplify DNA from Aspergillus fumigatus only, and ~s of no utility in idellliryillg other fungi.

Bl,~stomyces dermatitidis is present in the soil, usually in bird droppings and 25 animal feces. Infections often occur at construction sites and the ~nClling lung infiltration and pneumonitis are usually fatal in immunocoml).olllised patients.Diagnosis by culture may take weeks, and the organism is occasionally mict~ken for other fungi. Existing immlln~logical diagnostic tests are unreliable, and there is a need for rapid and reliable DNA based diagnostic tests. Similarly, Histoplasma - W O96/21741 PCTnB96/00026 . capsulatum exists in the soil and is known to have infected at le~t 20% Of the population of North Am~ric~ Most infections start in the lung and resolve ~ollL~leously, but may occasionally spread to other organs. AIDS patients - lc~ies~,lll a growing number of cases of Histopl~cmo~i~. Diagnosis is difficult as S immlln-~logical tests are often negative during the first 4-6 weeks of infection.
Coccidioides immitis is found in abl-n-l~nre in the soil in Southwestern United States. Dust storms, f~rming, building construction, earthquakes and even hikinghave been linked with outbreaks of ~ ç~e. Lung infection followed by cavitation and tli~sPmin~te~l miliary coccidioidomycosis are seen. ~eningiti~ is usually lethal, 10 and as with other fungi, mortality is highest in debilitated hosts. Four scloly~es of Cryptococcus neoformans cause disease in hllm~n~. These are Cryptococcus neoformans s~ .vLyl~e A, Cryptococcus neoformans var gatti scruLy~e B, Filobasidiella (Cryptococcus) neoformans var bacillispora selolyl,e C and - Filobasidiella (Cryptococcus~ neoformans var. neoformans serotype D. The - 15 incidence of this disease is growing rapidly, with up to 10% of HIV infected people developing cryptococcosis. DNA probes capable of detecting all 4 serotypes are required for the early diagnosis and tre~tment for life ll~e~le.,i.,g infections like ' cryptococcal m~nin~itis. A report by Stockman et. al. discusses commercial tests - for Histoplasma, Blastomyces, Coccidioides, and Cryptococcus based on the 1 8S
20 rRNA (Gen-Probe, Inc., San Diego, CA). The authors report sensitivities ranging from 87.8 to 100% and a specificity of 100%. One drawback ofthese probes is thatthese are used on rRNA extracted from fungal cultures. As some fungi may requireup to 3 weeks to grow in culture, this technique cannot be used to expedite diagnosis until a culture becomes available.
Candida albicans is one of the most common causes of fungal infection in hl-m~n~ It is present in the respiratory, gastrointestin~l and female genital tract of healthy individuals, and acts as an opportunistic pathogen in debilitated individuals on steroid or chemotherapy. Diabetes mellitus and indwelling catheters are other W O 96121741 PCT/lD~G/00026 predisposing causes. Tmmllnoco~l,p,o~ised hosts show rapid hematogenous spread of fungi. ~Iorbidity and mortality in u.,L,c;~Led cases is high. Candida glabrata, Candida g~lilliermondii, Candida kefyr, Candida h usei, Candida lusitaniae, Candida p~rapsilosis and Candida tropicalis are also known to cause disease in 5 h, ..n~ DNA probes capable of ide~iryillg these individual species would eli",i~ he need for multiple blood cultures and lengthy biochPmir~l speciation.

Re,-ent advances in molecular techniques have led to the approach of microbe ~letecti-)n and identification based upon the DNA sequence of ribosomal 10 genes. Cornml llly used ~lFtection techniques include either direct amplification of the ribosomal DNA (rDNA) genes by the polymerase chain reaction, or reverse transcription of the ribosomal RNA (rRNA) into complement~ry DNA (cDNA) followed b~y polymerase chain reaction amplification of the cDNA. Ribosomes are composites of unique rRNA and protein species that function in the translation of 15 mFss~n~er RNA into protein. Evolutionary studies are con~i~tent with the c ~l on that all extant life has evolved from a single org~ni~m Thus, all cellular orp~ni~m~ contain rRNA and these rRNAs are related by evolution. The evolutiona~y process is such that each species of organism appears to have unique regions of sequence in its ribosomal genes. The presence of these unique species20 specific regions allows one to design DNA probes that under conditions of hyhri(li7~tion will specifically bind to, and identify the polymerase chain reaction amplified ]DNA from only one species of fungus. For the purposes of this application, the word "primer" is used to mean a nucleotide sequence which can be exten~lP,~l by template-directed polymerization, and "probe" is used to mean a 25 nucleotide sequence capable of detecting its complemPnt~ry sequence by hybridization. Also, for the purpose of this application, the phrase "nucleotidesequence" is intPntlP~l to include either DNA or RNA forms or modification thereof.
- Furthermore, those versed in the art will recognize that primer sequences can be used as probes and vice versa. The use of nucleic acid hybridization to detect W O96121741 PCT/lb~GJC- 26 .
. . .
specific nucleic acid sequences of interest is also described by Kohne (U.S. Patent ~ . 4,851,330, 7/1989).
=

-~ In prokaryotes and eukaryotes, ribosomal RNA and the corresponding ~ ~ - S rDNA genes are i~lçntifie~l by the size of the RNA. The sizes are related in terms of se-lim~nt~tion velocity or S values. Thus, forprokaryotes the values are 5S, 16S, and 23S; and for eukaryotes the values are 5S, 5.8S, 1 8S and 28S. Because all ribosomes ~elrollll the same function which is essçnti~l for cell viability, ribosomal sequences are largely conserved, yet certain regions of each ribosomal species are subject to more variation without consequence to function. It is these hypervariable regions that allow one to identify diL~.e~ll species amongst members of the samegenus. As noted in the references, there are several reports where 5S, 1 8S and the hlter~llic spacer between 5.8S and 28S rDNA have been used for the detection and- identification of fungi (Holmes et. al., Hopfer et. al., Lott et. al., Maiwald et. al., ~kiml~ra et. al., Mitchell et. al., Nakamura et. al.). Holmes et. al. describe a PCR
'! test based on the co-amplification of the 5S rDNA and an adjacent nonkanscribed spacer region. This identifies only Candida albicans and detects other Candida ~ species without identifying individual or~ni~mc Hopfer et. al. and Maiwald et. al.
- ~ both use universal primers to amplify 1 8S rDNA ~om several fungi including - 20 Candida sp., ~spergillus fumigatus, Cryptococcus neoformans and Trichosporon sp. These amplicons are digested with restriction enzymes and the cut fr~gment~ are sized by gel electrophoresis. This restriction fragment length polymorphism pattern ' ~- enables them to identify most but not all org~ni~m~ This technique can be used on amplified DNA from a pure fungal culture. As clinical samples such as sputum usually contain multiple fungal or~ni.~m~, this technique has little utility in =i= .
diagnosis as multiple overlapping fr~pm~nt~ obtained from a mix of fungi would be - nearly impossible to interpret. Lott et. al. use the 5.8S RNA and the internal transcribed spacer (ITS2) to identify and speciate Candida albicans and related Candida species. ~kimllra amplifies a 687 bp fragment from the 1 8S rDNA of 25 W O96/21741 PCT/Lb5G/00026 medically importaIlt fungi and uses these in the ~ gnl)si~e of Candida albicans in clinical samples. Mitchell uses nested PCR to amplify 5.8S and int~ l transcribed spacer aTS) to identify Cryptococcus neoformans. No subsequent testing is done to verify the identity of the amplified DNA. N~k~mllra et. al. use 18S primers to detect 5 Aspergill2~s fumigatus infections of the lung. Most protocols given in these references can only be used to detect an extremely limited number of fungi from a clinical speçimen Hopfer et. al. and Maiwald et. al. can identify multiple org~ni~m~
from pure cultures, but their utility for clinical specimens co.l~;.;..;..g multiple fungal species is limited at best.
Ullited States patents have been issued to Weisburg et. al. for probes developecE for the detection of 18S small subunit ribosomal RNA sequences in fungi. These probes will detect fungi from many species, but cannot be used easily to identify any single species. United States patents have also been issued to 15 ~illim~n for probes developed for the specific detection of the bacteria Staphyloc~ccus aureus based on the 16S ribosomal sequences. Hogan et. al.
(E~uopean Pat. App. 0,272,009) describe one fungal probe for 18S rRNA and three fungal probes for 28S rRNA sequences. Two of these 28S probes detect several diLr~ l iungi while the third probe detects Candida krusei from a limited panel of 10 fungi. None of the 28S probes described by Hogan et. al. is related to any of the probes described in our invention. All probes claimed in our invention can be mapped vrithin the first 900 base pairs of a 28S gene. The probes described by Hogan et. al. are located further 3' on the 28S sequence, between base pairs 1000 and 2000 ~these numbers are comparable to the primary sequence of Saccharomyces cerevisiae 28S rRNA gene. Genbank accession number: J01355). Leclerc et. al.
have published reports analyzing the phylogenetic relationship between fungi based on partial DNA sequences of several fungal 28S genes sequenced by them. Some of - the org~ni cm~ claimed to have been sequenced by Leclerc are the same as some org~ni~m<. sequenced by us. These are Sporothrix schenckii, Pset~711escheria ~: =

W O96/21741 PCT/LD9G~'~f'~ 26 boydii, Blastomyces derm~ffiti(li~, Histoplasma capsulatum and Ch~fsosporium sp.Leclerc et. al. have not published any sequence data in their report, and to the best - - of our knowledge, they have not made these sequences publically available in the G~nR~nk The reverse-complement sequence of their seqllencing primer 401 (TCCC l-l l CAA CAA l l-l CACG) overlaps our SEQ ID NO: 1 (GTGAAATTGT
TGAAAGGGAA) by 19 nucleotides and their sequencing primer 636 (GGTCCGTGTT TCAAGACGG) overlaps our SEQ ID NO: 2 (GACTCCTTGG
TCCGTGTT) by 10 nucleotides. We are aware of no reports in the ~ dllll'e of variable regions from 28S rRNA genes of fungi being used as targets for the - 10 development of species specific diagnostic probes.

As discussed above, most present techniques for the molecular detection of fungi rely on the use of highly specific primers for the PCR amplification of only one fimgal species. Those that employ "Universal" primers for a PCR amplification ;- 15 of DNA from multiple org~ni~ms, use post-PCR amplicon identification techniques that are useful only on pure cultures of fungi. These are not be able to identify fungi from a clinical specimen co~ -g multiple fungal org~nisms Our first aim was to develop "Universal" primers for the 28S gene. These primers would be capable of amplifying in a PCR, 28S rDNA from most fungi. Our subsequent aim was to develop species specific probes for fungi of interest, that would be used to analyze our "Universal" 28S amplicon. These species specific probes would be able to detect the presence of fungi of interest even in situations cont~ining mixed fungal species.

One aspect of this invention is to provide nucleic acid primers capable of detecting 28S sequences from DNA or RNA of most fungi. These would be used as "Universal" primers in a polymerase chain reaction to amplify 28S sequences fromany fungus present in clinical, food, environmer.t~l or other samples. These ff ~
"Universal" primers would also be used to sequence the arnplified DNA. The wo 96/21741 PCT/~ ~26 sequence obtained would be used to identify the ~ungus by c~,.,.p~ with a -~ce of known fungal sequences.

A second aspect of this invention is to provide nucleic acid probes capable 5 of ~et~ctin~ and id~llliryillg, by nucleic acid hybri~i7~tion, the pathogens Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillusflavus, Aspergillu,s glaucus, Aspergillus niger, Aspergillus terreus, Candida glabrata, Candida g2lilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candid~ p,~rapsilosis, Candida tropicalis, Pseu~7l1escheria boydii, Sporothrix schenckii ~ nd other species by use of any of several dirr~.~llt folmats. Additionally, nucleotide sequence information is provided to identify these pathogens and other fungi by DNA sequence comparison (Figure 2) or by the construction of additionalprobes.
SUMMARY OF THE rNVENTION

Nucleic acid probes and primers are described for detectinp fungi that cause disease in humarls and ~nim~l~, as well as spoilage of food and beverages. These20 probes can detect rRNA, rDNA or polymerase chain reaction products from a majority oi fimgi in clinical, environment~l or food samples. Nucleic acid hybridizati3n assay probes specific for Aspergillus fumigatus, Blastomyces dermatitidls, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus flavus, Aspergillus glaucus, Aspergillus niger, 25 Aspergillus terreus, Candida glabrata, Candida guilliermondii, Candida kefyr,Candida h usei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Pseudallescheria boydii, Sporothrix schenckii and other species (Table 1 and Figure - ~ 2) are also described.

-W O96/21741 PCT/Lb~ ~~26 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents the relative position of the sequences described on the 28S subunit of fungi.
Figures 2A, B, C and D together represent the multiple sequence ~li nment for (SEQ ID NO: 24) through (SEQ ID NO: 74) .
DETAILS OF THE INVENTION

Our first objective was to develop nucleic acid primers for use in a polymerase chain reaction to amplify 28S genes from all fungi likely to be present in a clinical sample. This amplified DNA would then be arnenable to probing withseveral diLr~ -L species specific probes. Each one of these species specific probes would, under conditions of hybridization, anneal to 28S ribosomal DNA from only one species of fungus, thereby cletecting and identifying the species of fungus present in the clinical sample. The 28S gene was selected as a target because it had ~ regions that were conserved among fungi and these would provide potential annealing sites for "universal" fungal probes. The ribosomal 28S genes were alsoexpected to have hypervariable regions that would be unique enough to provide sites for species specific probes. The large rRNA gene is called the 23S rRNA gene in prokaryotes and 28S in eukaryotes. This ~ n~tion is based on the length and therefore the se-liment~tion coefficient of these rRNA molecules. Fungal large subunit rRNAs vary in size among different org~ni~m~ and are often referred to as being 25S, 26S or 28S. Since fungi are eukaryotes, and to m"int~in ullirollllity in this application, we shall refer to fungal large subunit rRNA as 28S rRNA.

Published sequences from Cryptococcus neoformans, two Candida albicans, Saccharomyces cerevisiae and two Schizosaccharomyces pombe 28S
genes are approximately 3.5 kilobases in length (Genbank accession numbers:

. - S~ tl~

W O 96/21741 PCT/lb5G/00026 L14068, L28817, X70659, J01355, Z19136 & Z19578). These four sequences were nefl, a~d a region of sequence variability was found clustered belw~en coordinates 200 and 700 from the 5' end of these genes. As an initial starting point, two nucleic acid primers P1 (ATCAATAAGC GGAGGAAAAG) and P2 5 (CTCTG(JCTTC ACCCTATTC) (see figure 1), capable of hybri~ in~ to all 4 of the above mentioned or~ni~m~ and not to human 28S sequences (GenBank accession number: M11167), were tl~ei~n~(l and used under low stringency hybridizalion conditions in a polymerase chain reaction to amplify a~l~x~ tely 800 base pairs of DNA ~ g this hypervariable region from the following 34 10 fungi that were obtained from the Mayo Clinic fungal collection: Acremonium sp., Aspergilll ~s clavatus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, ..~spergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauvaria sp., Bipolaris sp., Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, CandidaIrusei, Candidalusitaniae, Candidaparapsilosis, Candidatropicalis, Chrysosporium sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoforma~s serotype A, Curvularia sp., Geotrichum sp., Histoplasma capsulatum, Mucor sp., Penicillium sp., Pseu~71escheria boydii, Saccharomyces cerevisiae, Sporothri;~ schenkii and Trichosporon beigelii.
M~A was extracted from the fungi listed above by the following method. A
loopful of fungal culture was scraped off a culture plate using a sterile inoculation loop. The fungus was added one milliliter of sterile water in a 1.5 ml Sarsted (Newton, North Carolina) screw cap microcentrifuge tube. This tube was placed in25 a boiling ~,vater bath for 20 minlltes in order to lyse the fungus and release DNA
from the c ells. Two microliters of this whole cell lysate was used in a PCR to amplify 2,3S rDNA. All PCR amplifications were carried out as hot-start reactions in a 50 ul reaction volume using Perkin-Elmer (Norwalk, CT) 0.5 ml thin-wall polypropylene tubes and a Perkin-Elmer thermal cycler. Reagents added to the tube initic~ly were 2.5 ul of lOX PCR buffer (100 rnM tris pH 8.3, S00 mM KCl, 15 mM
MgC.12), S.0 ul of 50% glyc.,rol/1 mM cresol red, 8.0 ul of dNTP mix (1.25 ~I
each of dATP, dGTP, dTTP and dCTP), 12 picomoles of each mlclsi~ primer and sterile water to make up a volume oit'25 ul. A wax bead (Arnpliwax(~) Gem-100, S Per~n-Elmer) was added and the tubes heated to 77~C for I m~nute and cooled toroom temperature to form a wax barrier. 2.5 ul of 10X PCR buffer, S.~ ul of 50%
glyc~ rol/1 mM cresol red, 0.2 ul Taq polymerase (ArnpliTaq~) SU/ul, Perkin-Elmer) and 15.3 ul of sterile water was added to the tube along with 2.0 ul of DNA
from the fungal whole cell lysate described above. S0 cycles of therrnal cycling was 10 carried out at 94~C - 30 sec, 40~C - 1 min, 72~C - 2 min. The amplified DNA was electrophoresed and purified from a lo~ melt agarose gel by tris buffered phenol pH
8.0, phenol/chloroform/isoamyl alcohol (25:24:1 by vol.) and 3 ether extractions, followed by isopropanol precipitation and 70% ethanol wash.

We completely sequenced both strands of DNA amplified from the organisms listed above. All sequencing was carried out on an Applied Biosystems 3~3A sequencer. Every nucleotide in the sequences generated was verified and confirmed by e~mining the complementary nucleotide from the second strand sequence. We had now created a novel database consisting of nucleic acid sequences spanning a variable region of the 28S rDNA from a diverse collection of medically important fungi.

While the complete sequences for Candida albicans, Cryptococcus neofcirmans and Saccharomyces cerevisiae 28S genes had previously been published and deposited in GenBank, it was not obvious, nor had it been defined,whether any regions of sequence identity among these three org~ni.sm.~ would also be conserved among all fungi of interest. DNA sequences from all the fungi in our nove:r 28S database had to be analyzed in order to develop "Universal" 28S probes.
A11 sequences were subjected to extensive manipulation to identify optimal relative AMENDED SHE~

wo 96/21741 PcTns96/00026 nm~n~ in order to identify regions of similarity for use ~ "Universal" probes.
The selected probe sequences had to meet several important criteria besides the condition of being present in 28S genes from most fungal species. Each probe sequence required an al)l)ropliate thermal profile, secon-l~ry structure and utility in S a DNA amplification reaction. These probes were optimized to work for PCR
amplification in pure cultures of fimgus, as well as in the presence of DNA frommultiple sl~urces as in the case of clinical specimens. The probes were also designed t~ f~cilit~te direct sequencing of the amplified DNA. Our analysis led to the discovery of the oligonucleotide probes listed in (SEQ ID NO: 1~ and (SEQ ID10 NO;2). (F~r their location, see Figure 1.) The s~rces~fill identification ofthese two probes ((S~Q ID NO: 1) and (SEQ ID NO:2)) completed our first objective to develop mlcleic acid probes that would hybridize to, and detect 28S rRNA and rDNA from a majority of fungi (Figure 1 and Table 1). As shown later in this application, the novel sequence information generated by the use of our "Universal"
15 probes allowed us to develop species-specific probes ((SEQ ID NO:3) to (SEQ ID
NO:23)) capable of identifying 19 dir~~ L disease-c~using fungi.

Table 1:
20 Presence ~f hybridization sites for probes SEQ ID NO: 1 and SEQ ID NO: 2 in 28S
nucleic acid sequences.

SEQ ID NO: 1 SEQ ID NO: 2 Acremonium sp. + +
Aspergillus clavatus + +
Aspergillusflavus + +
Aspergillus fumigatus + +
Aspergillu., glaucus + +
Aspergillu., nidulans + +
Aspergillu., niger + +
Aspergillus, ochraceus + +

.-. CA 02209247 1997-07-02 -. W O96/21741 PCT/Lb~ C~26 ~ ' ; ~ Aspergillus terreus + +
~ Aspergillus unguis + +
=~ - . - Aspergillus ustus + +
Beauvaria sp. + +
-~ Bipolaris sp. + +
:-- Blastomyces dermatitidis + +
Blastoschizomyces sp. + +
~ Candida albicans + +
= Candida glabrata + +
=- Candida guilliermondii + +
- Candida kefyr + +
Candida krusei + +
-- Candida lusitaniae + +
Candida parapsilosis + +
Candida tropicalis + +
. . . ~
Chrysosporium sp. + +
Cladosporium sp. + +
Coccidioides immitis + +
Cryptococcus laurentii + +
Cryptococcusneoformans serotype + +
A
Cryptococcus neoformans var. + +
gattii serotype B
Cryptococcus terreus + +
Curvularia sp. + +
Filobasidiella (Cryptococcus) + +
neoformans var bacillispora serotype C
Filobasidiella (Cryptococcus) + +
neoformans var neoformans serotype D
Filobasidium capsuligenum + +
Filobasidium uniguttulatum + +
Fusarium sp. + +
Geotrichum sp. + +
Histoplasmacapsulatum + +
Malbranchea sp. + +
Mucor sp. + +
Paecilomyces sp. + +
Penicillium sp. + +
Pseudallescheria boydii + +
Rhizopus sp. + +

,~

: 14 W O 96/21741 ' PCT/lbsGlooo26 Saccharomyces cerevisiae + +
Scopulariopsis brevicaulis + +
~ Scopulariopsis brumptii + +
Sporothrix schenckii + +
Trichosporon beigelii + +
Human +

Probes SEQ ID NO: 1 and SEQ ID NO: 2 were used to s~lccçssfully amplify (Table 2) and sequence DNA (Figure 2) ~ .g this variable region from the followin~ 49 or~ni~m~: ~cremonium sp., Aspergillus clavatus, Aspergillusflavus, Aspergilll ~s fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, AspergiUus terreus, Aspergillus unguis, Aspergillus ustus, Beauvaria sp., Bipolaris sp., Blastomyces dermatitidis, Blastoschizomyces sp., Candida ,~lbicans, Candida glabrata, Candida guilliermondii, Candida kefyr, 0 Candidakrusei, Candidalusitaniae, Candidaparapsilosis, Candidatropicalis, Chrysosporium sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans serotype A, Cr,vptococcus neoformans var. gattii serotype B, Cryptococcus terreus, Cryptococcus laurentii, Curvularia sp., Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus) neoformans var neoformans serotype D, Filobasidium capsulige~um, Filobasidium unig~/ttu~atu~n, Fusarium sp., Geotrichum sp., Histoplas~na capsulatum, Malbranchea sp., Mucor sp., Paecilomyces sp., Penicillium sp., Pseudallescheria boydii, Rhizopus sp., Saccharomyces cerevisiae, Scopularivpsis brevicaulis, Scopulariopsis brumptii, Sporothrix schenkii and Trichosporon beigelii. This list contains all 4 serotypes (A, B, C and D) of Cryptococcus neoformans. This sequence information generated by the use of probes SF,Q ID NO: 1 and SEQ ID NO: 2 e~r~nrle~l the size of our ~l~t~b~e ~q consisting~ of fungal 28S sequences. All amplified DNA was sequenced across bothv strands fn~m a minimum of two dirr~lclll isolates of each organism to ensure accuracy of the data generated.

CA 02209247 l997-07-02 W O96/21741 PCT/lb~G/'~Ct26 Table 2:
Polymerase chain reaction amplification of 28S rDNA wi~ probes SEQ ID NO: 1 and SEQ ID NO: 2.

PCR with SEQ ID
NO: 1 & NO: 2 Acremonium sp. +
Aspergillus clavatus +
Aspergillus flavus +
Aspergillus fumigatus +
Aspergillus glaucus +
Aspergillus nidulans +
Aspergillus niger +
Aspergillus ochraceus +
Aspergillus terreus +
Aspergillus unguis +
Aspergillus ustus +
Beauvaria sp. +
Bipolaris sp. +
Blastomyces dermatitidis +
Blastoschizomyces sp. +
Candida albicans +
Candida glabrata +
Candida guilliermondii +
Candida kefyr +
Candida krusei +
Candida lusitaniae +
Candida parapsilosis +
Candida tropicalis +
Chrysosporium sp. +
Cladosporium sp. +
Coccidioides immitis +
Cryptococcus laurentii + r ' Cryptococcus neoformans serotype +
A
Cryptococcus neoformans va~. + t gattii serotype B
Cryptococcus terreus +

W O96/21741 PCT/lb~ 26 Curvular~a sp. +
Filobasidiella (Cryptococcus) +
neoformans var bacillispora serotype C
Filobasidiella (Cryptococcus) +
neoforma~2s var neoformans seroly~e D
Filobasidium capsuligenum +
Filobasidium uniguttulatum +
Fusarium sp. +
Geotrich~lm sp. +
Histoplasma capsulatum +
Malbranchea sp. +
Mucor sp. +
Paecilom~ces sp. +
Penicillium sp. ~
Pseudallescheria boydii +
Rhizopus sp. +
Saccharol~yces cerevisiae +
Scopulariopsis brevicaulis +
Scopulari~psis brumptii +
Sporothrix schenckii +
Trichospc?ron beigelii +
Human This list of fungi sequenced by us represents org~ni.~m~ responsible for most cases of subcutaneous and deep mycotic infections in hllm~n~ and also includes S saprophyles (non-pathogenic fungi) commonly encou,lL~l~d in clinical isolates.Since the two probes (SEQ ID NO: 1 and SEQ ID NO: 2) hybridize to 28S rDNA
from all tlle fungi listed above, they are capable of diagnosing the presence of a majority of fungi that are likely to be present in a clinical specimen. They arebelieved lo be primers for universally detecting fimgi.
Probes listed in SEQ ID NO: 1 and SEQ ID NO: 2 were also checked for - ~ their potential ability to hybridize to, and amplify (in a polymerase chain reaction) 23S sequences from bacteria by searching for hybridization sites among the 539 ;i CA 02209247 1997-07-02 W O96/21741 PCT/Lb~''C-_26 =:.
:
b~t~ri~l 23S genes listed in GenR~nk R~cteri~l 23S rDNAs do not have suitable hyhri-1i7~tion sites for SEQ ID NO: 1 and SEQ ID NO: 2 and these two probes should not be able to amplify b~cteri~l DNA under stringent conditions.

S Our second objective was to develop species specific probes, which under hybridization conditions, would detect Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus flavus, ~spergillus glaucus, Aspergillus niger, Aspergillus terreus, Candida glabrata, Candida guilliermondii, Candida ke~r, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Pseudallescheria boydii, and Sporothrix schenckii. We used our ~l~t~b~e of fungal 28S nucleic acid se~ e-lces to create a multiple sequence ~ nment of all the org~ni~m~ that we had sequenced. Every individual sequence was subjected to dl intensive comparison with all other sequences in our ~l~t~b~e in order to discover unique regions of sequence that would be present only in the fungus of interest, and would be absent in all other fungi. When unique stretches of sequence were identified, these were further analyzed for thermal profile and secondary structure.
Each probe constructed by us will, under conditions of hybridization, specifically - hybridize to and detect, nucleic acid sequence from the unique region of only one 20 specific target fungus. Those versed in the art will recognize that specification of a single-stranded DNA sequence implies the utility of the complementary DNA
- sequence, as well as the two equivalent RNA sequences. Furthermore, sequences incorporating modification of any of the moieties comprising the nucleic acid (i.e., the base, the sugar or the backbone) are functional equivalents of the sequence. It 25 should also be recognized that these additional sequences can potentially serve as - probes or primers. Finally, those versed in the art recognize that co~ ~;sons of extensive DNA sequences provides enough variability and uniqueness to speciate org~ni~m.~ (Figure 2).
;:~
.

-W O 96/21741 PCT/Lb,~ 26 Tb'e nucleic acid sequences for these species specific synthetic probes are listed in SEQ ID NO: 3 to SEQ ID NO: 23. There are two probes specific for Cryptococcus neoformans, two probes specific for Sporothrix schenckii, and one probe eacll forAspergillusfumigatus, Blastomyces dermafifidis~ Candida albicans,S Coccidioi,~es immitis, Histoplasma capsulatum, Aspergillusflavus, Aspergillus glaucus, ~ spergillus niger, Aspergillus terreus, Candida glabrata, Candida guilliermandii, Candida kefyr, Candida krusei, Candida lusifaniae~ Candida parapsilosis, Candida tropicalis and Pseu~nl~escheria boydii 28S rRNA and rDNA.
(See Tables 3 - 6 and further discussion below.) All species specific probes developed by us are novel and to the best of our knowledge have not been reported in the lileldlu~e. While all 28S genes sequenced by us had several regions that were dirr~r..l~ among the various species analyzed, the regions that would function best as species specific probes under conditions of 15 hybridization were not obvious. Fxt~neive analysis of each 28S sequence yielded several pctential probe sites. These were studied in detail to enable the selection of optimal unique sites for each probe, based on the need to obtain optimal hybridization characteristics under the test conditions. The highly specific hybridization characteristics of all probe sequences developed by us were then 20 v~ late~l by ~xp~l ;."ent~l results. The prior exi.et~n-~e in GenBank of sequences for Candida albicans and only one serotype of Cryptococcus neoformans 28S genes was in itself not sufficient to enable even an individual versed in this field to develop specific probes for either of these two org~niem~. We had to obtain novel 28S sequence from Candida albicans, Candida glabrata, Candida guilliermondii, 25 Candidak,efyr, Candidakrusei, Candidalusitaniae, Candidaparapsilosis, Candidatropicalis, Cryptococcus neoformans serotype A, Cryptococcus neoformans var.
gattii serotype B, Cryptococcus terreus, Cryptococcus laurentii, Filobasidiella - (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus~ neoformans var neoformans serotype D, Filobasidium '!i CA 02209247 1997-07-02 W O96/21741 PCTI~ ~~26 . . .

capsuligenum and Filobasidium uniguttulatum before we were able to identify . potential regions for the development of species specific probes for these two fungal - org~ni~m~ that would not cross react with the others listed above.
..;, b Our modification of the Chomczynski technique (see Example 2, below) allows us to obtain DNA from any clinical specimen, i~les~,e.;li~e of source (see ....
Table 8 for a variety of clinical specimens tested), within a 3 hour period. The PCR
amplification and subsequent probing can be accomplished with ease within a 24 hour period. The final identification is therefore possible in a day as opposed to ; 10 several days or weeks required by traditional methods. This speed and sensitivity of ~ - diagnosis can make a dir~.~"lce b~Lw~en life and death in debilitated patients - - battling fungal ~ e~es of lmtletçrmined cause. Rapid diagnosis will allow physicians to imm.orli~tely direct their therapy towards curing the identified causative fungus, rather than wait for days or weeks while the patient succumbs to - -- 15 an unknown fungus.
.
Our probes have the ability to pick out the correct target organism even in a mixed fungal infection because of their high level of specificity. The methods of Hopfer et. al. and Maiwald et. al., do not allow identification of individual species -- 20 in a mixed fungal infection because restriction fragment length polymorphism results are nearly impossible to interpret when multiple org~ni~m~ contribute to the restriction fr~gment.~ Their method can therefore only be used on a pure culture, and this also does not save any diagnostic time, because the fungus first has to be grown in culture.

The probes developed by us allow rapid species identification of a large number of pathogenic fungi by using multiple probes against only one PCR
amplified fragment of DNA. Coupled with our modified DNA extraction technique and our ability to accurately diagnose in the case of mixed org~ni~m~, this strategy :' -,, : ,.
=

CA 02209247 l997-07-02 W ~96J2174~ PCTllD5GlC~~26 can provide the ~ le~L amount of diagnostic information in the shortest amount of time. This diagnostic strategy is also ~men~ble to automation, which can result in even greater savings in time, money and effort.

The sequences and the complement of the sequences claimed in this disclosure, along with any modifications to these sequences, may potentially be utilized in assays for the identification of fungi based on several existing methodologies, as well as future improvements and alterations of this technology.
These teclmiques include, but are not limited to, assays based on hybridization,ligation, polymeri7~tion, depolymPri7~tion, seql1encing, ch~mi~ lPgr~ tion, enzymatie, digestion, electrophoresis, chromatography and amplification.
Furthermore, all such variations 11ltimzltely are based in some selection or amplifica1 ion process, some ligand or some nucleic acid moiety that recognizes or utilizes the sequences (SEQ ID NO: l) to (SEQ ID NO:23) claimed in this l 5 application. Such variations include but are not limited to use of a variety of linear or exponential target amplification scl~emec, such as, any of the myriad forms of PCR, the ~igase chain reaction, Q-beta repliase, etc.; direct detection of species-specific n~cleic acid purified or extracted from pure fungal culture using a probe selected ~~om the group (SEQ ID NO: 3) to (SEQ ID NO: 23), use of the complçm~nt~ry DNA forms of (SEQ ID NO: l) to (SEQ ID NO:23); use of the RNA
forms of 1hese sequences and their complement~, and use of derivatives of these DNA or RNA sequences by the addition of one or more reporter moieties from a variety of labels including nucleic acid sequences, proteins, signal generating ligands such as acridinium esters, and/or par~m~gnetic particles. These techniques may be u1ilized with DNA, RNA or modified derivatives used as either the target or the detection molecule.

- In addition to the 23 sequences SEQ ID NO: l to SEQ ID NO: 23, we also describe an additional 51 sequences SEQ ID NO: 24 to SEQ ID NO: 74. These 51 . CA 02209247 1997-07-02 - W O96/21741 PCT~B96/00026 sequences are inclusive of SEQ ID NO: 3 to SEQ ID NO: 23 and are shown as a - multiple sequence ~lignm~nt (Figure 2) with coordinate 1 corresponding to base #
431 of a l~eltince S. cerevisiae 28S rRNA gene. (The numbers are comparable to the primary sequence of S. cerevisiae 28S rRNA gene. Genbank accession number: b 5 J01355). These sequences were obtained by amplifying and seqll~ncing 28S rDNA
from various fungi with primers SEQ ID NO: 1 and SEQ ID NO: 2. (SEQ ID NO: 1 - col,csl,onds to coordinates 403-422 and the SEQ ID NO: 2 coll~,s~onds to coordinates 645-662 of the reference S. cerevisiae gene).

An analysis of these aligned sequences enabled us to develop the species - specific probes SEQ ID NO: 3 to SEQ ID NO: 23, and sites for these probes are shown underlined. These 51 aligned sequences contain sufficient variability, to enable a person versed in this art, to develop additional species specific hybridization probes in the 10-50 nucleotide length. Similarly, longer species 15 specific hybridization probes encompassing the entire 200+ nucleotide length can ' also be envisioned. Species identification may also be accomplished by direct DNA
sequence ~ tion of any DNA amplified with primers SEQ ID NO: 1 and SEQ ID NO: 2. If the derived sequence m~t~h~ a~l~xilllately 98% or more of any sequence in SEQ ID NO: 24 to SEQ ID NO: 74, then the identity of the organism 20 can be asc~llahled. Additionally, we recognize that parts of SEQ ID NO: 24 toSEQ ID NO: 74 may be specific for groups of fungi arranged phylogenetically at the level of genus or higher. SEQ ID NO: 24 to SEQ ID NO: 74, their ; complements, along with any modification to these sequences may also potentially be utilized in assays for the identification of fungi based on ç~i~tin~ methodologies 25 and future technologies as noted above for SEQ ID NO: 1 to SEQ ID NO: 23.

, ,. .

W O96121741 PCT/Lb~GtG~26 Legend to figure 2:

l~he multiple sequence ~lignm~nt shows the sequence of 28S ribosomal RNA gen.es ~mI~lified with primers SEQ ID NO: 1 and SEQ ID NO: 2. 21 species 5 specific probes (SEQ ID NO: 3 to SEQ ID NO: 23) are shown lln-lPrlin~(l Minor sequence variation among two isolate of the same organism are repl~i,e.lled by the ~,o~liate code (see key below). Major dirr~rellces among Rhizopus species are depicted by including 3 separate R~tizopus sequences in the ~ nm~nt (The org~ni~m~ in this figure are listed according to their sequence rel~te~lnes~ ) Key to symbols:

(.) gap in sequence to f~ilit~te ~ nment (R)Aor G
15 (W) A or T
(Y) T or (~
(M)Aor~
(K) T or ~J
(S) G or C
20 (B)T,GarC

Acremo Acremonium species A_clav Aspergillus clavatus A_flav Aspergillus flavus 25 A_filmi Aspergillus fumigatus A_glau Aspergillus glaucus A_nidu Aspergillus nidulans - A_nige Aspergillus niger A_ochr Aspergillus ochraceus SUBSTlTUTE SHEET (R1JLE 26 ~' ~ .
, - Wo 96/21741 Pcr/Isg6/00026 A_terr Aspergillus terreus : A_~ngu Aspergillus unguis ~~ A_ ustu Aspergillus ustus - Beauve Beauveriaspecies ;7 Bipola Bipolaris species Blasch Blastoschizomyces species B_derm Blastomyces dermatitidis -' Chryso Chrysosporium species Clados Cladosporium species - 10 Curvul Curvularia species C_albi Candida albicans C_glab Candida glabrata C~uil Candida guilliermondii C_im mi Coccidioides immitis - 15 C_kefy Candida kefyr C_krus Candida krusei C_laur Cryptococcus laurentii C_lusi Candida lusitaniae -- C_neob Cryptococcus neoformans var gattii se~vlype B
,-- 20 C_neof Cryptococcus neoformans serotype A
C_para Candida parapsilosis ; C_terr Cryptococcus terreus - ~ C_trop Candida tropicalis .
. Fusan Fusarium species F caps Filobasidium capsuligenum - F_neoc Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C
F_neod Filobasidiella (Cryptococcus) neoformans var neoformans serotype D

SUBSTITUTE SI~EET ~RULE 26 W O 96/21741 PCT/L~ 26 F~ g Filobasidium uniguttulatum Geotri Geotrichum species H_caps Histoplasma capsulatum Malbra Malbranchea species Mucor_ Mucor species Paecil Paecilomyces species Penici Penicillium species P_boyd Pse~ lescheria boydii Rhizol Rhizopus species isolate #1 Rhizo2 Rhizopus species isolate #2 Rhizo3 Rhizopus species isolate #3 Sporot Sporothrix schenkii S_brev Scopulariopsis brevicaulis S_brum Scopulariopsis brumpti S_cere Saccharomyces cerevisiae T_beig Trichosporon beigelii Fllrther variations of the invention that utilize any of the named sequences 20 will be a~,l,~elll to those with Ol lhl~ skill in the art. The following examples illustrate various aspects of the invention but are not intPntle-l to limit its usefulness.

EXAMPI,E 1. Testing probes SEQ ID NO: 3 to SEQ ID NO: 23 for hybridization specificit~Y.
Probes listed in SEQ ID NO: 3 to SEQ ID NO: 23 were tested for specificity against their target org~ni~m~. Probe SEQ ID NO: S for Candida albicans was the - first one lested against a panel of fungi taken from the Mayo Clinic collection. 28S
rDNA frc,m Acremonium sp., Aspergillus clavatus, Aspergillusflavus, Aspergillus SuBsTlTuTE ,C~HFFT ~ c q~

~ =
' CA 02209247 1997-07-02 -~ W O 96/21741 PCT/lD'"00026 ~. .

, f~umig~tus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus -- ~ ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Aspergillus : - sp., Beauvaria sp., Bipolaris sp., Blastomyces dermatitidis, Candida albicans, ~-- Candidaglabrata, Candidaguilliermondii, Candidakefj~r, Candidahusei, = ~ . S Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium sp., . .
. ~ Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans sel(,ly~e A, .. ......... .
-: Curvularia sp., Fusarium sp., Geotrichum sp., Histoplasma capsulatum, Mucor sp., '~-. Penicillium sp., Pseudallescheria boydii, Rhizopus sp., Saccharomyces cerevisiae, Scopulariopsis brevicaulis, Sporothrix schen*ii and Trichosporon beigelii was : 10 amplified in a polymerase chain reaction using oligonucleotide probes SEQ ID NO:
,; 1 and SEQ ID NO: 2. All PCR amplifications were carried out as hot-start ,- - reactions in a 50 ul reaction volume using Perkin-Elmer (Norwalk, CT) 0.5 ml thin-~- wall poly~ ylene tubes and a Perkin-Elmer thermal cycler. Reagents added to the - tube initially were 2.5 ul of 10X PCR buffer (100 mM tris pH 8.3, 500 mM KCl, 15 15 mM MgCk), 5.0 ul of 50% glycerolll mM cresol red, 8.0 ul of dNTP mix (1.25 ~ i mM each of dATP, dGTP, dTTP and dCTP), 11 picomoles of each nucleic acid -- primer and sterile water to make up a volume of 25 ul. A wax bead (Ampliwax "
Gem-100, Perkin-Elmer) was added and the tubes heated to 77~C for 30 seconds and cooled to room temperature to form a wax barrier. 2.5 ul of 1 0X PCR buffer, . .
20 5.0 ul of 50% glycerol/1 mM cresol red, 0.2 ul Taq polymerase (AmpliTaq SU/ul, ''= Perkin-Elmer) and 15.3 ul of sterile water was added to the tube along with 2.0 ul of ~ -- DNA from the fungal whole cell boiled lysate described above. 50 cycles of =; . .
- thermal cycling was carried out at 94~C - 30 sec, 50~C - 1 min, 72~C - 2 min. Five -r-- - . microliters of polymerase chain reaction mix from each sample was run on a 5%

= - - 25 polyacrylamide gel to visually confirm the successful amplification of 28S rDNA
from each fungus listed above. 40 ul of the r~m~ining amplified 28S rDNA was -~~ denatured in 1 N NaOH, and half of this denatured rDNA was slot blotted on to a -~ positively charged polysulphone based membrane equilibrated in 0.5 N NaOH. The ~ = . . .
-- membrane was air dried for 15 minlltes and baked in a vacuurn oven at 80~C for 30 ;

--'~- SUBSTITUTE S,~EET(RULE 26) .. ~.~, , .

W O96/21741 PCT/Lb~G~'~CC26 ,.,i"l-~es. .~mplified rDNA from each species was now bound and immobilized at aseparate spot on the membrane. The free binding sites on the membrane were blocked by in~llh~tin~ the m~mhr~n.? for 3 hours at 40~C in hybridization buffer(100 ml of hyhri(li7~tion buffer was made using lg non-fat milk powder, 6g 5 NaH2PO~, 7g SDS, 200 ul 0.5M EDTA and adjusted to pH 7.2 with NaOH). The specific probe for Candida albicans (SEQ ID NO: 5) was end-labeled with radioacti~re phnsphorus using 32p ATP and T4 polynucleotide kinase. 50 picomolesof this probe was added to 70 millilit~r.s of hybridization buffer and the membrane was probed at 40~C overnight. The membrane was washed in hybridization buffer 10 at 40~C for 15 ~ es followed by a wash in 2X SSC at 40~C for 15 minl~tes The membran- was then exposed on x-ray film for at least 1 hour. The oligonucleotideprobe SEQ ID NO: 5 only hybridized to amplified 28S rDNA from Candida albicans (~see Table 3) Under these hybridization conditions, probe SEQ ID NO: 5is extremely specific for Candida albicans. The sequence of oligonucleotide probe 15 SEQ ID ~O: 5 differs from the sequences of other species of Candida by as few as 1 or 2 bases, but these mi~m~tches are sufficient to prevent stable hybrids fromforming with the other Candida species.

Probes SEQ ID NO: 3 to SEQ ID NO: 23 were tested for specificity, as 20 described above for the Candida albicans probe SEQ ID NO: 5, against the samepanel of fungi listed in the prece~ling paragraph. The positively charged polysulphone based membrane probed with Candida albicans probe SEQ ID NO: 5 was washed in 0.5 N NaOH at 40~C for 10 mintltes to remove all bound Candida albicans probe. The membrane was sequentially probed with all probes listed in 25 SEQ ID ~O: 3 to SEQ ID NO: 23. For each subsequently tested probe, the membran~ was blocked for at least 30 mimltes, probe hybridization was carried out -at 40-42~C for at least 3 hours, and post-hybridization washes were done in 2X SSC
for 20 minllte~ The membrane was stripped between probings by washing in 0.5 to 1.0 N NaOH at 40-42~C. Results are listed in Tables 3 to 6.

SUBSTITUTE SHEFT ~R~

e CA 02209247 1997-07-02 ~ WO 96/21741 PCT/IB96/00026 ;~

As shown in Tables 3 to 6, each probe listed in SEQ ID NO: 3 to SEQ ID
NO: 23 specifically hybridizes to only one target fungal 28S nucleic acid sequence.
- This specificity is essenti~l for id~;nliryillg a given species of fungus in clinical . ' i' !
ee;~ C~ mixed fungal or~ni~m~ with a_igh level of reliability. The 5 39 org~ni~m~ listed in these Tables re~res~;l.l a majority of or~ni~m~ that are ~ . , .
commonly isolated from clinical samples. While we have developed 21 species ~.=--specific probes (SEQ ID NO: 3 to SEQ ID NO: 23) that identify a total of 19 individual org~ni~mc, the additional org~ni~m~ listed in the test panel were used to ~ ensure that our probes did not have any cross-reactivity with other fungi likely to be - ~- 10 present in a clinical specimen. The ability to accurately and reliably diagnose, and ~~ identify to a species level, this large a number of pathogens is nnm~teh~cl by any ; other report. The fact that we can achieve this by probing DNA amplified by a .
single pair of "Universal" probes (SEQ ID NO: 1 and SEQ ID NO: 2) is highly advantageous as it saves time, money and effort by providing the ability to test a 15 single amplified target with 21 dirrelellt probes (SEQ ID NO: 3 to SEQ ID NO: 23).

.
--. .
- ~ A GenBank search was carried out with all probes listed in SEQ ID NO: 3 to SEQ ID NO: 23 in order to determine whether similar gene sequences were present =~ in the ~l~t~ba~e 28S sequences for Candida albicans and one serotype of 20 Cryptococcus neoformans are already present in GenBank, and as expected, the probes for Candida albicans and Cryptococcus neoformans correctly identified the- 28S sequences from these two o.~a,.~;.. ~ Ten other probes also matched DNA
sequences from a variety of genes not related to the 28S gene (Table 7). This was expected because short stretches of sequence identity can often be found for any~= 25 query sequence in unrelated genes from the same or a dirr~l~n~ org~ni~m This observation is known to those versed in this art. In all cases, sequences that matched - a probe sequence were not located within the 28S rRNA genes. Our probes are used . ~, - to analyze 28S DNA that has been previously amplified in a polymerase chain '. reaction with our probes SEQ ID NO: 1 and SEQ ID NO: 2. Under stringent .~j.

....
- = 28 -~ SUBSTITUTE SHEET (RULE 26) .- - ; - ~.

W O96/21741 PcTlLbr~o~26 conditions, these two probes only amplify DNA from fungal 28S rRNA genes.
The~rorc no amplified DNA from the non-28S genes listed in Table 7 will be available for the hybAdization of probes SEQ ID NO: 3 to SEQ ID NO: 23. The presence of related sequences in non-28S, unamplified genes will not be detected5 and will, thus, not have any effect on the sensitivity or the specificity of our detection and identification strategy.

SUBSTITUTE SHEET (RIJLE 26) ~=- CA 02209247 1997-07-02 W O 96/21741 PCTnB~6~C-26 . ~ .
- =:
.-~.
, . Table 3:
- ~ Detection of species specific 28S sequence wi~ probes SEQ ID NO: 3 to SEQ ID NO: 8 .
-FUNGUS SEQ ID: 3 SEQ ID: 4 SEQ ID: S SEQ ID: 6 SEQ ID: 7 SE;Q ID: 8 Acremonium sp.
Aspergillus clavatus Aspergillusflavus Aspergillus f o.. ~ . +
Aspergillus glaucus Aspergillus nidulans Aspergillus niger Aspergillus ocA, ~ce~
Aspergillus terreus Aspergillus unguis Aspergillus ustus Aspergillus sp.
Beauvaria sp.
Bipolaris sp.
Blastomyces ~, la~it~ - +
Candida albicans - - +
Candida glabrata Candida guilliermondii Candida kefyr Candida krusei Candida lusitaniae Candida parapsilosis Candida tropicalis Chrysosporium sp.
Cladosporium sp. - - - - - -Coccidioides immitis - - - +
CryrtQcOCCI'~ ne~,/u,.. ,~ - - + +
Curvularia sp.
Fusarium s p.
C2.,~ .~... sp.
Hi~tc~p/~ 7 CnrS1t~t7'~.7 Mucor sp.
Penicillium sp.
Pseu~ le~t.~heria boydii Rhizopus sp.
Saccharomyces cerevisiae Scopulariopsis brevicaulis Sporothrix sch~,.ck.i Trichosporon beigelii S

. --SUBSTITUTE SHEET (RULE 26 r~

W O 96/21741 PCT~B96/00026 + Positive Negative a~er 20 minute wash in 2X SSC

Table 4:
Det~ctinn of species specific 28S sequence with probes SEQ ID NO: 9 to SEQ ID NO: 14 S' FUNGUS SEQ ID: 9SEQID: 10SEQ ID: 11SEQ ID: 12 SEQ ID: 13 SEQ ID: 14 A.,_ ' sp.
Aspergillus clavatus - -Aspergillusflavus Aspergillus Ji o~t~
Aspergillus glaucus - +
Aspergillus nidulans Aspergillus niger - +
Aspergillus v. h. a. e..~
Aspergillus terreus - - - +
,~ spergillus unguis ~spergillus ustus spergillus sp.
. eauvariasp.
Bipolaris sp.
. Iastomyces c~, '' -Candida a,'bicans ~andida g 'abrata - - - +
C~mdida guill,'~, .lii - - +
C'andida kefyr Candida krusei Candida lusitaniae Candida parapsilosis Candida lr~iculi.~
Ch,~f~v~", sp.
Cladci.",."' sp.
Cocci~ioi les immitis CryFtococc~ neoformans Curvularia sp.
Fusarium sp.
Geut,.. ~. sp.
Histoplasma c~lrs~ +
Mucor s p.
PeniciWum sp.
Ps j'nllo~cheria boydii Rhizopus sp. - - - - - -.,accha, ~ yc~s cerevisiae ~copulariopsis brevicau.'is ~p-Jr vll~ ~ s~.. ,.~ii - -Tric~lo~ v~vn beigelii C~l IRCTI~I ITF Cl-IFFT r~ll 11 F

~ =
-~ CA 02209247 1997-07-02 -' , :
-. - W O96/21741 PCT/lb5~ 26 ~, :
.
~ .; . . .
; ~--F -= _, .
- '"
:i .............................................................................. .
- - - i + Positive - I Negative after 20 minute wash in 2X SSC
.

5 Table 5:
=,, .-. Detection of species specific 28S sequence with probes SEQ ID NO: 15 to SEQ ID NO: 20 ~:
; = ~ . .
- - - FUNGUSSEQ ID: 15SEQ ID: 16SEQ ID: 17 SEQ ID: 18 SEQ ID: 19 SEQ ID: 20 .=~; ~ A-" ~ sp.
Aspergillus clavatus Aspergillusflavus ' Aspergillush~ u~ - - - - - -Aspergillus glaucus Aspergillusnidulans Aspergillus niger Aspergillus o.,h, a, ~u~ - - - - - -Aspergillus terreus Aspergillus unguis Aspergillus ustus Aspergillus sp.
Beauvaria sp.
Bipolaris sp.
Blastomyces dermatitidis Candida albicans Candida glabrata Candida guilli~, ,.1.i - - -Candida kefyr +
Candida krusei - +
Candida lusitaniae - - +
Candida parapsilosis - - - +
Candida tropicalis - - +
Chry..~s~u, sp.
Clad~,v, .u .. sp.
Cocc'i~ D~ immitis Cryptococcus neoformans Curvularia sp.
usarium sp. - - - - - -Geut,.cl sp.
Hi~tQpl~ 7 c~
Mucor sp.
Penicillium sp.
Pseu~llo~cl7eria boydii - - - - +
Rhizopus sp.
SaCC~ .. .yces cerevisiae - - - - - -, ~ ,;
,, ~' ' .- ~

W O 96/21741 ~CT/lb,'~0~26 Scopulariopsis b,~v,cu..lii, Spu, vlh, ~ sc/~,..,h--'r". I~u~,uu, ~". beigelii + Positive Negative a~er 20 minute wash in 2X SSC

5 Table 6:
Detection of species specific 28S sequence with probes SEQ ID NO: 21 to SEQ ID NO: 23 FUNGUS SEQID:21 SEQID:22 SEQID:23 A., , sp.
Aspergillus clavatus - -Aspergillus flavus +
Aspergillus f ,~ulu~
Aspergillus glaucus ~spergillus nidulans spergillus niger ~spergillusoch,ac~
~spergillus terreus Aspergillus unguis Aspergillus ustus Aspergillus sp.
Beauvaria s p.
Bipolaris sp.
y. ~ d~, ~fic Candida albicans Candida glabrata Candida guilliermondii Candida kefyr Candida krusei Candida lusitaniae Candida parapsilosis Candida t, vr;culi, Cl~y~v,~uu~ ;..,.. sp.
Clad~,u, sp.
Cocci~ioi ~ immitis Gyf~lococ~ u~ neoformans Curvularia s p.
Fusarium sp.
Geulricl ùm sp.
Hi~toFI ~7 cnrs2/1~7tU~2 Mucor sp.
Penicillium sp.

SUB~TITI ITF ~ T IDI 11 r ~~ - CA 02209247 1997-07-02 . . . ~; . .
h W O96121741 PCTAB96/00026 ~, ..
.. ..

.
~ .
p~ Q~!heria boydii Rhizopus sp.
~,'' .. 'ac.,k.. , ~ c~ cerevisiae .- - "copulariopsis l"e~
- ~r70~ vlh ~ scl.~,.cl ii + +
r;e~lJ~o,O,. beigelii .

. ~ Positive ~- . - Negative after 20 minute wash in 2X SSC
_ 3 . : ~
. A
S Table 7:
~- GenR~nk search results listing genes from other or~ni~m~ having 100% identit,v to probes SEQ
D NO: 3 to SEQ ID NO: 23 -- , ~- ~ PROBEORGANISM GENE ACCESSION
= ~ SEQ IDMATCHED MATCHED* NUMBER
~-13 '=. ' NO: (see note below) ' 3, Aspergillus f- g~ lu~ 3 -7 - Blastomyces d~,~ 7titj~i~ 4 Streptomyces verticillus bleomycin acetyl L26955 .~ t 4 Giardia muris upstrearn of rRNA X65063, S53320 genes 4Aspergillus nidulans uric acid-xanthine X71807 - - - 4 HomosapiensT-cellsurface X16996 gl~,u~)lut~
-: ~ 4Homo sapiens MIC2 M 16279, M22557, J03841, M22556 Candida albicans 5 Candida albicans 28S r~A L28817 ,~ Cocci~ioi~P~ immitis 6 - -'. - Cryptncocc2~ neoformans 7 Cryptococcus neoformans28S rR~A L14067, L14068, Cryptncoccu~ neoformans 8 Cryptococcus ne~,.,.~28S rR~A L14067, L14068, = L20964 - . 8Escherichiacoli 0111 cld Z17241 Hi~topl, ~7 ~nr/S1/lnt7~ 7 9 - : Aspergillus glaucus 10 Pse~ nas cob genes M62866 - ~ denitrif cans Aspergillus niger 11 spergillus terreus 12Human cytomegalovirusgenome X17403 12Homo sapiens GABA receptor L08485 . Candidaglabrata 13Homosapiens Class I MHC X03664,X03665 ~ ~ . Candida guilliermondii 14 ~. Candida keJj~r 15 . ~ . . ..

~ -, . .

-=;, ~
~ 34 ., ~ SUBSTITUTE SHEET (RULE 26) W O96/21741 PCT/LbYG~'~OC26 Candida h~use~ 16 P~~ c~nas syringaep~nir~ nbinding L28837 prote~n Candida l~itaniae 17 Chicken AK1 D00251 17 Mouse IL10 M84340 Candida parapsilosis 1 Polytomella agilisbeta-2 hlbulin~33' 73 ~Tobacco chlo}oplast genome Z000~4, 54304 Aedesae~p~i amylase L~3~ 40 Homosapiens ~,1.l.. ~5~.. c 13ql4 L14473 Candida Ir~ ~ ' 1~
P~ C~ ~eria boydii 20 Drosophila mel~ g~_ ., AcTr66B X71789 Cow actin 2 D12816 Aspergillusflavus 21 Spu~v~h~LA s~,h.~ ii 22 Spu, vlh, ~ sch_,.. ,~ii 23 Sulfate reducing bacteria protein D21804 23Equine h~ I genome M86664 * Note: As ~ cllssec earlier in this document, the presence of sequences similar to probes SEQ
ID NO:3 to SEQ ID ~O: 23 in genes not related to 28S does not have any effect on the specificity or sensitivity of our d iagnostic strategy. Our species specific probes are used to analyze 28S DNA
S that has been previously amplified in a polymerase chain reaction ~,vith our probes SEQ ID NO: 1 and SEQ ID NO:2. l~ese two probes will not amplify DNA from any gene other than 28S in column #4 (GENE M ATCHED), and therefore no amplified DNA from these non-28S genes will be available for the hybridization of probes SEQ ID NO: 3 to SEQ ID NO: 23.

10 EXAMPLE 2. Use of method in example 1 to test clinical specimens for specific fungal org~ni~m~

Clinical samples taken from the respildlory and ga~lloi-,le~ l tract of healthy individuals almost always contai n some fungal flora. Most of these fungi are non-pathogenic, but may give 15 false positives on traditional immunnchemical diagnostic tests for pathogenic fungi.

We obtained 44 clinical specimens from diverse sources ranging from sputum and incision drainage tubes, to inb~rvertebral disc and lung biopsies. Traditional smear and culture results showed that all 44 specimens contained at least 1 type of fungus. In order to test the efficacy of SUBSTITUTE SHEET (RULE 26) ~ CA 02209247 1997-07-02 . ~ ~

., , our probes, we extracted DNA from all 44 clinical samples and used probes SEQ ID NO: 1 & 2 in a polymerase chain reaction to amplify fungal 28S sequences present in these samples.

Y ~ DNA was extr~ ted from all clinical samples by our modification of the technique of S Chomczynski and Sacchi which originally described the use of acid gl-~nic1inium thiocyanate-.
phenol-chloroform to pl~,r~lclllially extract RNA from cells and tissues. We replaced room = - t~ dlule cell lysis by boiling lysis, and acid g~l~nidinium thiocyanate-phenol-chloroform = -~ extraction by ~lk~lin~ phenol-guanidine thiocyanate to ~ r~,r~llLially extract DNA from cells. 1.5 : ~ ml Sarsted (Newton, North Carolina) polypropylene screw cap tubes with o-ring seals were used - 10 for the extractions. 200 ul of specimen was added to 500 ul of GPT reagent (6 M gll~ni~line - - thiocyanate dissolved in 50 mM tris pH 8.3 mixed with an equal volume of phenol buffered in tris - pH 8.0). This was mixed by vortexing and im m~ tely placed in a boiling water bath for 15 J~ es. The tubes were spun in a microcentrifuge for 5 seconds and 250 ul of chloroform/iso-amyl alcohol (24:1 by volume) was added and mixed by vortexing. The liquid phases were 15 separated by centrifugation for 10 minlltes and 450 ul of aqueous (upper) phase was transferred to a fresh tube. The aqueous phase was mixed with 500 ul of 100% isoprol)allol and placed at -20~C
for at least 1 hour. At the end of this period the tubes were centrifuged for 15 minutes and the - supçrn~t~nt removed without disturbing the nucleic acid pellet. The pellet was washed with 500 ul - of ice-cold 70% ethanol to remove traces of GPT reagent by gently inverting 2 times and then '- 20 c~ ;ruged for 5 minlltt?c The ethanol was removed and the pellet dried in a speed vac for 10 I..i,.,-~es. The pellet was resuspended in 25 ul of sterile deionized water and 5 ul was used in a 50 ul PCR amplification. The PCR was carried out as a hot-start reaction using the thermal cycling - conditions for probes SEQ ID NO: 1 and SEQ ID NO: 2 described in example 1. Gel electrophoresis showed that probes SEQ ID NO: 1 and SEQ ID NO: 2 successfully amplified - 25 DNAfromall 44 specimens.

-~ - The amplified DNA from each specimen was transferred to a positively charged .; .
~ ' polysulphone based membrane. We radioactively labeled our species specific probes SEQ In ~--, SUBSTlTlJTE SHEET (RULE 26) . .; ~

WO 96/21741 PCT/lk5GlO0026 NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and sequentially probed the membrane to test for the presence of 28S rDNA firom Aspergillus fumigatus, Candida albicans, Coccidioides immitis and C7yptoccccus neoformans respectively. Membrarle blocking, probe hybridization and washes were done ex~ctly as described in example 1. The results are shown in Table 8.
s No false posi~ives were observed, indicating a specificity of 100% for these 4 probes in the clinical spe~im~n~ te~ted. 10 out of 12 culture positive samples forAspergillusfumigatus, and 11 out of 13 samples of 'andida albicans were identified, in-li5~tin~ a detection sensitivity of about 85% for ~ese two pn~bes. Additionally, two out of two Coccidioides immitis and two out of two 10 Cryptococcus neoformans were correctly identified (detection sensitivity of 100%). As seen by these results, ~e probes described in this invention can be used on a diverse variety of clinical specimens with exce]lent efficacy.

Table 8.
Detection of Aspergi,~lus fumigatus, Candida albicans, Coccidioides immitis and Cryptococcus neoformans in clinical specimens using species specific probes.

Specim~n typesmear and culturePCR withSEQ ID: 3SEQ ID: 5 SEQ ID: 6 SEQ ID: 7 results SEQ ID: 1, 2 U035 sputumA. flavus +
U069 pleuraA. fimnig~ c + +
U070 bronchial wash A. flavus +
M019 bronchial wash A. fi-rnig~tl-c + +
M020 sputummixed fiungal flora + - +
X35254 sputumC. albicans + - +
M20910 sputumA. fiunnig~l~lc + +
M055 sputumC. albicans + - +
M056 ahfl~min~l mixed fungal flora +
M057 drainagetube C. albicans + - (-M059 ind. sputum c. aibicans + - +
M060 ind. sputum mixed fungai flora +
M083 bronchialwash C. albicans + - +
M084 sputumA. fumigatus + (-) SUBSTITUTE SHEET (RULE 26) - - WO 96/21741 PCr/IB96/00026 . .
~ .
M085 throatC. albicans +
A001 sputum A. r~.. ig,.,-.~ +
~: A002 leg Blastomyces +
~= A003 leg Blastomyces +
: ~ - A005 discA. fi~mi~;~hlc +
-- - A037 disc A. r... ig,.l.. c +
i~ A039 tracheaC. albicans +
-- A040tracheaC. albicans +
~ ~ A102 ~ en.a A. r,.. ;g~l~,c +
~: : Y004 sputum C. albicans +
- Y016 induced sputum Cocc;dioides +
~~ : Y028 sputum Coccidioides +
- J003 chest Aspergillus sp. +
J045 bronchial wash C. albicans +
~- J046 ethmoid yeast +
- J047 chest A. filmi~ lc +
J048 sputum C. albicans +
y J073 lung Aspergillussp. +
'~ 7 - J074 lung A. r... i~ +
~' U017 lipA. filmi~tllc +
- - U033 sputummixedfungalflora +
U071 sputumC. albicans +
~ U072 BA lavage Spulull.. i~ +
-~. U073 knee Hi~lo~ .. .a +
U074 Il-alldil)l~ Cr~,~,lucoc~ s +
- ~ U075 CSF Cry~l-,coc.u~ +
U076 knee Histoplasma +
U077 soft tissue Histoplasma +
~, U051 buccal A. filmig~tllc +
~ ,~ Y055 sputum mixed fungal flora +
- + Positive - Negative (-) Missed - ~.

.
. ,. - .

. .

, =. .

: . ~
, . .

=.
~.

~:, =
.

SUBSTITUTE SHEET (RIJLE 26 .
. ~ . . i W O96/21741 PCTlLDr~ 26 EXAMPLE 3. DNA sequence based identific~tion of unlcnown fungal org~ni~m.c.

Ar~other utility of our probes is in the rapid DNA sequence based identification of a pure cullure of fungus. Probes SEQ ID NO: 1 and SEQ ID NO: 2 are used in a 5 polymerase chain reaction to amplify 28S rDNA from an unknown fungus. Probes SEQ
ID NO: 1 or SEQ ID NO: 2 are then used as seqllPncinp primers to obtain DNA sequence from this ~1mplifiçd 28S DNA belonging to the unknown fungus. This DNA sequence is co~ d ed to the fungal 28S DNA sequences in our ~l~t~b~e, and a sequence match at, or overlappir.g any one of the probe sequences in SEQ ID NO: 3 to SEQ ID NO: 74 will 10 co~ -ll the identity of the fungus. This technique cannot be used directly on clinical samples, as these usually contain DNA from more than one fungus, and the DNA
sequence p,~;lwldl~d will consist of overlapping sequences of several org~ni~m~. This technique ~as utility in rapidly and reliably identifying colonies of a single fungus on culture plates, clinical specimens, food, ph~ eutical, environm~nt~l or other samples 15 cc)~ p only one species offungus.

EXAMPLIE 4. Capture and identification of target DNA or RNA

All primers and probes described in this invention disclosure may be labeled with 20 any detect~ble reporter or signal moiety including, but not limited to radioisotopes, enzymes, ;mtigens, antibodies, chemilllmin~scent reagents and fluorescent chemicals.
Additionally, these probes may be modified without ch~nping the substance oftheir purpose by terrnin~l addition of nucleotides designed to incorporate restriction sites or other usefill sequences. These probes may also be modified by the addition of a capture 25 moiety (ineluding, but not limited to para-magnetic particles, biotin, fluorescein, dioxigenin, antigens, antibodies) or ~ttzl~hed to the walls of microtiter trays to assist in the solid phase capture and purification of these probes and any DNA or RNA hybridized to ~ these prob~s. Fluorescein may be used as a signal moiety as well as a capture moiety, the latter by interacting with an anti-fluorescein antibody.

.Cil IRRTITI ITF ~F~T ~ q~

}- CA 02209247 1997-07-02 .. :. . - i ~ W O96/21741 PCT/LD~'JG~26 :
. ~, . .
- ~- A typical utility of these modifications would be as follows. Primers SEQ ID NO:
. .
1 and SEQ ID NO: 2 would be utilized to amplify 28S rDNA from a sample, if present, as described previously. Primers would be modified so as to contain a biotin moiety at their - ~ ~ 5' ends. A streptavidin solid phase, such as a par~m~nPtic particle, would be used to 5 s~dl~ PCR products, if present, from the reaction mi~ C. The amplified target may ~- be subsequently hybridized to a third probe ((SEQ ID NO: 3) to (SEQ ID NO: 74) or - their complen ~nt.c) attached to a detectable moiety to ~l~te. ~-~;ne which species of fungus is present in the given sample. Multiple probes, each labeled with a diLr~.enl detectable ; ~ ~ moiety may be used at one time to analyze the amplified target.
'~ 10 . .
-~ Alt~rn~tively, Primers SEQ ID NO: 1 and SEQ ID NO: 2 would be utilized to . ;
' amplify 28S rDNA from a sample, if present, as above. In a separate reaction, individually, either SEQ ID NO: 1 or SEQ ID NO: 2 would be modified by ~ rhment to a solid phase capture moiety, such as a par~m~nPtiC particle, and SEQ ID NO: 3 to SEQ
15 ID NO: 74 (or their complements) would be modified by addition of a detectable moiety.
- Alternately, in the amplicon, any sequences delimited by SEQ ID NO: 1 and SEQ ID NO:

2, including but not limited to SEQ ID NO: 3 to SEQ ID NO: 74, may be used in the 1 ,.
- design of a capture probe. One of the probes ~q1tz~ch~l to a solid phase (SEQ ID NO: 1 and SEQ ID NO: 2) or any other ~ l;ately designed sequences and one of the probes 20 modified by ~ çhment to a detectable moiety (SEQ ID NO: 3 to SEQ ID NO: 74 or their . ~ complements) would be hybridized together, in solution, to products of the PCR, if they w; had been generated. The hybrids, if present, would be captured from the solution, and analyzed by a method a~l).o~.iate to the detection moiety. ~etection of the hybridized ~E probe would indicate which species of fungus was present in the given sample. Multiple = . ,.
:- =" - .~ 25 probes, each labeled with a dir~r~ detectable moiety may be used at one time to analyze the amplified target.

.

. ...
~!~4 ' ..
~_~; 30 :.
, ; -~, SUBSTlT~ SHEET(RUL~ ~Ç) W O96/21741 PCT/Lb9G~0026 EXAMPl,E 5. Species-specific amplification of fungal DNA

Another utility of the probes described in this invention is their usage as primers in the direct rietection of a specific fungal species by virtue of a nucleic acid amplification S reaction. fn this embo-lim~nt, one primer is a u~iv~l~al one, such as (SEQ ID NO: 1) or (SEQ ID ]~0:2), and the other is a species-specific primer selectecl from the group cnn~i~tin~ of (SEQ ID NO:3) to (SEQ ID NO: 23) or the complement~ thereof. One variation of this approach is the substitution of (SEQ ID NO: 1) or (SEQ ID NO:2) with any functional sequence located in proximity to the species-specific primer. Another 10 variation of this approach is the selection of any a~lol";ate species specific primer pair from SEQ ID NO: 24 to SEQ ID NO: 74.

SUBSTITIITF .SHFFT ~RI 11 R

-~ CA 02209247 1997-07-02 -W O96/21741 PCT/lb5G~o~-26 ~ . .
SEQUENCE LISTING

' (1) GENERAL INFORMATION:
:' ~ v .- 5 (i) APPLICANT:
- (A) ~n~lhll, Gu~preet S.
(B) Kline, Bruce C.

.
- (ii) TITLE OF INVENTION:
Nucleic Acid Probes for the Det~ction and Identification of Fungi .
.~ (iii) NUMBER OF SEQUENCES: 23 ..
-~
.=
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Ciba Corning Diagnostics COIP.
- (B) STREET: 63 North Street (C) CITY: Medfield - (D) STATE: M~c~chlleett~
~ .
(E) COUNTRY: USA
-~ 20 (F)ZIP: 02052 .
- "~ (v) COMPUTER READABLE FORM:
-~ (A) MEDIUM TYPE: Diskette 3.5 inch, 1.44 Mb storage ~; (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS 6.2 - (D) SOFTWARE: Word 6.0 -- .
(vi) CURRENT APPLICATION DATA: 2?
,rc: ~ (A) APPLICATION NUMBER:
-- . 30 (B) FILING DATE:

.~
.
- ,. .
;; 42 TUlE Sr~
.... , ~ ~

W O 96/21741 PCT/lb_''nO026 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(viii) ATTORNEY INFORMATION:
(A) NAME: M~ t~,lll, Arthur S.
(B) REGISTRATION NUMBER: 28,244 (C) DOCKET NUMBER: CCD-180 (ix~ TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 508 359-3836 (B) TELEFAX: 508 359-3885 (2) INFO~MATION FOR SEQ ID NO 1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for fungal org7lni~m~

(iii) HYPOTHETICAL: No (ivj ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

U~E~

= ~ ~

W 096/21741 PCT~B96/00026 =
. t, ..
:.' '~ ~ (3) INFORMATION FOR SEQ ID NO 2 .
-- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 , :-~ S (B) TYPE: nucleic acid - (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
- - (ii) MOLECULE TYPE: Nucleic acid probe for fungal or{~ni~m~
~ 10 ~, -~ (iii) HYPOTHETICAL: No ~- (iv) ANTISENSE: No ' ' ~- 1 15 (v) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

~- GACTCCTTGG TCCGTGTT 18 (4) INFORMATION FOR SEQ ID NO 3:
; 20 --- (i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single - 25 (D) TOPOLOGY: linear -. t - = ~ (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus fumigatus : -- (iii) HYPOTHETICAL: No ~ 30 .~

SUBSTITUTE SHEET (RULE 26) W 096~21741 PCTILD,.'~OJ26 (iv) ANTISENSE: No (v' SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(5) INFORMATION FOR SEQ ID NO 4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii 3 MOLECULE TYPE: Nucleic acid probe for Blastomyces dermatitidis (iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v~ SEQUENCE DESCRIPTION: SEQ ID NO: 4:

25 (6) INFORMATION FOR SEQ ID NO 5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single SUBSTITUTE SHFFT IRI ll F

-~ ' '; CA 02209247 1997-07-02 .' W O96/21741 PCT/lb~<'J~ 26 ; ~, .
_ i ~ . .
. ' . ~ (D) TOPOLOGY: linear , (ii) MOLECULE TYPE: Nucleic acid probe for Candida albicans ., - 5 (iii) HYPOTHETICAL: No , ~. .
~~ ~ (iv) ANTISENSE: No i . .
. .
(v) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
~'''' 10 t~ ' : CCTCTGACGA TGCT 14 ....
.~--= -...3;
- (7) INFORMATION FOR SEQ ID NO 6:

~ . .
(i) SEQUENCE CHARACTERISTICS:
...... .
(A) LENGTH: 14 .
(B) TYPE: nucleic acid -f;- ' (C) STRANDEDNESS: single ~- (D) TOPOLOGY: linear ~ .

- ; ~ 20 - i ~- (ii) MOLECULE TYPE: Nucleic acid probe for Coccidioides immitis .
- -~ (iii) HYPOTHETICAL: No . . .
; .
-~ 25 (iv)ANTISENSE: No _ r , ~ (v) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

-~~ ' TCTGGCGGTT GGTT 14 ~. 30 (8) INFORMATION FOR SEQ ID NO 7:

. .
~ . .

St~BSTITUTE StlEET (RULE 26) .~ .

W O96/21741 PCT/lb~ ~~26 i)i SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for Crypfococcus neoformans (iii) HYPOTHETICAL: No ) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(9) INFO]RMATION FOR SEQ ID NO 8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for Cryptococcus neoformans (iii) HYPOTHETICAL: No (i~J) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

SUBSTITUTE SHEET (RUI E 26) - . W O96/21741 PCT/Lb56/00026 :

.
~ AGTTCTGATC GGTG 14 . . ~
. (10) INFORMATION FOR SEQ ID NO 9: ~
~. .~ =
- -- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 - . (B) TYPE: nucleic acid - (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
~ -~
~ - (ii) MOLECULE TYPE: Nucleic acid probe for Histoplasma capsulatum ., ~
(iii) HYPOTHETICAL: No (iv) ANTISENSE: No ~' (v) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
-:- -.
-- ; (11) INFORMATION FOR SEQ ID NO 10: ~
.:
(i) SEQUENCE CHARACTERISTICS-. .
(A) LENGTH: 14 (B) TYPE: nucleic acid -~i - (C) STRANDEDNESS: single . .
(D) TOPOLOGY: linear ~ -, (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus glaucus ,~.
~ -:
., SUBSTITUTE SHEET ~RULE 26) wo s6r2l741 PCT/IB96/00026 (iii;) HYPOTHETICAL: No (ivj ANTISENSE: No S
(v) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

10 (12) INFORMATION FOR SEQ ID NO 11:

(i) ~EQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus niger (iii'~ HYPOTHETICAL: No (iv'l ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

(13) INFO.~MATION FOR SEQ ID NO 12:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 SuBsTlTuTE ~HFFT ~?111 r W O96/21741 PCT/lD,.~__26 .

- (B) TYPE: nucleic acid = (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus terreus , ~ ;~ (iii) HYPOTHETICAL: No , .
~ . .
-~ ~ (iv) ANTISENSE: No ~,, 10 _ e .
(v) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

.
., ' - 15 (14) INFORMATION FOR SEQ ID NO 13:

-'~ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 .D .
~; (B) TYPE: nucleic acid (C) STRANDEDNESS: single -~ (D) TOPOLOGY: linear .
r - - - (ii) MOLECULE TYPE: Nucleic acid probe for Candida glabrata -~- 25 (iii)HYPOTHETICAL: No ; (iv) ANTISENSE: No , Y

~ . (v) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

:~- 30 CTTGGGACTC TCGC 14 i -SUBSTITUTE SHEET (~llLE 2~i) W O 96~21741 PCTAB96/00026 (15) INFC.RMATION FOR SEQ ID NO 14:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii'g MOLECULE TYPE: Nucleic acid probe for Candida guilliermondii (iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v'g SEQUENCE DESCRIPTION: SEQ ID NO: 14:

(16) INFORMATION FOR SEQ ID NO 15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: Nucleic acid probe for Candida kefyr (iil) HYPOTHETICAL: No Sl IR~TlTl ITL c~LI~rT ,nl .. _ ~.

=,-.. ~ CA 02209247 1997-07-02 ~ W O96/21741 PCT/Lb~5.'~5~26 . ~ .

-- - .
... :
.
(iv) ANTISENSE: No ~, ~ . 4 .
~' (v) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
- -... ..
.. . , --~ TTCGGCTTTC GCTG 14 =-~=. (17) INFORMATION FOR SEQ ID NO 16:
. . , ~ T..i;
~ . .
: 10 (i~ SEQUENCE CHARACTERISTICS:
.- j---. = . .
(A) LENGTH: 14 (B) TYPE: nucleic acid . . ~ . . .
~. - (C) STRANDEDNESS: single . . .
=- (D) TOPOLOGY: linear ~ ~- (ii) MOLECULE TYPE: Nucleic acid probe for Candida krusei ,, f ~, , (iii) HYPOTHETICAL: No , 20 (iv) ANTISENSE: No ~ . c -= - - (v) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
.

= . .
~' GGGATTGCGC ACCG 14 ,~ ~
c-~ ~ - 25 (18) INFORMATION FOR SEQ ID NO 17:
= ~ ~
~~- (i) SEQUENCE CHARACTERISTICS:
- R - (A) LENGTH: 14 e .a~F
~ -~ (B) TYPE: nucleic acid ,~ ., .
- ~ 30 (C) STRANDEDNESS: single .
,, . i .
~. . l 52 . = .~, / .

W O 96/21741 PCT/lb5G~ C26 (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: Nucleic acid probe for Candida lusitaniae (ii~) HYPOTHETICAL: No (iv) ANTISENSE: No (vi SEQUENCE DESCRIPTION: SEQ ID NO: 17:

(19) INFORMATION FOR SEQ ID NO 18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for Candida parapsilosis (iii) HYPOTHETICAL: No ) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

30 (20) INFORMATION FOR SEQ ID NO 19:

SUBSTITUTE SHEET (RULE 26) _ . . t ~ ,_ -- . W O 96/21741 PCT/l~ C~26 .: :
'~
. .

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid =
~;.; . 5 (C) STRANDEDNESS: single ~'- (D) TOPOLOGY: linear :; ~.. , (ii) MOLECULE TYPE: Nucleic acid probe for Candida tropicalis (iii) HYPOTHETICAL: No ~, ~
~ lV) ANTISENSE: No , ' ~.'J --: (v) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
.. ~ .
~ . 15 _~, .

. ., = ~
... .
~- . (21) INFORMATION FOR SEQ ID NO 20:

., .
=,. , -- .
- 20 (i) SEQUENCE CHARACTERISTICS:
- ~ ~ (A) LENGTH: 14 (B) TYPE: nucleic acid - - (C) STRANDEDNESS: single = . (D) TOPOLOGY: linear . . .
(ii) MOLECULE TYPE: Nucleic acid probe for Pseudallescheria boydii , j, .
. = .
~. = ~
; (iii) HYPOTHETICAL: No . . ~
.. - ~
(iv) ANTISENSE: No . ~ ,.
~ j~3 30 -;
~ . .
. _ -': 54 SUBSTITUTE SHEET (RULE 26) , . .

(v) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GCGATGl~GAA TGTG 14 (22) INFO~MATION FOR SEQ ID NO 21:

(i) .3EQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillusflavus (iii1 HYPOTHETICAL: No (iv ~ ANTISENSE: No ~ (v) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

(23) INFORMATION FOR SEQ ID NO 22:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single - (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe for Sporothrix schenckii StJBST~TUTE SHEET (RULE 26) = =

W O96/21741 PCT/L~ J~_26 . .~j , ' .
~ (iii) HYPOTHETICAL: No ~ 4 .-- . ' . (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

. . , ~ i 1 CGGACCACCC GGCG 14 .
.
. . .
(24) INFORMATION FOR SEQ ID NO 23:

= ~
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 -- (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear , (ii) MOLECULE TYPE: Nucleic acid probe for Sporothrix schenckzi -(iii) HYPOTHETICAL: No = (iv) ANTISENSE: No -~ (v) SEQUENCE DESCRIPTION: SEQ ID NO: 23:. .~ ,.

.

7' 25 . ---~ (25) INFORMATION FOR SEQ ID NO 24:

; ~ (i) SEQUENCE CHARACTERISTICS:
~ ~: (A) LENGTH: 208 : 30 (B) TYPE: nucleic acid .
_ !

-'; SUBSTITUTE SHEET (RULE 26) 7 ,5 ._ .
.. .
.

W O96/21741 PCT~B96/00026 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Acremonium species specific region of 28S gene.
s (iii) HYPOTHETICAL: No (iv~ ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

GAI~CAGACTT GGG~1CG~1G AATCATCCGG C~L1~1CGCC GGTGCACTTT
GCI'~1CC~AG GCCAGCATCA GTTCGCGCCG GGGGATAAAG GTTTCGGGAA
TGIrAGCTCCT TCGGGAGTGT TATAGCCCGT TGCGTAATAC CCTGGCGTGG
ACrGAGGTCC GCGCTCTGCA AGGATGCTGG CGTAATGGTC ATCAGTGACC
CGTCTTGA

20 (26) INFORMATION FOR SEQ ID NO 25:

. (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Aspergillus clavatus specific region of 28S gene.

r (iii) HYPOTHETICAL: No SUBSTITUTE SHEFT ~Rl 11 .
-- W O96121741 PCT/lb9GJ'~C26 .
~ . .
~ ~ (iv) ANTISENSE: No ';
_ . t =: (v) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

~ . .
.,~ . 5 GACCAGACTC GCTCGCGGGG TTCAGCCGGC ATTCGTGCCG GTGTACTTCC
CC~lGGGCGG GCCAGCGTCG GLllGGGCGG CCG~l~AAAG GCCTCCGGAA
TGTATCACCT ~lCGGG~ l'~'L CTTATAGCCG GGGGTGCAAT GCGGCCTGCC
~' TGGACCGAGG AACGCGCTTC GGCTCGGACG CTGGCGTAAT GGTCGTA~AT
0 GACCC~l~ll GA
.:
.
~.
., - - (27) INFORMATION FOR SEQ ID NO 26:
. J ~

;, - 15 (i) SEQUENCE CHARACTERISTICS:
~= (A) LENGTH: 212 1 - (B) TYPE: nucleic acid - --- (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~- 20 (ii) MOLECULE TYPE: Aspergillus flavus specific region of 28S gene.
-s -~ (iii) HYPOTHETICAL: No .

~- (iv) ANTISENSE: No :- - 25 , . (v) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
. ~ T ~
' i ' GACCAGACTC GCCTCCAGGG TTCAGCCGGC ATTCGTGCCG GTGTACTTCC
-. ~ CTGGGGGCGG GCCAGCGTCG GTTTGGGCGG CCGGTCAAAG GCTCCCGGAA
_e, 30 TGTAGTGCCC TYCGGGGCAC CTTATAGCCG GGAGTGCAAT GCGGCCAGCC
_- TGGACCGAGG AACGCGCTTC GGCACGGACG CTGGCATAAT GGTCGYAAAC
.!,,i -- GACCC~l~ll GA
:. ., ;.
.~ ' . , ~- - SUBSTITUTE SHEET ~RULE 26) =

wo 96/21741 Pcr/Isg6l00026 (28) INFORMATION FOR SEQ ID NO 27:

(i) ~EQUENCE CHARACTERISTICS:
(A) LENGTH: 212 S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Aspergillus fumigatus specific region of 28S gene.
(iii' HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

GAC'CAGACTC GCCCGCGGGG TTCAGCCGGC ATTCGTGCCG GTGTACTTCC
CCGTGGGCGG GCCAGCGTCG GTTTGGGCGG CCGGTCA~AG GCCCTCGGAA
TGI'ATCACCT CTCGGGGTGT CTTATAGCCG AGGGTGCAAT GCGGCCTGCC
TGGACCGAGG A~CGCGCTTC GGCTCGGACG CTGGCGTAAT GGTCGTA~AT
GAC'CCGTCTT GA

25 (29) INFORMATION FOR SEQ ID NO 28:

(i) ',EQUENCE CHARACTERISTICS:
(A) LENGTH: 212 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET ~RII! F ~fi~

~ CA 02209247 1997-07-02 _ . W O96/21741 PCT/lb5G~ 26 ~ .--~ .
(ii) MOLECULE TYPE: Aspergillus glaucus specific region of 28S gene.
, . ,,~, ~ .
. .;
~.. .
~ ~ ~. (iii) HYPOTHETICAL: No , ,.~ . , , . ~
, . .
(iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
.
. ~ .

. . CCGGGGGCGG GCCAGCGTCG GTTTGGGCGG CCG~L~AAAG GCCCCTGGAA
: . ' . TGTAACGCCT CTCGGGGCGC CTTATAGCCA GGGGTGTCAT GCGGCCAGCC
, ~ ' TGGACCGAGG AACGCGCTTC GGCACGGACG CTGGCATAAT GGTCGTA~AC
.. - GACCC~L~lL GA
, .
(30) INFORMATION FOR SEQ ID NO 29:
;=; !
= .
~ -. (i) SEQUENCE CHARACTERISTICS:
~ ;
(A) LENGTH: 213 ."
~-_ (B) TYPE: nucleic acid . _~

.- ; (C) STRANDEDNESS: single ~ (D) TOPOLOGY: linear ; - .
.

(ii) MOLECULE TYPE: Aspergillus nidulans specific region of 28S gene.
f -, . .
.
- (iii) HYPOTHETICAL: No -, ~ .
... .
~- ~ (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

~ ., ~
.
- SUBSTITUIE SHEET (RULE 26) , .-, :

W O96121741 PCT/LD~ 26 GAi~CAGACTC GGCCCCGriGGG TTCARCCAGC A~ G~lG GTGTACTTCC
CCtaGGGGCGG GCCAGCGTCG ~LllGGGCGG CC'G~l~AAAG GCCCCAGGAA
TGTATCGCCC lCCGGG~llG TCTTATAGCC lGGG~lGCAA TGCGGCCAGC
CC-JGACCGAG GAACGCGCTT CGGCACGGAC GCTGGCGTAA TGGTCGCA~A
CG:~CCCGTCT TGA

10 (31) INFC~RMATION FOR SEQ ID NO 30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii,i MOLECULE TYPE: Aspergillus niger specific region of 28S gene.

(iii) HYPOTHETICAL: No .

(iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
GA~,CAGACTC GCCCGCGGGG TTCAGCCGGC ATTCGTGCCG GTGTACTTCC
CCGTGGGCGG GCCAGCGTCG ~LllGGGCGG CCGGTCA~AG GCCCCTGGAA
TGTAGTRCCC TCCGGGGYAC CTTATAGCCA GGGGTGCAAT GCGGCCAGCC
TG(,ACCGAGG AACGCGCTTC GGCACGGACG CTGGCATAAT GGTCGTA~AC
GACCCGTCTT GA

SUBSTITUTE SHEET (RULE 26) A' W O96/21741 PCT/lk~G~ 26 -. (32) INFORMATION FOR SEQ ID NO 31:

~ (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 212 S (B) TYPE: nucleic acid (C) STRANDEDNESS: single - (D) TOPOLOGY: linear : (ii) MOLECULE TYPE: Aspergillus ochraceus specific region of 28S gene.
., 10 ~- (iii) HYPOTHETICAL: No ~ s - . (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

- GACCAGACTC GCCCGCGGGG TTCAGCCGGC ATTCGTGCCG GTGTACTTCC
.~ CCGCGGGCGG GCCAGCGTCG GTTTGGGCGG CCGGTCA~AG GCCCCCGGAA
, -- TGTAGCACCC TTCGGGGTGC CTTATAGCCG GGGGTGCAAT GCGGCCAGCC
- -. 20 TGGACCGAGG AACGCGCTTC GGCACGGACG CTGGCATAAT GGTCGTAAAC
, GACCC~l~Ll GA

(33) INFORMATION FOR SEQ ID NO 32:
~-~ 25 -- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH:
~. (B) TYPE: nucleic acid -~- (C) STRANDEDNESS: single -, ~ 30 (D)TOPOLOGY: linear .
, SUBSTITUTE SHEET ~RULE 26) W O96121741 PCT/LD, ~0~26 (ii' MOLECULE TYPE: Aspergillus terreus specific region of 28S gene.

(iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

AA/~Q GACTC GCTCGCGGGG TT Q GCCGGG CTTCGGCCCG GTGTACTTCC
0 CC(~CGGGCGG GCCAGCGTCG ~l-llGGGCGG CCG~l~AAAG GC~lCCG~AA
TG'~AGCGCCC TTCGGGGCGC CTTATAGCCG GGGGTGCA~T GCGGCCAGCC
TG(,ACCGAGG AACGCGCTTC GGCACGGACG CTGGCATA~T GGTTGTAAAC
GA~C~ L~'l"l' GA

(34) INFORMATION FOR SEQ ID NO 33:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Aspergillus unguis specific region of 28S gene.

(iii) HYPOTHETICAL: No (iv~ ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

SUBSTITUTE SHEET (~UE~ 26) -- W O96/21741 PCTnB96/00026 .
;.
, "
j ~C~q~CTC GGCLlCGGGG TTCAGCCAGC ALlCLlG~LG GTGTACTTCC
~ CCGGGGGCGG GCCAGCGTCG GTTTGGGCGG CCGL'lLAAAG GCCCCAGGAA
--' TGTATCACCC TCCGGGGTTG TCTTATAGCC TGGGGTGCAA TGCGGCCAGC
CTGGACCGAG GAACGCGCTT CGGCACGGAC GCTGGCATAA TGGTTGCAAA
= 1 - CGACCC~l~l TGA
', ~ .
.
.
~-- (35) INFORMATION FOR SEQ ID NO 34:
' 10 ----. (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 ' (B) TYPE: nucleic acid (C) STRANDEDNESS: single .
(D) TOPOLOGY: linear - ~. (ii) MOLECULE TYPE: Aspergillus ustus specific region of 28S gene.

=.
~ ~ ~ (iii) HYPOTHETICAL: No , ..
-. 20 (iv)ANTISENSE: No - (v) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
. ~
.
.....
.
.
;~ 25 GACCAGACTC GGCCCCGGGG TTCAGCCAGC ACTCGTGCTG GTGTACTTCC
_=
:r5 CCGGGGGCGG GCCAGCGTCG GTTTGGGCGG CCGGTCAAAG GCCCCAGGAA
- T~l~lCGCCC TCCGGGGCGT CTTATAGCCT GGGGTGCAAT GCGGCCAGCC
~ r . CGGACCGAGG AACGCGCTTC GGCACGGACG CTGGCGTAAT GGTCGCA~AC .
.
~ GACCCL-lLLl GA
. 30 .
,-=;
. . .

SUBSTITUTE SHEET (RULE 26) .. . i W O96/21741 PCT/Lb,.~D26 (36) INFC~RMATION FOR SEQ ID NO 35:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii' MOLECULE TYPE: Beauveria species specific region of 28S gene.
(iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v)~ SEQUENCE DESCRIPTION: SEQ ID NO: 35:

GACCAGACTT GGGCTTGGTT GATCATCCGG G~ll~lCCCC GGTGCACTCT
. TC_GGCCCAG GCCAGCATCA GTTCGCCCTG GGGGACAAAG GCTTCGGGAA
CGrGGCTCTC TCCGGGGAGT GTTATAGCCC GTTGCGTA~T ACCCTGTGGC
GG~CTGAGGT TCGCGCATTC GCAAGGATGC TGGCGTAATG GTCATCAGTG
ACCCGTCT

25 (37) rNFC3RMATION FOR SEQ ID NO 36:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 - (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET (RULE 2 -~ CA 02209247 1997-07-02 ~ .
W O96/21741 PCT/Lb,''~0026 : ,, i. ~i . ~ , :' =~;
.~,;.~
. .
(ii) MOLECULE TYPE: Bipolaris species specific region of 28S gene.
.
.
-- . (iii) HYPOTHETICAL: No f .- 5 - (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

.
~ -' 1 0 -~ AGCCAGACTT GCTTGCAGTT GCTCATCCGG GCTTTTGCCC
. . .
-' GGTGCACTCT TCTGCAGGCA GGCCAGCATC A~llldGGcG
.~_ GTGGGATAAA G~l~l~l~lC ACGTACCTTC ~llCGG~llG
. ..
- GCCATATAGG GGAGACGTCA TACCACCAGC CTGGACTGAG
., 15 GTCCGCGCAT CTGCTAGGAT GCTGGCGTAA TGGCTGTAAG
=' CGGCCC~l~l TGA

.

~-: . (38) INFORMATION FOR SEQ ID NO 37:
=: 20 -~ (i) SEQUENCE CHARACTERISTICS:
,......................... .
-~- (A) LENGTH: 105 :. .
~ (B) TYPE: nucleic acid ; 1 .
=~ (C) STRANDEDNESS: single - 25 (D) TOPOLOGY: linear .

.
=' - (ii) MOLECULE TYPE: Blastoschizomyces species specific region of 28S
-= gene.
s -~ 30 (iii) HYPOTHETICAL: No .
~ ; (iY) ANTISENSE: No . .

. 66 SU8STITUTE SHEET (RULE 26 = ., .

W O96/21741 PCTlLv~G~V~a26 (v) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

S TG~AATTGTT GA~AGGGAAG GCGATGGTAG GA~TAAGAGG CTGCGGTTTG
AAATAATTGT ~L'l L lCGGGCC ACG~l~lC~-l GAGCCTGCTT TCGCACCCGT
CTIGA

10 (39) INFORMATION FOR SEQ ID NO 38:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Blastomyces dermatitidis specific region of 28S
gene.
(iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

GACCAGAGTC GGCCGTGGGG GTTCAGCGGG CAllC~lGC CCGTGCACTC
CCCCACGGGC GGGCCAGCGT CG~LLLC~AC GGCCGGTCAA AGGCCCCCGG
AAL(iL~L~GC ~LClCGGGGC GTCTTATAGC CGGGGGTGCA ATGCGGCCAG
TCGGGACCGA GGAACGCGCT TCGGCACGGA CGCTGGCTTA ALG~lC~lAA
GCGl~CCCGTC TTGA

S~S~l~Ll~ RUI~ ~

r CA 02209247 1997-07-02 WO 96/21741 PCT/lbSGJ~ 26 (40) INFORMATION FOR SEQ ID NO 39:

:- - (i) SEQUENCE CHARACTERISTICS:
- 5 (A) LENGTH: 213 .= (B) TYPE: nucleic acid (C) STRANDEDNESS: single .
(D) TOPOLOGY: linear ' ' 10 (ii) MOLECULE TYPE: Chrysosporium species specific region of 28S gene.

- (iii) HYPOTHETICAL: No .
.
-~ (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
=

- AACCAGACTT GCGCGCGGCC GATCATCCGG l~ll~l~ACC GGTGCACTCG
GCCGTGCTCA GGCCAGCATC GGTTTTGGCG GCTGGATA~A GGCCCTAGGA
ATGTGGCTCC TCTCGGGGAG TGTTATAGCC TAGGGTGCAA TGCAGCCTGC
-- TGGGACCGAG GACCGCGCTT CGGCTAGGAT GCTGGCGTAA TGGTTGTAAG
CGGCCCGTCT TGA

; 25 (41) INFORMATION FOR SEQ ID NO 40:

(i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 207 - 30 (B) TYPE: nucleic acid (C) STRANDEDNESS: single ....
. ~:
:

SUBSTITUTE SHEET (RULE 26) W O96121741 PCT/lb,"J~26 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Cladosporium species specific region of 28S gene.

(iii'? HYPOTHETICAL: No (iv,, ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

AAC-~CAGACTT GCTCGCGGTG TTCCGCCGGT ~LL~LGACCG GTCTACTCGC
CGCGTTGCAG GCCAGCATCG 'L~''LG~'l GCCG CTGGATAAGA CTTGAGGAAT
GT~GCTCCCT CGGGAGTGTT ATAGCCTCTT GTGATGCAGC GAGCGCCGGG
CG~bGGTCCGC GCTTCGGCTA GGATGCTGGC GTAATGGTCG TAATCCGCCC
~11..'1 l~:;A

(42) INFO~MATION FOR SEQ ID NO 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Curvularia species specific region of 28S gene.

(iii'? HYPOTHETICAL: No (iv'l ANTISENSE: No SUBSTITIJTE SHEET (RU.E 2fi) ~- CA 02209247 1997-07-02 W O96/21741 PcT/~ c~26 -(v) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

. . ~
AGCCAGACTT GCTTGCAGTT GCTCATCCGG GCTTTTGCCC GGTGCACTCT
TCTGCAGGCA GGCCAGCATC A~lllGGGCG GTGGGATA~A G~l~l~lGAC
- AC~llC~llC ~llCGG~lLG GCCATATAGG GGAGACGTCA TACCACCAGC
CTGGACTGAG GTCCGCGCAT CTGCTAGGAT GCTGGCGTAA TGGCTGTAAG
CGGCCC~l~l TGA
'~ 10 - (43) INFORMATION FOR SEQ ID NO 42:

;~ - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 (B) TYPE: nucleic acid ~ (C) STRANDEDNESS: single - (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Candida albicans specific region of 28S gene.
:' , - (iii) HYPOTHETICAL: No -(iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

:' GATCAGACTT GGTATTTTGC ATGCTGCTCT CTCGGGGGCG GCCGCTGCGG
TTTACCGGGC CAGCATCGGT TTGGAGCGGC AGGATAATGG CGGAGGAATG
TGGCACGGCT TCTGCTGTGT GTTATAGCCT CTGACGATGC TGCCAGCCTA
-~ GACCGAGGAC TGCGGTTTTT AACCTAGGAT GTTGGCATAA TGATCTTAAG
':i ;.j SUBSTITUI~ SHEET (RULE 26) =~:

CA 02209247 l997-07-02 W O96/21741 PCT~B96/00026 TCGCCC~ L ~'-1' TGA
(44) INFORMATION FOR SEQ ID NO 43:

(i) SEQUENCE CHARACTERISTICS:
S (A) LENGTH: 223 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: Candidaglabrata specific region of 28S gene.

(iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

GATCAGACAT G~l~llllGC GCCCCTTGCC ~l~lC~lGGGC TTGGGACTCT
CGC~GCTCAC TGGGCCAGCA TCGGTTTTGG CGGCCGGAAA AAACCTAGGG

CCAGCCGGGA CCGAGGACTG CGATACTTGT TATCTAGGAT GCTGGCATAA
TGGTTATATG CCGCCCGl~l TGA

(45) INFO~MATION FOR SEQ ID NO 44:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 (B) TYPE: nucleic acid (C) STRANDEDNESS: single Tm ~F ~H

~- CA 02209247 1997-07-02 , W 096/21741 PCT~B96/00026 r ~'_ ' (D) TOPOLOGY: linear ' ~ (ii) MOLECULE TYPE: Candida guilliermondii specific region of 28S gene.

.. ~
~- 5 (iii) HYPOTHETICAL: No (iv) ANTISENSE: No -- .
~=~ (v) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
:~ 10 GAT Q GACTC GATATTTTGT GAGCCTTGCC 'll~ GCGG GGTGACCCGC
. AGCTTATCGG GCCAGCATCG GTTTGGGCGG TAGGATAATG GCGTAGGAAT
GTGACTTTRC llC~L~AAG TGTTATAGCC TGCGTTGATG CTGCCTGCCT
AGACCGAGGA CTGCGATTTT ATCAAGGATG CTGGCATAAT GATCCCA~AC
-- CGCCC~l~ll GA
:

(46) INFORMATION FOR SEQ ID NO 45:
-; 20 - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 (B) TYPE: nucleic acid -- (C) STRANDEDNESS: single (D) TOPOLOGY: linear .-; (ii) MOLECULE TYPE: Coccidioides immitis specific region of 28S gene.

-(iii) HYPOTHETICAL: No ~- 30 .~ .
~ (iv) ANTISENSE: No ,: .

~ t .
JBSTITUTE SHEET (RULE 26~

W O96121741 PCTIL~5G'~--26 (v) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

S A~ rTC G~lC~lGGGG GCTCAGCGGG CATGAGTGCC CGTGTACTCC
CC(~TGCTCC GGGCCAGCAT CA~L~lGGC G~~ lAA AGGC~l~lGG
AA'~GTATCGT C~lCCGG~AC GTCTTATAGC CAGGGGCG~ ATGCGGCCAG
CCC~GGACTGA GGAACGCGCT TCGGCACGGA TGCTGGCATA Al~Ll~lAA
GCGGCCCGTC TTGA
10 .

(47) INFORMATION FOR SEQ ID NO 46:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 187 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Candida ke~r specific region of 28S gene.

(iii? HYPOTHETICAL: No (iv3 ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
.

GATCAGACAT GGCGTTTGCT LCGG~LllCG CTGGGCCAGC ATCAGTTTTA
GCG~lLG~AT AAA'LC~LCGG GAATGTGGCT ~LG~LLCGGT AGAGTGTTAT
AGCCC~L~GG AATACAGCCA GCTGGGACTG AGGATTGCGA ~Ll l--L~-l-~AA
GG~TGCTGGC GTAATGGTTA AATGCCGCCC ~L~lLGA

C~l t~TlTl ITl~ T fRI 11 F ~f-~

'-- CA 02209247 1997-07-02 . I .
-i ' W O96/21741 PCTAB96/00026 .
, --- .
.
= . .
A:
- (48) INFORMATION FOR SEQ ID NO 47:
~ ~ ~
.. -. ~
-- S (i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 213 --~ (B) TYPE: nucleic acid ~~ (C) STRANDEDNESS: single ~- (D) TOPOLOGY: linear -......... 10 (ii) MOLECULE TYPE: Candida husei specific region of 28S gene.
..

(iii) HYPOTHETICAL: No ., r~ . 15 (iv) ANTISENSE: No _ .. 1 - (v) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
_ . .
~ r ~

: ~- 20 CGCCCGACAT GGGGATTGCG CACCGCTGCC l~LC~lGGGC GGCGCTCTGG
1-_ '; ' ' r ~ . G~ 1"L 1 CC~ LG GGCCAGCATC G~l"L~Lla~l GCAGGAGAAG GG~Ll~lGGA
-, ~- -- AC~lGG~l~L TCGGAGTGTT ATAGCCAGGG CCAGATGCTG CGTGCGGGGA
~ CCGAGGACTG CGGCC~l~ lA GGTCACGGAT GCTGGCAGAA CGGCGCAACA
CCGCCC~L~L TGA

(49) INFORMATION FOR SEQ ID NO 48:

. , ~- - (i) SEQUENCE CHARACTERISTICS:
--- 30 (A) LENGTH: 236 . ~. .
~ (B) TYPE: nucleic acid ,.
.
.
.
~ -- 74 Slt8STITUTE SHEET (RVLE 26 wo 96121741 pcTlIs96looo26 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Cryptococcus laurentii specific region of 28S gene.
s (iii,. HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

AGTCAGTCGT GTCTGGGAGG CTCAGCCGGT TCTGCCGGTG TATTCCTCTC
AGACGGGTCA ACATCAGTTT TGTCCGACGG ATAATGGCGG CGGGA~AGTA
GCAC~lCCGG ~~ lATA GCCCGCTGTC GCATACGCCG GATGAGACTG
AGGCATGCAG CTCGCCTTTA TGGCAGGGGT TCGCCCACTT TCGAGCTTAG
GAT~TTGACG TAATGGCTTT A~ACGACCCG TCTTGA

20 (50) INFORMATION FOR SEQ ID NO 49:

~i) S EQUENCE CHARACTERISTICS:
(A) LENGTH: 173 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
(ii) ]~OLECULE TYPE: Candida lusitaniae specific region of 28S gene.

(iii) HYPOTHETICAL: No RCTITI ITC ~ r,~ ,~

~- W O96/21741 PCT~B~ JC26 '' -(iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
.;, .
,~.- S
~- AAGCAGACAC G~lllLACCG GGCCAGCGTC GAAAAGGGGG GAGGAACAAG
' AACTCGAGAA TGTGGCGCGC ACCTTCGGGY GCGC~l~LlA TAG~LC~l~l TGAC'GC~lCC AlCC~ ~"L'L'l'C GAGGCCTGCG ATTCTAGGAC GCTGGCGTAA
TGGTTGCAAG CCGCCC~L~l TGA
... . 10 .:
(51) INFORMATION FOR SEQ ID NO 50:

(i) SEQUENCE CHARACTERISTICS:
. i 15 (A) LENGTH: 238 ~- (B) TYPE: nucleic acid (C) STRANDEDNESS: single ' (D) TOPOLOGY: linear f' 20 (ii) MOLECULE TYPE: Cryptococcus neoformans var gattii (serotype B) specific region of 28S gene.

(iii) HYPOTHETICAL: No , (iv) ANTISENSE: No . . .

r (v) SEQUENCE DESCRIPTION: SEQ ID NO: 50: ~ ~

AGTCAGTCGT GTCTATTGGG TTCAGCCAGC TCTGCTGGTG TATTCCCTTT
AGACGGGTCA ACATCAGTTC TGATCGGTGG ATAAGGGCTG GAGGAATGTG
GCA~l~llCG GG~l~l~llA TAGCCTCCTG TCGCATACAC TGGTTGGGAC
- TGAGGAATGC AGCTCGCCTT TATGGCCGGG GTTCGCCCAC GTTCGAGCTT

'-. SUBSTITUTE SHEET (RULE 26) W O96121741 PCTILD~W026 AGGATGTTGA CAAAATGGCT TTA~ACGACC C~L~ A

(52) INFC)RMATION FOR SEQ ID NO 51:

S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 238 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii MOLECULE TYPE: Cryptococcus neoformans (serotype A) specific region of 28S gene.

(ii~) HYPOTHETICAL: No (iv) ANTISENSE: No (v' SEQUENCE DESCRIPTION: SEQ ID NO: 51:

AGrCAGTCGT GTCTATTGGG TTCAGCCAGT TCTGCTGGTG TATTCCCTTT
AG.~CGGGTCA ACATCAGTTC TGATCGGTGG ATAAGGGCTG GGGGAATGTA
GC~CTCTTCG GA~1~-1~11A TAGC~1C~1G TCGCATACAC TGGTTGGGAC
TG.AGGAATGC AGCTCGCCTT TATGGCCGGG GTTCGCCCAC GTTCGAGCTT
AG,ATGTTGA CAAAATGGCT TTA~ACGACC C~1~11GA

(53) INFC RMATION FOR SEQ ID NO 52:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 SuB~ ITF CI I~I~T ~DI 11 1 : CA 02209247 1997-07-02 W O96/21741 PCT~B96/00026 , . .

- (B) TYPE: nucleic acid ~= (C) STRANDEDNESS: single (D) TOPOLOGY: linear . . ~
- 5 (ii) MOLECULE TYPE: Candida parapsilosis specific region of 28S gene.

. (iii) HYPOTHETICAL: No ~-. (iv) ANTISENSE: No ~, 10 (v) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

, GATCAGACTT GGTATTTTGT ATGTTACTCT ~lCGG~GGTG GCCTCTACAG
-~ 15 TTTACCGGGC CAGCATCAGT TTGAGCGGTA GGATAAGTGC AAAGA~ATGT
GGCACTGCTT CGGTAGTGTG TTATAGTCTT TGTCGATACT GCCAGCTTAG
ACTGAGGACT GCGGCTTCGG CCTAGGATGT TGGCATAATG ATCTTAAGTC
-~- GCCC~L~llG A

--. 20 (54) lNFORMATION FOR SEQ ID NO 53:

:' (i) SEQUENCE CHARACTERISTICS:
rl (A) LENGTH: 238 (B) TYPE: nucleic acid - 25 (C) STRANDEDNESS: single ~. (D) TOPOLOGY: linear ; ; (ii) MOLECULE TYPE: Cryptococcus terreus specific region of 28S gene.

(iii) HYPOTHETICAL: No ~ .

- . SUBSTITUTE SHEET (RULE 26) W O96/21741 ~ /lL,5.'~_Z6 (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

S AG'rCAGTCAT GTCTATTGGA CTC'AGCCGGT l~GCCG~lG TA~llC~l-''L
AG~TGGGGTC AACATCAGTT TTGATCGCTG GAAAAGGGCA GGAGGAATGT
AGGACTCTCG GGTGA~CTTA TAGC~ a TCGTATACAG 'l'~lLGGGAC
TG~GGAACGC AGCAlGC~ll TAlGGCC'GGG ~llCGCC~AC GTACATGCTT
AGi~ATGTTGA CATAATGGCT TTA~ACGACC C'~l~Ll~A

(55) INFORMATION FOR SEQ ID NO 54:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Candida tropicalis specific region of 28S gene.

(iii) HYPOTHETICAL: No (iv~ ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

GAq'CAGACTT GGTATTTTGT ATGTTACTTC TTCGGGGGTG GCCTCTACAG
TTTATCGGGC CAGCATCAGT TTGGGCGGTA GGAGAATTGC GTTGGAATGT
GGCACGGCTT C'G~ll~L~lG TTATAGCCTT CGTCGATACT GCCAGCCTAG
ACTGAGGACT GCGGTTTATA CCTAGGATGT TGGCATAATG ATCTTAAGTC
GCCC~l~llG A

SUBSTITUTE StlEET (RULE 26) -: - CA 02209247 1997-07-02 '~:
. W O96t21741 PCT~B96/00026 ' ~ ~.
(56) INFORMATION FOR SEQ ID NO 55:
. .
. .
- (i) SEQUENCE CHARACTERISTICS:
- ~- (A) LENGTH: 211 ., - ~ 5 (B) TYPE: nucleic acid (C) STRANDEDNESS: single - ~ (D)TOPOLOGY: linear . . .
~, ~ (ii) MOLECULE TYPE: Fusarium species specific region of 28S gene.
;'-'' 10 ~- (iii) HYPOTHETICAL: No ., . .
(iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
--:~ .
- GACCAGACTT GGG~llG~ll AATCATCTGG G~ll~l~Ycc AGTGCACTTT
TCCAGTCCAG GCCAGCATCA ~llllCSCCG GGGGATAAAG RCTTCGGGAA
TGTGGCTCYC YYCGGGGAGT GTTATAGCCC ~ll~Y~lAAT ACCCTGGBGG
~'- GGACTGAGGT TCGCGCWTCT GCAAGGATGC TGGCGTAATG GTCATCAACG
ACCC~l~llG A

25 (57) INFORMATION FOR SEQ ID NO 56:

.

-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 238 (B) TYPE: nucleic acid (C) STRANDEDNESS: single . (D) TOPOLOGY: linear ,, , , SUBSTITUTE SHEET (RULE 26) W O 96/21741 PCTnB96/00026 (ii, MOLECULE TYPE: Filobasidium capsuligenum specific region of 28S
ge~le.

S (iii) HYPOTHETICAL: No (iv3 ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

AG'~CAGTCAT GTCTATTGGA CTCAGCCGGT TCTGCCGGTG TAlllC~ll"l AGi~LGGG~lC AACATCAGTT TTGACCGTTG GATAAAGGCA GGAAGAATGT
AG~.ACTCTCG GGTGA~CTTA TAG~ll~llG TCACATACAA TGGTTGGGAC
TGi-~GGAACGC AGCATGCCTT TATGGCCGGG A'1"1'C~'1'C~AC GTACATGCTT
AG~,ATGTTGA CATAATGGCT TTAAACGACC ~'l'~'l''l'~A

(58) INFORMATION FOR SEQ ID NO 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 238 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Filobasidiella neoformans var bacillispora (serotype C) specific region of 28S gene.

(iii ~ HYPOTHETICAL: No SUBSTITUTE SHEET (RULE 26) -, CA 02209247 1997-07-02 W O96/21741 PCT~B96/00026 =;

- - (iv) ANTISENSE: No -~ (v) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
~; ~
~ . S
AGTCAGTCGT GTCTATTGGG TTCAGCCAGC TCTGCTGGTG TAllCC~lll AGACGG~A ACATCAGTTC TGATCGGTGG ATAAGGGCTG GAGGAATGTG
GCA~.~llCG GG~-L~l~ l"l'A TAGC~l~ L'G TCGCATACAC lG~llGGGAC
TGAGGA~TGC AG~lCGC~ll TAlGGCCGGG GTTCGCCCAC GTTCGAGCTT
0 AGGATGTTGA CAAAATGGCT TTA~ACGACC C~l~ ~"L~A

(59) INFORMATION FOR SEQ ID NO 58:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 238 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Filobasidiella neoformans var neoformans (sero~pe D) specific region of 28S gene.

(iii) HYPOTHETICAL: No (iv) ANTISENSE: No - (v) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

f, AGTCAGTCGT GTCTATTGGG TTCAGCCAGT TCTGCTGGTG TATTCCCTTT
: ' ... .

- S~BSTITUTE SHEET (RULE 26) W ~961~1741 PCTnB96/00026 G~ A ACATCAGTTC TGALC~Gl~G ATAAGGGCTG GAGGAATGTG
GC~ CG GG~L~l~l lA TAGC~lC-lG TCGCATACAC lGGllGG~AC
TG.~GGAATGC AG~.CGC~l~ TATGGCCGGG ~l"lC~G~AC GTTCGAGCTT
AG3ATGTTGA CA~AATGGCT TTAAACGACC C~l~l l ~A

(60) ~NFORMATION FOR SEQ ID NO 59:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 236 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Filobasidium uniguttulatum specific region of 28S
gene.

(iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) 3EQUENCE DESCRIPTION: SEQ ID NO: 59:

AGTCAGTCGT GCTCAATGGA CTCAGCCGTT CTGCGGTGTA TTTCCATTGG
GTGGGGTCAA CATCAGTTTT GATCGCTGGA TAAAGGCAGG AGGAATGTAG
CACCCCCGGG TGAACTTATA GC~"l~ll~lC ACATACAGTG GTTGGGACTG
AGGi~ACGCAG CATGCCTTTA TGGCCGGGAT TCGTCCACGT ACATGCTTAG
GATt7TTGACA TAATGGCTTT AAACGACCCG TCTTGA

(61) INFORMATION FOR SEQ ID NO 60:

SUBS~ E S~E~ ~RLl!~ ~B~

i CA 02209247 1997-07-02 ; .W O 96/21741 PCTAB96/00026 .' ' .

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 (B) TYPE: nucleic acid S (C) STRANDEDNESS: single (D) TOPOLOGY: linear ,~ .

(ii) MOLECULE TYPE: Geotrichum species specific region of 28S gene.

(iii) HYPOTHETICAL: No "

(iv) ANTISENSE: No - (v) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
1~
~''' -.- AATCAGACTT GGTGCTGTTG TTCAACTRTG TTTCGGCATA GTGTACTCAG
CAGTACTAGG CCAAGGTGGG ~l~lllGGGA GTGA~AAAGA AGTAGGAACG
TAACTCTTCG GAGTGTTATA GCCTACTTTC ATAGCTCCTC AGGCGCCTCA
GGACTGCGCT TCGGCAAGGA CCTTGGCATA ATGATTCTAT ACCGCCCGTC
TTGA

25 (62) lNFORMATION FOR SEQ ID NO 61:
.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) W O 96/21741 PCT/Lb5C~'U~_26 (ii) MOLECULE TYPE: Histoplasma capsulatum specific region of 28S gene.

(ii:.) HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

GAfCAGAGTC GGC~Y~GGG GTTCAGCGGG CA~ C CCGTGCAATC
CCOCGCGGCC GGGCCAGCGT CG~lllCGAC GGCCGGTCAA AGGCCCCCGG
AA'~ CGC ~l-~lCGGGGC GTCTTATAGC CGGGG~LGCA ATGCGGCCAG
TCGGGACCGA GGAACGCGCT CCGGCACGGA CGCTGGCTTA AlG~l~L~'A
lS GCGACCCGTC TTGA

(63) lNFORMATION FOR SEQ ID NO 62:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 ~ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Malbranchea species specific region of 28S gene.

(iii) HYPOTHETICAL: No (iv) ANTISENSE: No SUBSTITUTE SHFFT r~l It .li W O96/21741 PcT/lL~ ~A26 . .
-;

= (v) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

..
. ~ ~

AGACAGACTC GAGCGCGGGG GCTCAGCGGG TATTGTTATG CCCGTGCACT
CCCCCGCGCC CGGGCCAGCA TCA~ L 111GG CGGCCG~1~A AAGGCCCTTG
; ~
GAATGTATCG TCCTCCGGGA C~l~LlATAG CCAAGGGTGC AATGCGGCCA
GCC~GGACTG AGGAACGCGC 11CGG~ACGG ATGCTGGCGT AATGGCTGTA
- AGCGGCCCGT CTTGA

-;
' 10 (64) INFORMATION FOR SEQ ID NO 63:
.
(i) SEQUENCE CHARACTERISTICS:
; ~- (A) LENGTH: 237 (B) TYPE: nucleic acid . (C) STRANDEDNESS: single (D) TOPOLOGY: linear . .
. .
~ . (ii) MOLECULE TYPE: Mucor species specific region of 28S gene.

. . (iii) HYPOTHETICAL: No . .
(iv) ANTISENSE: No ~ 25 (v) SEQUENCE DESCRIPTION: SEQ ID NO: 63:

.
-- AGCCAGACTG GTTTGACTGT AATCAACCTA GAA11C~L1C TGGGTGCACT

TGCAGTCTAT ACCTGCCAAC AACAGTTTGA TTTGGAGGAA AAAATTAGTA
~- GGAATGTAGC ~1~1CGAGGT GTTATAGCCT ACTATCATAC TCTGGATTGG
.= 30 ACTGAGGAAC GCAGCGAATG CCWTTAGGCR AGATTGCTGG GTGCTTTCGC
TAATA~ATGT TAGAATTTCT GCTTCGGGTG GTGCTAA
r -SUBSTITUTE SHEET (RULE 26) W O96/21741 PCT/lk5G~ C26 (65) INF(:)RMATION FOR SEQ ID NO 64:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 209 S (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii'~ MOLECULE TYPE: Paecilomyces species specific region of 28S gene.
(iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

GACCAGACTT GGGCCCGGTG GATCATCCAG C~ CGCT GGTGCACTCC
GCCGGGTTCA GGCCAGCATC AGTTCGCCGC GGGGGA~AAA GGCTTCGGGA
ACGTGGCTCC TACGGGAGTG TTATAGCCCG TTGCATAATA CCCTGGGGCG
GAC'TGAGGTT CGCGCTCCGC AAGGATGCTG GCGTAATGGT CATCAGCGAC
CCC i'l'~'l 'L~A

25 (66) INFO]~LATION FOR SEQ ID NO 65:

(i) ',EQUENCE CHARACTERISTICS:
(A) LENGTH: 199 ~, (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Sl~.~Tln ITF Cl~ T rDI 11 r W O96/21741 PCT~B96/00026 (ii) MOLECULE TYPE: Penicillium species specific region of 28S gene.

(iii) HYPOTHETICAL: No ., . S
(iv) ANTISENSE: No , -: (v) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
.
'', 10 ':
GACCAGACTC GCCCACGGGG TTCAGCCGGC ATTCGTGCCG GTGTACTTCC
CCGCGGGCGG GCCAGCGTCG ~llLG~KcGG CCGGTCA~AG GCCCTCGGA~
TRTAACGCCC CCCGGGGCGT CTTATAGCCG AGGGTGCCAT GCGGCCAGCM
CAGACCGAGG AACGCGCTTC GGCTCGGACG CTGGCATAAT G~lC~lAAA

(67) INFORMATION FOR SEQ ID NO 66:

- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear , .
(ii) MOLECULE TYPE: Pse1/~7~771escheria boydii region of 28S gene.

(iii) HYPOTHETICAL: No (iv) ANTISENSE: No ~- (v) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

SUBSTITUTE SHEET (Rl 11 F ~1 w~ 96/21741 PCT/L~>~G~ 26 GACCAGACTT GTGCCG~lCG AATCAGCCGC C~ GGG CGGCGCACTT
CGGCGGGCTC AGGCCAGCAT CA~l~CG~lG CAGGGGGAGA AAGGCGATGG
GAATGTGGCT ~llGG~AGTG TTATAGCCCG CCGCGCAATA CCCCTCGGCG
GACTGAGGAC CGCGCATCTG CAAGGATGCT GGCGTAATGG TCGTCAGCGA
CCC~ ;l L~A

10 (68) INFO~MATION FOR SEQ ID NO 67:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 244 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~IOLECULE TYPE: Rhizopus species (NO: 1) specific region of 28S
gene.
(iii) ]~YPOTHETICAL: No (iv) I~NTISENSE: No (v) S'EQUENCE DESCRIPTION: SEQ ID NO: 67:

AGCC~GACTG GCTTGTCTGT AATCAATCTA G~lllC~lGC CTGGATGCAC
-~ TTGC~GACTA TTTGCCTGCC AACGACAATT ~l"l"l"l"l"lGAGT GTA~AAACTA
TTGG~AATGT GGCCAATATT TATTTATTGG TGTTATAGTC CTTTAGA~AA

3 0 TACCTTGAAT TGGATTGAGG AACGCAGCGA ATG~Ll~l~l TTnGAGGCAA
A~l~LllAT TGGGATTTAC GGATCAGACT GTGGCATTGT CACA

RcTlTI ~r ~ rrT ,.

W O96/21741 PCT/lb,~ ~26 (69) INFORMATION FOR SEQ ID NO 68:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 - ~ 5 (B) TYPE: nucleic acid - (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Rhizopus species (NO: 2) specific region of 28S
gene.

~, .
(iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

AGCCAGACTG G~ll~l~l~l AATCAATCTA GGCTTCGGCC TGGATGCACT
TGCAGGCTAT GCCTGCCAAC GACAATTTGA CTTGAGGGAA A~AACTAGGG
- 20 GA~ATGTGGC CCACTTGTGG GTGTTATAGT CCCTTAGA~A ATACCTTGGG
TTGGATTGAG GAACGCAGCG AATGCTTATT GGCGAGTTTT CCAGGAAGGT
'l"L'l ~l~AGGT ACTAC

- (70) INFORMATION FOR SEQ ID NO 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 - (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET (RVLE 26) CA 02209247 l997-07-02 W O96J~1741 PCT/Lb5C/00026 (ii) MOLECULE TYPE: Rhizopus species (NO: 3) specific region of 28S
gerle.

(iii ~ HYPOTHETICAL: No s (iv) ANTISENSE: No (v) ~EQUENCE DESCRIPTION: SEQ ID NO: 69:

AGC~AGACTG G~L1~1~1~1 AATCAGTCTA AG~11~GG~1 TGGATGCACT
TGCAGGCTAT GCCTGCCA~C GACAATTTGG CTTGAGGGAA AAAACTAAGG
GAA~TGTGGC CCA1~C~1~G GTGTTATAGT CCCTTAGA~A ATACCTTGGG
CTG~ATTGAG GTACGCAGCG AATGCTATTT GGCGAGTTGG CTGGGAATAT
TTT~TGAGGT GCTTT

(71) INFOF~MATION FOR SEQ ID NO 70:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: Sporothrix species specific region of 28S gene.

(iii) ]~YPOTHETICAL: No (iv) ANTISENSE: No SUBSTITUTE StlEET (RULE 20 W O96/21741 PCT/lb3G~-026 .
- (v) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
' I

GACCAGACTT GCGCCYCGCG GACCACCCGG C~l-l~lCGCC GGTGCACTCT
GCGKKGCGCA GGCCAGCATC G~11~1CC~'A GGGGGACAAA GGCCGCGGGA
ACGTAGCTCC TTCGGGAGTG TTATAGCCCG CGGCGGCATG CCCCTGGGGG
GACCGAGGAC CGCGCTTCGG CAAGGATGCT GGCGTAATGG TCACCAGCGA
ACC~l~ L'l'~A

'~ 10 - ' (72) INFORMATION FOR SEQ ID NO 71:
, (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 - 15 (B) TYPE: nucleic acid (C) STRANDEDNESS: single - (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Scopulariopsis brevicaulis specific region of 28S
~- 20 gene.

(iii) HYPOTHETICAL: No (iv) ANTISENSE: No -(v) SEQUENCE DESCRIPTION: SEQ ID NO: 71:

GAC Q GACTT GCGCCCGTCG GATCAACCGT CGCTTGCGGC GGCGCACTCC
- GGCGGGCTCA GGCCAGCATC A~llC~lCCG GGGGGAGA~A GGCGGCGGGA
ATGTGGCTCT TCGGAGTGTT ATAGCCCGCC GTGTAATACC CTCGGGTGGA

' ' C~l~:l L~A

SUBSTITUTE SHEET (RIJLE 26) W O96121741 PCT~B96/00026 (73) INF~RMATION FOR SEQ ID NO 72:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Scopulariopsis brumptii specific region of 28S gene.

(iii ~ HYPOTHETICAL: No (iv, ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO: 72:

GACCAGACTC GCGCCCGTCG GATCAGCCGT CGCTCGTCGG CGGCGCACTC
CGGCGGGCTC GGGCCAGCAT CAGTTCGCCT CGGGGGGAGA AAGGCGGCGG
GAATGTGGCT CTACGGAGTG TTATAGCCCG CCGCGTAATA CCCCCGGGCG
GACTGAGGAC CGCGCGTATG CAAGGATGCT GGCGTAATGG TCGTCAGCGA
CCC~l~l~A

(74) INFORMATION FOR SEQ ID NO 73:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ W O96/21741 PCT/Lb5G/00026 ~ .

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Saccharomyces cerevisiae specific region of 28S
gene.
. , (iii) HYPOTHETICAL: No (iv) ANTISENSE: No ~
-- 10 (v) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
b, GATCAGACAT G~'l ~ l l L l'~'L GCC~l~LGCT CCTTGTGGGT AGGGGAATCT
- CG QTTTCAC TGGGCCAGCA TCAGTTTTGG TGGCAGGATA AATCCATAGG
AATGTAGCTT GC~lCG~lAA GTATTATAGC CTGTGGGAAT ACTGCCAGCT
GGGACTGAGG ACTGCGACGT AAGTCAAGGA TGCTGGCATA ATGGTTATAT
- GCCGCCCGTC TTGA
:
;.. - 20 (75) INFORMATION FOR SEQ ID NO 74:

. .
.- . (i) SEQUENCE CHARACTERISTICS:
: (A) LENGTH: 236 . (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Trichosporon beigelii specific region of 28S gene.

(iii) HYPOTHETICAL: No i-- 94 . SUBSTITUTE SHEET (RULE 26) W ~96/ZI741 PCT/1b,.'~CO26 (iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SE;Q ID NO: 74:
!

AGTCAGTCGT ~'L'l'~"ll 1G~A TTCAGCCAGT TCTGCTGGTC TA~1C~L1G
GAACGGGTCA ACATCAGTTT TGTCCGGTGG ATAAAGGTAG TAGGAATGTG
A~1,~1CCGG AAGTGTTATA GCCTATTATC ACATACACTG GGTGAGACTG
AGG;~CTGCAG ~1CGC~111~A TGGCCGGCCT TCGGGCACGT TCGAGCTTAG
GATGTTGACA TAA1GG~1-L1- AAACGACCCG TCTTGA

SL)BST¦nlTF .C~ tT ~Dl 1I r ",~

Claims

We claim:

1. An oligonucleotide probe for a region of the 28S subunit of fungi, said region being bracketed on one side by the hybridization site of a universal oligonucleotide probe having the nucleotide sequence of SEQ ID NO: 1 and on the other side by the hybridization site of a universal oligonucelotide probe having the nucleotide sequence of SEQ ID NO:2, which probe is able to identify one species selected from the group consisting of Acremonium sp., Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria sp., Bipolaris sp., Blastoschizomyces sp., Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var gattii serotype B, Cryptococcus neoformans serotype A, Cryptococcus laurentii, Cryptococcus terreus, Curvularia sp., Fusarium sp., Filobasidium capsuligenum, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus) neoformans var neoformans serotype D, Filobasidium uniguttulatum, Geotrichum sp., Histoplasma capsulatum, Malbranchea sp., Mucor sp., Paecilomyces sp., Penicillium species, Pseudallescheria boydii, Rhizopus sp., Sporothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae, and Trichosporon beigelii.

2. An oligonucleotide probe for a region of the 28S subunit of fungi, said region being bracketed on one side by the hybridization site of a universal oligonucleotide probe having the nucleotide sequence of SEQ ID NO:1 and on the other side by the hybridization site of a universal oligonucelotide probe having the nucleotide sequence of SEQ ID NO:2, which probe is able to identify one species selected from the group consisting of Acremonium sp., Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria sp., Bipolaris sp., Blastoschizomyces sp., Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var gattii serotype B, Cryptococcus neoformans serotype.A, Cryptococcus laurentii, Cryptococcus terreus, Curvularia sp., Fusarium sp., Filobasidium capsuligenum, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus) neoformans var neoformans serotype D, Filobasidium uniguttulatum, Geotrichum sp., Histoplasma capsulatum, Malbranchea sp., Mucor sp., Paecilomyces sp., Penicillium species, Pseudallescheria boydii, Rhizopus sp., Sporothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae, and Trichosporon beigelii, said probe comprising all or part of any one of the sequences (SEQ ID NO: 3) through (SEQ ID NO: 74), or any functional equivalent thereof, which is able to uniquely identify the corresponding species of fungus.

3. An oligonucleotide probe for the 28S subunit of fungi having the nucleotide sequence GTGAAATTGT TGAAAGGGAA (SEQ ID NO: 1) or any functional equivalent thereof.

4. An oligonucleotide probe of claim 3, said probe being capable of specifically hybridizing under stringent conditions to the nucleotide sequence or to the complement of the nucleotide sequence of (SEQ ID NO: 1).

5. An oligonucleotide probe for the 28S subunit of fungi having the nucleotide sequence GACTCCTTGG TCCGTGTT (SEQ ID NO: 2) or any functional equivalent thereof.

6. A method of determining whether one or more fungal species selected from a group of fungi is present in a sample comprising the following steps:
a. extracting the nucleic acid material from the fungi contained in said sample, b. adding two known primers, (SEQ ID NO 1) and (SEQ ID NO 2), or the functional equivalent thereof, bracketing the areas of interest on the 28S rDNA or rRNA present in said group of interest, c. amplifying the sequence between said primers, and d. using one or more third labeled probes to determine which of said fungi is present, wherein said third probes are selected from the group consisting of (SEQ ID
NO 3) through (SEQ ID NO 74), any portion thereof and functional equivalents thereof.

7. A method of claim 6 in which said amplifying procedure is the polymerase chain reaction.

8. A method of claim 6 which, following said amplification, comprises the following step:
d. using two or more third probes to determine which of said fungi is present, one of said third probes being attached to a moiety which allows separation of said probe and one or more third probes connected to labeled moieties, wherein said third probes are selected from the group consisting of (SEQ ID NO 3) through (SEQ ID NO 74), any portion thereof and functional equivalents thereof.

9. A method of claim 6 which excludes said adding step and said amplification step.

10.A method of claim 6 wherein said fungal species is selected from the group consisting of Acremonium sp., Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria sp., Bipolaris sp., Blastoschizomyces sp., Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var gattii serotype B, Cryptococcus neoformans serotype A, Cryptococcus laurentii, Cryptococcus terreus, Curvularia sp., Fusarium sp., Filobasidium capsuligenum, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus) neoformans var neoformans serotype D, Filobasidium uniguttulatum, Geotrichum sp., Histoplasma capsulatum, Malbranchea sp., Mucor sp., Paecilomyces sp., Penicillium species, Pseudallescheria boydii, Rhizopus sp., Sporothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae, and trichosporon beigelii.

1l. A method of claim 6 wherein more than one third probe is used, each said third probe connected to a different signal moiety or moiety which allows separation of said third probe.