WO2001023616A2 - Method of identifying pathogenic cryptococci - Google Patents

Abstract

Novel DNA sequences isolated from pathogenic yeast provide a basis for discriminating between different genotypes. The invention provides probes and primers that are useful in differentiating among pathogenic yeast and assays for facilitating such differentiation. Methods of differentiating among such yeast are provided; the intergenic spacer nucleic acid sequences of 91 strains in electronic form are also provided.

Description

METHOD OF IDENTIFYING PATHOGENIC CRYPTOCOCCI

BACKGROUND OF THE INVENTION

Opportunistic fungal infections are emerging as a significant cause of morbidity and mortality in immune-compromised patients. Successes in the treatment of malignancies and in organ transplantation, as well as the adverse impact of the HIV/ AIDS pandemic present a population of immune compromised patients extremelysusceptible to opportunistic infections. Successful antibiotic prophylaxis of bacterial infections in this population often is accompanied by the emergence of fungal infections, particularly those featuring one or more species of yeast. The situation is worsened by the increased incidence among yeast pathogens of strains with antifungal resistance. The opportunistic character of these fungal pathogens and the increasing resistance to antifungals have contributed to a rise in the frequency and severity of human fungal infections, many refractory to antifungal therapy. Mortalities caused by opportunistic fungal pathogens range from 15% to 40% for the frequently encountered yeast Candida albicans to over 80% for recently emerged Trichosporon species. Systemic mycoses such as candidiasis, cryptococcosis, histoplasmosis and aspergillosis often are associated with suppression of the immune system associated, for example, with chemotherapy for cancer, particularly leukemia, organ or bone marrow transplants, AIDS, and drug abuse (Viscoli & Castagnola 1999, Gozdasoglu et al. 1999, Pfaller et al. 1999, Krcmery et al. 1999a, Krcmery et al. 1999b; Lascaux et al. 1998, Warnock, 1998, Kawakami et al. 1998, Muller et al. 1999),

The increased severity in diseases caused by known pathogenic yeasts such as Candida albicans, Cryptococcus neoformans, Malassezia furfur and Trichosporon cutaneum presents a major challenge to medical mycology. To make matters worse, serious pathogens are emerging from among species associated with soil, food, and plants and species previously considered to be harmless animal and human saprophytes have become opportunistic pathogens. The majority of these opportunistic yeasts reside among species of Candida, Cryptococcus, Malassezia, Rhodotorula, Sporobolomyces, and Trichosporon. Among the well-known infections, candidiasis, caused by C. albicans, is complicated by the emergence of species such as Candida parapsilosis, Candida. glabrata, Candida tropicalis and Candida krusei as serious pathogens (Pfaller et al. 1999, Huang et al. 1999, and Darwazah et al. 1999). Another important opportunistic pathogen, Cryptococcus (Filobasidiella) neoformans, causes fatal systemic and central nervous system infections in immunocompromised patients (Buchanan & Murphy 1998). Estimates of the rates of incidence of C. neoformans in AIDS patients range from 5 to 30% . In immunodeficient, viral-seropositive individuals, an infection with C. neoformans indicates progression to AIDS. The problem is aggravated by the emergence of cryptococcal strains that have become resistant to some of the widely used antifungal agents (reviewed by Boekhout et al. 1997).

The incidence of trichosporonosis, a fungemia caused by Trichosporon spp., has increased during the past 10 years (Krcmery et al. 1999b). Trichosporonosis is nearly always fatal, especially in immunosuppressed patients with neutropenia (Warnock 1998). Malassezia yeasts, members of the normal human cutaneous flora, are emerging as important opportunistic pathogens capable of initiating systemic infections. Under the influence of predisposing factors, Malassezia yeasts cause a variety of diseases. Severe forms of dermatitis occur in patients with neurological disorders such as Parkinson's disease, multiple sclerosis, stroke, and mood depressions, while systemic, life-threatening infections occur in patients receiving corticosteroid or immumosuppressive treatment and in patients with AIDS. (See Faergemann 1997, Gueho et al. 1998 for reviews).

A common difficulty encountered in treating yeast diseases, including those produced by lesser-known yeasts (Rhodotorula. Sporobolomyces, non-neoformans Cryptococcus, and non-albicans Candida, for example), is their differential response to antifungal agents. Kawakami et al. 1998 found, for example, that while Candida albicans infections decreased over the past 10 years, concomitant with the introduction of fluconazole treatment, fungemias caused by other yeast species that exhibit fluconazole resistance increased. Differences in reactions to other antibiotics have been reported; Brummer et al. (1998), for example, reported that the wide-spectrum antifungal triazole, voriconazole, was active in serum against C. neoformans var. neoformans but not C. neoformans var. gattii. Survival from systemic fungal infectons depends upon the prompt initiation of appropriate antifungal therapy. The variable responses exhibited by different serotypes and genotypes of the fungal pathogens signal a need for a rapid and reliable method of differentiating among serotypes and genotypes (Brummer 1998; Lopez-Ribot 1999). Case reports indicate that, despite lengthy illnesses, the causative agent often is identified too late for proper therapy, resulting in the death of the patient (Krcmery et al. 1999a, Muller et al. 1999, Hsu et al. 1998, Olesen, et al., 1997, Slavoski and Tunkel 1995).

Rapid and dependable identification of pathogens is of critical importance for the prompt treatment of diseases and the determination of their epidemiology. Rapid tests capable of resolving yeast strains, serotypes and varieties would provide objective diagnoses on which to base antifungal therapy. For decades, yeast identification has depended upon classical biochemical, physiological and morphological analyses taking days to weeks to complete (Kurtzman and Fell 1998). Commercial products based on the classical methods are widely used. These tests are often inaccurate due to physiological variability between strains within species, and therefore are incapable of distinguishing clinically important species and strains (Hoppe & Frey 1999, Wadlin et al. 1999, Paugam et al. 1999, Espinel-Ingroff et al. 1998, and Milan et al. 1997).

Highly specific tests have been developed; such as the germ tube test for C. albicans, the CGB medium test for C. neoformans (Kwon-Chung 1998) and antigen tests for various species (Hamilton 1998). Cryptococcus neoformans strains have been divided into serotypes (A, B, C, D, and AD) on the basis of antigenic differences in polysacchararide components of the cells. Serological classification uses rabbit antisera that are commercially available from Iatron Laboratories (Tokyo). This serotype classification is useful for understanding global epidemiology of cryptococcal infections and the serological relationship of clinical strains (Casadevall and Perfect 1998).

Various DNA-based molecular techniques are being developed and applied to meet the need for rapid tests. Several molecular fingerprinting techniques, using the polymerase chain reaction (PCR) in combination with electrophoresis, have been devised for the identification of yeast species, varieties and inter-specific strains and genotypes. Van Belkum, et al. (1994) and Boekhout, et al. (1998) used randomly amplified polymorphic DNA (RAPD) to readily discriminate strains within species of Malassezia. Boekhout, et al. (1998) and Gueho, et al. (1998) found, in contrast, that karyotyping with pulsed field gel electrophoresis (PFGE) did not resolve strains of Malassezia species, an indication that molecular tests, per se, are not uniformly useful for the identification of serotypes, varieties and strains. Significant RAPD results were obtained for C. neoformans. Boekhout et al. 1997 discriminated individual C. neoformans strains and revealed geographical relationships of C. neoformans var. neoformans and C. neoformans var. gattii using a combination of PFGE and RAPD. Pernice, et al. (1998) observed intra variety differences in C. neoformans serotype A using RAPD analyses. Numerous other PCR-based fingerprinting techniques have been used to identify yeast (for example, Kohno et al. 1994, Morace et al. 1999, Sorrell et al. 1996 a & b, and Viviani et al. 1997). Reiss, et al. (1998) reported that fingerprinting by restriction enzyme analysis and probe hybridization can resolve strains within species. They further determined that fingerprinting by this method was more sensitive for fungal infection diagnosis than were blood culture methods. Fingerprinting techniques are often limited to variability within an individual species, however, and require considerable technical expertise. These tests also are time-consuming and quite labor intensive, allowing only a low throughput in potentially high volume testing.

Tests based on gene amplification and species-specific hybridization probes provide a more selective analysis (Jordan & Durso 1996). Reiss, et al. (1998) generically amplified the ITS2 region of Candida isolates and used DNA probes designed to recognize resolving species-specific sequences within the amplified products to identify 16 species of Candida in a microp late-based assay. The entire assay process, from sample preparation to detection of amplified product, was accomplished in 7 hours, 2.5 days faster than conventional culture methods. Another promising system is the use of species-specific PCR primers (Fell 1993 & 1995, Haynes et al. 1995, Mannarelli & Kurtzman 1998, Nagai, et al. 1999, Prariyachatigul et al. 1996). These PCR-based analyses are highly effective. Nagai et al. 1999 detected as little as 5 fg of Trichosporon DNA, a sensitivity that allowed them to find a specific fragment of Trichosporon asahii DNA in sera from patients with early to late disseminated trichosporonosis. Reiss et al. (1998) noted that different laboratories adapt different molecular tests to identify pathogemc yeasts, because a "gold standard" method for dependable identification and discrimination of yeasts is not available.

A prerequisite for dependable molecular tests is the availability of comprehensive sequence databases that will allow differentiation at the species, strain, genotype, or other subspecies level. Prior to the present invention, however, no such database existed for the Cryptococcus pathogens.

SUMMARY OF THE INVENTION The invention, therefore, relates to the discovery that IGS1, the intergenic spacer region lying between the large ribosomal DNA (LrDNA) and the small ribosomal DNA (SrDNA) genes, of certain pathogenic yeasts, can be used to identify specific serotypes and genotypes.

It is an object of the invention to provide DNA sequences useful in designing amplification primers and hybridization probes for discriminating among pathogenic yeast and a database containing these sequences. According to this object of the invention, the DNA sequences of the intergenic spacer region of 91 strains of Cryptococcus are provided (depicted in Figure 1) and these same sequences are provided in electronic form, suitable for database applications. It is yet another object of the invention to provide primers and probes useful for discriminating among pathogemc yeasts. According to this object of the invention, universal and discriminating primers and probes, based on the inventive Cryptococcus sequences, are disclosed.

In one embodiment, the primers of the invention are adapted from a suitable portion of a sequence selected from the group consisting of, with reference to Figure 1, positions 1-65, 105-190, 172-191, 184-241, 337-369, 423-440, 423-449, 586-603. 586- 600, 683-161, 875-92, 960-988, 1056-1088, 1082-1122, 1108-1140, 1178-1264, 1284- 1449, and includes minor variants of said primer and complements of said primer. In another embodiment, the universal primers are based on the group consisting of: GGTCTCGGGGGGCTTCCTCT,GGCTTCCTCTAGAGACTTGG, GGTCAAGCAAAGTCTAGAAAAG,GGTGAGTATGTGATGTGA, ACAAGACAAGTAGGGAA, and include complements of that group; and minor variants of that group.

In still another embodiment, the discriminating primers and probes of the invention are adapted from a suitable portion of a sequence selected from the group consisting of:

sr CTCTAACATGTTGGGTCTCGGGGGGCTTCCTCTAGAGACTTGGATGTAAGGGGC

TTTACGCATTC;CATCAGTCTTCAGCTTGGGT;

TGGGGGACTTGGGAGCTGGTGCTTGTGTCGCAT;

GGGGGACTTGGGGTAAGACGCCTTGCA;TATGTATATAGTTGA; GGTGAGTATGTGATGTGAGAAGGATGGTACCCCATCCTG;

CTTATGAAGTGTGGATGACTCTAGGAGGGGTCTGAGAATGTGCTGTGCCAGCCA

GGCTGATAATAGTTTAATTGTTAGCTTGGACTTGTACACAGTCTCATCAGTCTTC

AGCTTGGGTCATTGATATTCTGTAAGTCAAAACTTATCCATTCACTGTAGTAGGT

TGCGGAGGACTTGAAAGTTCTCTCTGCAATTGAGACACTTACCAGGCCATCGCA AGTTGGCAGT;

GAGAATGTGCTGTGCCAGCCAGGCTGATAATAGTTTAATTGTTAGCTTGGACTT

GTACACAGTCTCATCAGTCTTCAGCT;TAGGTTGCGGAGGACTT;

GTTGCGGAGGACTTG AAAG ;

CAGCTTTCTATGCATGCAGCCTCGGCGCCGACAGTTGAAAAAAATGTATAGTCT CTGGACCATGAGAGA;TTGAATGTGTAATAGCTCAAGTGCCAGGAG;

GTCAATAGCCACCTGGGTGAGTATGTG;AACCGACTTGAGGGAGTGTCAGGATG

CATG;

GCAGACTCAGCCTTTTATCTTCGGCCTCACTGGCACACGCTTGAAGATCA;

TGGAAGCGGCGCTAGGGCCAAGCGGCTTGTCGCTGTC;ATCTTATCGACAGGCTG GTGTTTCCTTCGTTCCCTTTCGTCTTTCAACTCGTCTTAG;

AAGGAAACAAGATAAAGTAGGACAAGACAAGTAGGGAGGTTAGTG;

GTGTGCTGTGCGAGCCCGGCTGATAATTGTTTTCATTGTTAGCTTGGATTGCACA

CACAGTCTCATCAGTCTTCAGCTTGGGTTATCGATATTCTGTAAGTCCAAACTTA

TCCATTCACATTCACTGTAGTGTAGGTTGCAGAGAGGACTTGGAAAGTTCTCTCT CTGCACTGG;

TGGGTTATCGATATTCTTCTGTAAGTCCAAACTTATCCATTCACATTCACTGTAG

TGTAGGTTGCAGAGAGGACTTGGAAAGTTCTCTCTCTGCACTGG;

GGTTGCAGAGAGGACTTGG;GCATACTACTGATTT;

GCATACAGCCTTGGCGCCGGCAGCCGAAAAAAATGTATGGTCTTTAGAC; TGAGAGATTTCGATGTGTTATAGCTCAAGTACCAGGAGCAGTAGTCATGATCTG

AAATCATGGATCTGTTTCCACGCCTGGTGTGAGCAGGAGTAAGACTTTGAAGTT

GACCTTGGGTGACAAAAAAATGGGGTGGTGTCAGGAACCACCCAGGTGAGTATG

έ ;TGAGAGATTTCGATGTGTTATAGCTCAAGTACCAGGAGCAGTAGTCATGATCTG

AAATCATGGATCTGTTTCCACGCCTGGTGTGAGCAGGAGTAAGACTTTGAAGTT

GACCTTGGGTGACAAAAAAATGGGGTGGTGTCAGGAACCACCCAGGTGAGTATG

;GGTGGTGTCAGGAACCACCCAGGTGAGTATGTG; GCTGGCGAAGATGCAGTCAGCAACCAAAAAATTCTGGTAAGCACTCTGAAACGA

CTTGGGCGAGTCTCAGGATGCATGAGAGGCTTGCCGATCACAATTTTATCGAGC

AGGCGATGTTTCCTTCATTCCATTTCATCTTTCAGCTGGCCTTAGGATACGGCAG

AACAAGACAAGTAGGGAAGTTAGTGTTATAATCTT

AGCCAGGCTGATAATAGTTTAA;GTACGCTACTGATTT;GTGACTTCTTTTAGTCG TGGG;

GTAATCGTGAACCTGGGGGTCATGGATCTGTTTCTACACCTGATGTGAGCTGGA

GCAAGACTTTGGAGTTGACCTTGGGTGACAAAAAAT;

GGGTGGTGTCAATAGCCACCTGGG;GTACACTACTGATTTTACGGT;

GGAGCAGTAATTGTGAAACCTGAGGTCATGGATCTGTTTCCACACCTGATGTGA GCTGGAGCAAGAGTTTGGAGTTGACTTTGGGCGACAAAAAATGGGATGGTGTCA

;CCAACAAAAAAACCCGGTAAGCAC;

TGAAGATCATCAGAGGCACTCATTTAACCCTGTTCCCATTATCAG;

TTGTCAGCTTGGACTTGT;CTCTGGACCATGAGAGAGTTGAATGTGAATAGC;

TGGACCATGAGAGAGTTGAATGTGTAATAGCTCAAG;GTGAACCTGAGGTCATG G;TGGGGTGATGTCAATAGCCACCTG;GATGCATGGGCAGACTCAGCC;

ATTTAACCCTGTCCCCATTATCAG:ATCGATATTCTTCTGTAAGTC;

ATTCTTCTGTAAGTCCAAACT:TACGCATTCACATAATATTAGTTGGGGACTGAG

G;GCAAGTAGAGTCAAACAG;AGGAGAGAGGTCGGGCTGGTAATTAGTAATATC

AGTCAGTCATTTCAGCT;GCTGGCGCCATCGATACTTTATAAGTCAAACTTAACC ATTTCACTGTGTAGTA;GTTATGCGGCAAGCAAG;TGTGCTTATAGCATAGTC;

GCTGGTCAAGCAAACGTTTAAGTTGAGTCAAGCT;CAGTCTCGATGTCGCTGG;

AGTAGCTGTGATCTGAGGCCATGGATC;CAAAAAGTTGGGGCGGTGTCAGCAGC

GGACACGGTGAGT;TGGTACCCGATCTTGTTGGTGAAGGTGCAGTCA;

TTGGAAGAGTCTCGGGATGCAGGAGCGGATTCAGCGTTTTCTTTGGCCTTACTTA TCGAGGGA;TTCCCTTTTCGTTTTTCAACCGGTCATAGAAACAAGATTCTTAAGA

CGTGATTTGATTAGA;TGCTTTTTTTTATGACGATGACTTGTCAAAGTGTGG;

AGTAGTAGGCTCTGAATTACTAGAGACACTTGC;CAAGTTGGCAGGCAGGCAGG; GCAGGACACACATACTATTGATTT;TGCTGGTGCTTGAGTTGCATA; GAGAGAGTATATGCATGTCGCGGGGGGGGGACTTGGCT;TTAAAGTATTTAG; GTTTGACCCGACCTGACGGTG;ATCGCTAAGAATTACTCCGGTCGCGGGGGGCTT GCAACTTGTCT;GTCTTTGGACGATGTGAGATTTCATTGTGTAATAGC; GTTTCCATACCTGGTATAAGCTCGAGTGAGACTGTGCAAGTT;

GAGGGACTGGGTGTCCCCTTCGTTCCC;GGTCATAAAAACAAGATTCTAAGACGT ; GGGAAATTAGTGTTATATTCTT;TGCTTTTTTACGATGACTTGTCAAAGTGTGG; TCGTACACATACTATTGATTT;GGCTACTGGTACTGTGTTGCAT; TACTGGTACTGTGTTGCATA;TTATATAGTATT;GTTGACCCGACCTGATGGTG; ATCACTAAGAATTACTCCAGTCGCGGGGGGCTTGTAACTTCTCT;

GACGATGTAAGATTTCATCGCGTAATAGC;GGATCTGGTTCCACACCTGGTATGA GCTCGAGTGAG;GGGAAATTAGTGTTATGTTGTT;TGGATACAAGAGGCTTTGCT TT;TGCTTTTTGACGATGAACTTGTCAAAGTGTGG;TGACGGGTACTAGAGACACT TGC;TCGTCATACACATCCTATTGATTT;TACTGGTGCTTGTGTTGCCTA; TTATAGTATTGG:TAGTATTGGTTACTTGCC;ATGAAAAACAGGTAAA; AACAGGTAAATGTGGTATGG;GTTGACCCAACCTGACAGTG; ATCACTAAGAATGACTCCAGTCGCGGGGGACTTGGAACTTCTCT; GTCTTTGGAGGATGTAAGATTTCATCGTGTCATAGCTCAAGT; CATGGATCGGGTGCCACCCCTGGTATGAGCTCGAGTGAGACTGTGCAAGGTCAA TTTGGCCTTGGG; GGGAAATTAGTGTCATGTTCTT; and their complements; and minor variants thereof.

It is an additional object of the invention to provide methods for use in discriminating among pathogenic yeasts. According to this object, a method is provided that generally entails contacting a suspected yeast-containing sample with at least one of the probes or primers of the invention. Further to this object, one embodied method involves amplifying the intergenic spacer region of a sample and bringing the resultant amplified product into contact with an inventive discriminating primer.

In another embodiment the sample is amplified using polymerase chain reaction (PCR) with at least two inventive universal primers. This embodiment preferrably further entails bringing the resultant amplified product into contact with at least one inventive discriminating primer. In further embodiments the discriminating primer may be extended in a polymerase chain reaction or it may be immobilized. Still another

7 embodiment provides a method calling for amplifying a sample with a pair of primers, including at least one inventive discriminating primer, and detecting the resultant amplification product.

It is yet another object of the invention to provide kits useful in discriminating among pathogenic yeasts. Further to this object, an assemblage is provided that contains at least two inventive universal primers. In a preferred embodiment, the assemblage also contains a discriminating primer. In another preferred embodiment, the discriminating primer is immobilized on a solid matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a representative alignment of 91 IGS sequences from strains of Cryptococcus neoformans var. neoformans and C. neoformans var gattii, also known respectively as Filobasidiella neoformans var. neoformans and F. neoformans var bacillispora (Kwon-Chung 1998). Figure 2 shows a cladogram of genotypes and serotypes of Cryptococcus neoformans, which graphically demonstrates the relationships established by the present methods, based on partial IGS sequence analysis (PAUP parsimony analysis). The annotation shows the relationship between the present groupings and the classical serotypes (A, B, C, D and AD). Figure 3 shows a portion of the IGS region for five different strains of

Phaffia rhodozyma: (a) ATCC24228; (b) ATCC24230: (c) CBS6938; (d) VKMY2786; (e) CBS5905.

DETAILED DESCRIPTION OF THE INVENTION In view of the deficiencies in the art, the inventors provide a comprehensive sequence database for identification of yeasts. This database is a feasible and reliable source of probes for differentiation of yeasts to the species level and to subspecies levels.

To meet the need for a rapid test, in one embodiment, the invention provides an assay based on the microplate format, employing PCR amplified product capture by specific probes. Such gene -based assays are suitable for rapid differentiation of species, strains and genotypes of pathogenic yeasts.

i The invention provides a series of novel DNA sequences that are derived from the Intergenic Spacer One (IGS1) region of certain pathogemc yeasts. Based on sequence conservation and variability the inventors have used this information to identify genotypes within the two varieties of Cryptococcus neoformans: C. neoformans var. neoformans and C. neoformans var gattii, also known respectively as Filobasidiella neoformans var. neoformans and F. neoformans var bacillispora (Kwon-Chung 1998).

The invention further provides materials, methods and kits useful in identifying various C. neoformans genotypes. Genotypes la, lb, lc, 2a, 2b, and 2c represent C. neoformans var. neoformans and genotypes 3, 4 and 5 are C. neoformans var. gattii. Although considerable controversy exists regarding the taxonomic status of the two varieties, mating reactions indicate that the two taxa represent sexually independent species. Genotypes la. lb, lc and 2a. 2b, and 2c represent opposite mating types of C. neoformans var. neoformans.

Published information indicates that the opposite mating types are represented by Serotypes A and D. Mixed presence of serotypes in Genotypes la, lb ad lc and 2a, 2b and 2c is indicative of cross hybridization. Similarly, Gentoypes 3 and 4 represent one mating type of C. neoformans var. gattii, whereas Genotype 5 is the opposite mating type. Mixed serotypes among the genotypes of var. gattii also indicates cross hybridization.

The representative strains in each genotype are listed below:

Genotype la: cbs_1143_; cbs_1144_; cbs_1932_; cbs_1933_; cbs_4572_; cbs_4868_; cbs_6961_; cbs_7779_ cbs_879_; cbs_880_; cbs_886_; cbs_887_; cbs_889_; cbs_916_; RV_55447_; rv_66025_; rv_58145_; rv_61790; rv_65662; rv_59379_; cbs_7812;Av_B10_; rv_61756_; rv_55446_; Av_B7_; Av_B5_; Av_B4_; Av_B3_; Av_B12 Av_B2_; Bd_2_; and rv_64610.

Genotype lb: rv_46115_; rv_55451_; Av_Bl_; Av_B13_; Av_Bll_; rv_59351_; rv_59369_; and rv_62210.

Genotype lc: rv_58146.

Genotype 2a: cbs_4194_; cbs_6886_; cbs_888_; Av_B6_; cbs_7822_; rv_62992_; and J9.

Genotype 2b: cbs_6900_; cbs_6901_; cbs_7000_; cbs_7816_;cbs_7824_; cbs_7825_; cbs_7826_; and rv_62692.

Genotype 2c: cbs_132_;cbs_5467_; cbs_5474_; cbs_950_; cbs_6885_; cbs_5728_; cbs_7815_; cbs_7814_; cbs_939_; cbs_918_; cbs_1584_; Ba_3_; Ba_4_; cbs_131_; BA_1_; and cbs_464.

Genotype 3 : cbs_1930_; cbs_6956; cbs_7750_; and imh_1658.

Genotype 4 : cbs_5757_; cbs_6998_; cbs_6992_; cbs_6290_; cbs_7229_; cbs_7748_; cbs_919_; rv_5265_; 48A_; 52A_; 55A_; 56A_; and 59A.

Genotype 5 : cbs_5758_: cbs_6994_; and cbs_6996.

Nucleic Acids

General

One strand of each inventive DNA is provided in Figure 1 ; the complement of each inventive DNA is also included in the present invention. The numbers at the left in Figure 1 designate known strains of Cryptococcus neoformans. Further details relating to these strains may be found in Table 1 of Boekhout et al. 1997. Int'l J. Systematic Bacteriol. 47(2): 432-442, which table is hereby incorporated by reference.

In addition to the full-length DNAs of the invention, which themselves find application in identifying pathogenic yeasts, the invention contemplates probes and primers, which are generally shorter sequences derived from those in Figure 1. All sequences herein are designated with reference to the consensus numbering presented at the top of Figure IThe inventive DNAs do not include the sequences reported by Fan, et al. 1995. J. Med.Mycol. 33(4): 215-221 ; GenBank Accession Nos. L27028 and L27029.As used herein, the terms "probe" and "primer" are used interchangeably. One skilled in the art will understand that primers are generally used for amplification procedures, whereas probes are typically employed in classical hybridization methods. In the present invention, all of the disclosed probes may be used in the classical sense, or in many cases they may be used as primers in amplifications. As used herein, "amplified product" designates the nucleic acid amplification product.

As used herein "minor variants" of probes or primers are those that have sequence deviations from a suitable portion of those sequences shown in Figure 1. but retain at least 70% sequence identity to those shown in Figure 1. Preferred minor variants

// retain at least 80% identity, while others retain at least 85% or 90% identity. Even more preferred variants retain at least 95% identity. Variants include molecules that are shortened or lengthened at the 3' and/or 5' end(s), with reference to suitable portions of sequences in Figure 1. While such variations may readily be measured by hand, they can also be measured using the Blast 2 algorithm, as implemented at the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST), using default parameters. Such minor variants, however, still function as described, i.e., as universal or discriminating probes. The artisan will be aware of how much deviation may be present so as to conserve the essential character of these variants. Moreover, they may be tested empirically, as set out in detail below.

As used herein, a "suitable portion" used with reference to an inventive nucleic acid sequence means a portion of an inventive sequence (or variants thereof) that is of sufficient length and conservation to confer the requisite functional characteristic. Thus, "universal" probes and "discriminating" probes contain sufficient sequence to fall within the definitions of these terms, as set out below. Therefore, a "suitable portion" should be read with reference to the probe design section of this disclosure and the sections on universal and discriminating probes, set out below.

Universal Probes and Primers As used herein, a "universal probe" or "universal primer"is one that, under standard hybridization conditions, will hybridize specifically to all members of a predetermined taxon, e.g., one or more pathogenic yeast genotypes. Standard hybridization conditions include those of standard PCR and LCR assays. Typical PCR and LCR reaction conditions are presented below in the Examples. As indicated below, temperature and ionic strength are the primary driving forces in a hybridization reaction. As the artisan will appreciate, techniques like PCR and LCR entail hybridization at an elevated temperature, typically at least about 55°C. The artisan will also appreciate that some standard buffers contain about 5 to about 30 mM Tris-HCl (or other suitable buffer), about 10 -75 mM salt (KC1 or NaCl, typically), about 1 to about 15 mM divalent cation (typically MgC12) and may contain about 0.05% to about 0.5% detergent (like Triton X- 100 or NP-40). Accordingly, those conditions should be taken into account in probe design and optimization. Generally, universal primers are used as amplification primers in, for example. PCR. Accordingly, they will be useful in pairs. Moreover the universal primers near the ends of the IGS usually are designed so as to amplify the sequences between them. However, when sequence coordinates are provided herein, it should be understood that both the sense sequence and the antisense (complementary) sequence are contemplated. Where only the sense or only the antisense (complementary) sequence is meant, it is specified. This applies equally to the discriminating probes, described below.

Universal primers typically are designed from sequences within about 100 nucleotides of the 5' and 3' ends of the IGS, as shown in Figure 1, or they are just outside it, like LrDNA primer LRU (5'-TTA CCA CAG GGA TAA CTG GC-3') and 5SR primer (5'-GGA TCG GAC GGG GCA GGG TGC-3'). Primer 5SR represents the antisense complement of nucleotides 24 to 44 from the 5' end of the secondary structure of the Cryptococcus neoformans 5S region (Fan et al (1995). In general, any other conserved regions are suitable for the design of umversal primers. Particular regions of interest for universal probes include sequences around positions 14-43of Figure 1 (preferably the sense sequence), 718-739, 1082-1099. and 1450- 1465 of Figure 1 (preferably the antisense complementary sequence). Thus, in these regions, there are only relatively isolated single-base alterations among species, which can be accounted for in designing probes. For instance, while the internal portion of the probe may contain one to a few non-conserved positions, the 3' end (at least about 3 nucleotides) should be made up of conserved sequences. Another set of universal probes contemplated have the capability of differentiating between Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii . These probes will generally include positions around 1-65, 105-190, 172-191, 184-241. 337-369, 423-440, 423-449, 586-603. 586-600, 683-716, 875-92, 960-988, 1056-1088. 1082-1122, 1108-1140, 1178-1264, 1284-1449 of Figure 1. These regions are useful in generating universal probes for C. neoformans var. neoformans ( 1-65, 172-191, 337-369, 423-449, 586-600 and 1082-1122) or var. gattii ( 105-190. 184-241, 423-440, 586-603, 683-716, 875-892, 960-988, 1056-1088, 1108-1140, 1178-1264 and 1284-1449). Accordingly, these probes will be useful in differentiating C. neoformans var. neoformans and C. neoformans var. gattii and in providing universal amplification primers for further identification within those groups.

/3 The following are preferred sequences that may be used for designing universal probes for all genotypes of Cryptococcus neoformans. All sequences are shown 5' to 3', per conventional usage. It will be understood that minor variants of these sequences, as described herein, will also yield suitably universal probes. FORWARD:

• SEQUENCE 14 - 33 GGTCTCGGGGGGCTTCCTCT • SEQUENCE 24 - 43

GGCTTCCTCTAGAGACTTGG

REVERSE: • SEQUENCE -718 - 739

GGTCAAGCAAAGTCTAGAAAAG

• SEQUENCE 1082 - 1099 GGTGAGTATGTGATGTGA

• SEQUENCE 1450 - 1465 ACAAGACAAGTAGGGAA

Probe length and exact identity may be ascertained as described below. In addition, for non-conserved, variable positions, non-conventional bases, like inosine, may be employed in place of the native base. Universal probes will generally be based on the sequence presented by the ranges set out. but they can extend up to about 10 nucleotides outside these ranges. Variants, like 1. 2. 3. 4, 5 and 6 nucleotides. are specifically contemplated.

Discriminating Probes

As used herein, a "discriminating" probe is one that, under a given set of hybridization conditions, can be made to anneal specifically to one genotype or serotype of pathogenic yeast DNA, but not to others. The terms "hybridization probe" and "capture probe" are used to denote a discriminating probe that is adapted or used in a capture-based assay.

Such probes will generally be based on the sequence presented by the ranges set out in the present application, but they can extend up to about 10 nucleotides outside

/4 these ranges. Variants, like 1, 2, 3, 4, 5 and 6 nucleotides, are specifically contemplated. Again, non-conventional bases may be used to account for a limited number of non- conserved positions.

The artisan will recognize that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions see, for example, Sambrook et al., 1989, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Green Publishing Associates and Wiley Interscience. NY.

Structural relatedness between two polynucleotide sequences can be expressed as a function of "stringency" of the conditions under which the two sequences will hybridize with one another. As used herein, the term "stringency" refers to the extent that the conditions disfavor hybridization. Stringent conditions strongly disfavor hybridization, and only the most structurally related molecules will hybridize to one another under such conditions. Conversely, non-stringent conditions favor hybridization of molecules displaying a lesser degree of structural relatedness. Hybridization stringency, therefore, directly correlates with the structural relationships of two nucleic acid sequences. The following relationships are useful in correlating hybridization and relatedness (where Tm is the melting temperature of a nucleic acid duplex): a. Tm = 69.3 + 0.41(G+C)% b. The Tm of a duplex DNA decreases by loC with every increase of 1 % in the number of mismatched base pairs. c. (Tm)μ2 - (Tm)μl = 18.5 Iogl0μ2/μl where μl and μ2 are the ionic strengths of two solutions.

Hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Thus, discriminating probe design should be undertaken with these considerations in mind.

The following sequences, including the sense and antisense sequences, have been identified as the most likely portions of the intergenic spacer regions to yield probes

y that discriminate among the species, varieties and genotypes identified. All sequences are shown 5' to 3', per conventional usage. It will be understood that certain minor variants of these sequences, as described herein, will also yield suitably discriminating probes.

Cryptococcus neoformans var. neoformans GENOTYPES la, lb, lc, 2a, 2b, and 2c • SEQUENCE - 1 - 65 CTCTAACATGTTGGGTCTCGGGGGGCTTCCTCTAGAGACTTGGATGTAAGGGGC

TTTACGCATTC

SEQUENCE - 172 - 191 CATCAGTCTTCAGCTTGGGT

SEQUENCE - 337 - 369

TGGGGGACTTGGGAGCTGGTGCTTGTGTCGCAT

SEQUENCE - 423 - 449

GGGGGACTTGGGGTAAGACGCCTTGC SEQUENCE - 586 - 600

TATGTATATAGTTGA

SEQUENCE 1082 - 1122

GGTGAGTATGTGATGTGAGAAGGATGGTACCCCATCCTG

Cryptococcus neoformans var. neoformans GENOTYPES la, lb, & lc

SEQUENCE 72-309

CTTATGAAGTGTGGATGACTCTAGGAGGGGTCTGAGAATGTGCTGTGCCAGCCA

GGCTGATAATAGTTTAATTGTTAGCTTGGACTTGTACACAGTCTCATCAGTCTTC

AGCTTGGGTCATTGATATTCTGTAAGTCAAAACTTATCCATTCACTGTAGTAGGT TGCGGAGGACTTGAAAGTTCTCTCTGCAATTGAGACACTTACCAGGCCATCGCA

AGTTGGCAGT

SEQUENCE - 105 - 186

GAGAATGTGCTGTGCCAGCCAGGCTGATAATAGTTTAATTGTTAGCTTGGACTT

GTACACAGTCTCATCAGTCTTCAGCT SEQUENCE - 240 - 257

TAGGTTGCGGAGGACTT

SEQUENCE - 243 - 262

GTTGCGGAGGACTTGAAAG

l<* SEQUENCE 859 - 927

CAGCTTTCTATGCATGCAGCCTCGGCGCCGACAGTTGAAAAAAATGTATAGTCT

CTGGACCATGAGAGA

SEQUENCE 929 - 958 TTGAATGTGTAATAGCTCAAGTGCCAGGAG SEQUENCE 1067 - 1093

GTCAATAGCCACCTGGGTGAGTATGTG

SEQUENCE 1171 - 1200

AACCGACTTGAGGGAGTGTCAGGATGCATG SEQUENCE 1202 - 1251

GCAGACTCAGCCTTTTATCTTCGGCCTCACTGGCACACGCTTGAAGATCA

SEQUENCE 1333 - 1369

TGGAAGCGGCGCTAGGGCCAAGCGGCTTGTCGCTGTC

SEQUENCE 1371 - 1428 ATCTTATCGACAGGCTGGTGTTTCCTTCGTTCCCTTTCGTCTTTCAACTCGTCTTA

G

SEQUENCE 1429 - 1473

AAGGAAACAAGATAAAGTAGGACAAGACAAGTAGGGAGGTTAGTG

Cryptococcus neoformans var. neoformans GENOTYPES 2a, 2b, & 2c

SEQUENCE -109 - 305

GTGTGCTGTGCGAGCCCGGCTGATAATTGTTTTCATTGTTAGCTTGGATTGCACA

CACAGTCTCATCAGTCTTCAGCTTGGGTTATCGATATTCTGTAAGTCCAAACTTA

TCCATTCACATTCACTGTAGTGTAGGTTGCAGAGAGGACTTGGAAAGTTCTCTCT CTGCACTGG

SEQUENCE - 187 - 305

TGGGTTATCGATATTCTTCTGTAAGTCCAAACTTATCCATTCACATTCACTGTAG

TGTAGGTTGCAGAGAGGACTTGGAAAGTTCTCTCTCTGCACTGG

SEQUENCE - 253 - 279 GGTTGCAGAGAGGACTTGG

SEQUENCE - 310 - 328

GCATACTACTGATTT SEQUENCE 870 - 918

GCATACAGCCTTGGCGCCGGCAGCCGAAAAAAATGTATGGTCTTTAGAC

SEQUENCE 921 - 1091

TGAGAGATTTCGATGTGTTATAGCTCAAGTACCAGGAGCAGTAGTCATGATCTG AAATCATGGATCTGTTTCCACGCCTGGTGTGAGCAGGAGTAAGACTTTGAAGTT

GACCTTGGGTGACAAAAAAATGGGGTGGTGTCAGGAACCACCCAGGTGAGTATG

SEQUENCE 1061 - 1093

GGTGGTGTCAGGAACCACCCAGGTGAGTATGTG

SEQUENCE 1122 - 1483 GCTGGCGAAGATGCAGTCAGCAACCAAAAAATTCTGGTAAGCACTCTGAAACGA

CTTGGGCGAGTCTCAGGATGCATGAGAGGCTTGCCGATCACAATTTTATCGAGC

AGGCGATGTTTCCTTCATTCCATTTCATCTTTCAGCTGGCCTTAGGATACGGCAG

AACAAGACAAGTAGGGAAGTTAGTGTTATAATCTT

Cryptococcus neoformans var. neoformans GENOTYPE la

SEQUENCE - 121 - 143

AGCCAGGCTGATAATAGTTTAA

SEQUENCE - 310 - 328

GTACGCTACTGATTT SEQUENCE - 475 - 495

GTGACTTCTTTTAGTCGTGGG

SEQUENCE 961 - 1058

GTAATCGTGAACCTGGGGGTCATGGATCTGTTTCTACACCTGATGTGAGCTGGA

GCAAGACTTTGGAGTTGACCTTGGGTGACAAAAAAT SEQUENCE 1060 - 1083

GGGTGGTGTCAATAGCCACCTGGG

Cryptococcus neoformans var. neoformans

GENOTYPE lb

SEQUENCE - 310 - 334 GTACACTACTGATTTTACGGT

SEQUENCE 955 - 1070

If GGAGCAGTAATTGTGAAACCTGAGGTCATGGATCTGTTTCCACACCTGATGTGA GCTGGAGCAAGAGTTTGGAGTTGACTTTGGGCGACAAAAAATGGGATGGTGTCA SEQUENCE 1141 - 1165 CCAACAAAAAAACCCGGTAAGCAC SEQUENCE 1243 - 1287

TGAAGATCATCAGAGGCACTCATTTAACCCTGTTCCCATTATCAG

GENOTYPE lc

SEQUENCE 144 - 162

TTGTCAGCTTGGACTTGT SEQUENCE 911 - 944

CTCTGGACCATGAGAGAGTTGAATGTGAATAGC

SEQUENCE 914 - 949

TGGACCATGAGAGAGTTGAATGTGTAATAGCTCAAG

SEQUENCE 967 - 985 GTGAACCTGAGGTCATGG

SEQUENCE 1058 - 1081

TGGGGTGATGTCAATAGCCACCTG

SEQUENCE 1193 - 1213

GATGCATGGGCAGACTCAGCC SEQUENCE 1264 - 1287

ATTTAACCCTGTCCCCATTATCAG

Cryptococcus neoformans var. neoformans

GENOTYPE 2a

SEQUENCE - 451 - 472 GCAAGTAGAGTCAAACAG

Cryptococcus neoformans var. neoformans

GENOTYPE 2b

SEQUENCE - 57 - 97

TACGCATTCACATAATATTAGTTGGGGACTGAGG Cryptococcus neoformans var. neoformans

GENOTYPE 2c

SEQUENCE - 193 - 213

11 ATCGATATTCTTCTGTAAGTC

SEQUENCE - 199 - 219

ATTCTTCTGTAAGTCCAAACT

Cryptococcus neoformans var. gattii GENOTYPES 3, 4 and 5

SEQUENCE - 105 - 190

AGGAGAGAGGTCGGGCTGGTAATTAGTAATATCAGTCAGTCATTTCAGCT

SEQUENCE - 184 - 241

GCTGGCGCCATCGATACTTTATAAGTCAAACTTAACCATTTCACTGTGTAGTA SEQUENCE - 423 - 440

GTTATGCGGCAAGCAAG

SEQUENCE - 586 - 603

TGTGCTTATAGCATAGTC

SEQUENCE - 683 - 716 GCTGGTCAAGCAAACGTTTAAGTTGAGTCAAGCT

SEQUENCE 875 - 892

CAGTCTCGATGTCGCTGG

SEQUENCE 960 - 988

AGTAGCTGTGATCTGAGGCCATGGATC SEQUENCE 1056 - 1088

CAAAAAGTTGGGGCGGTGTCAGCAGCGGACACGGTGAGT

SEQUENCE 1108 - 1140

TGGTACCCGATCTTGTTGGTGAAGGTGCAGTCA

SEQUENCE 1178 - 1264 TTGGAAGAGTCTCGGGATGCAGGAGCGGATTCAGCGTTTTCTTTGGCCTTACTTA

TCGAGGGA

SEQUENCE 1284 - 1449

TTCCCTTTTCGTTTTTCAACCGGTCATAGAAACAAGATTCTTAAGACGTGATTTG

ATTAGA Cryptococcus neoformans var. gattii

GENOTYPE 3

SEQUENCE - 57 - 96

IG TGCTTTTTTTTATGACGATGACTTGTCAAAGTGTGG

SEQUENCE - 242 279

AGTAGTAGGCTCTGAATTACTAGAGACACTTGC

SEQUENCE - 290 - 309 CAAGTTGGCAGGCAGGCAGG

SEQUENCE - 305 - 328

GCAGGACACACATACTATTGATTT

SEQUENCE - 350 - 370

TGCTGGTGCTTGAGTTGCATA SEQUENCE - 374 - 415

GAGAGAGTATATGCATGTCGCGGGGGGGGGACTTGGCT

SEQUENCE - 458 - 475

TTAAAGTATTTAG

SEQUENCE - 637 - 672 GTTTGACCCGACCTGACGGTG

SEQUENCE - 747 - 790

ATCGCTAAGAATTACTCCGGTCGCGGGGGGCTTGCAACTTGTCT

SEQUENCE 909 - 944

GTCTTTGGACGATGTGAGATTTCATTGTGTAATAGC SEQUENCE 990 - 1031

GTTTCCATACCTGGTATAAGCTCGAGTGAGACTGTGCAAGTT

SEQUENCE 1254 - 1304

GAGGGACTGGGTGTCCCCTTCGTTCCC

SEQUENCE 1340 - 1389 GGTCATAAAAACAAGATTCTAAGACGT

SEQUENCE 1462 - 1483

GGGAAATTAGTGTTATATTCTT

Cryptococcus neoformans var. gattii

GENOTYPE 4 SEQUENCE - 57 - 96

TGCTTTTTTACGATGACTTGTCAAAGTGTGG

SEQUENCE - 305 - 328

XI TCGTACACATACTATTGATTT

SEQUENCE - 347 - 369

GGCTACTGGTACTGTGTTGCAT

SEQUENCE - 350 - 370 TACTGGTACTGTGTTGCATA

SEQUENCE - 458 - 473

TTATATAGTATT

SEQUENCE - 637 - 672

GTTGACCCGACCTGATGGTG SEQUENCE - 747 - 790

ATCACTAAGAATTACTCCAGTCGCGGGGGGCTTGTAACTTCTCT

SEQUENCE 916-944

GACGATGTAAGATTTCATCGCGTAATAGC

SEQUENCE 984 - 1019 GGATCTGGTTCCACACCTGGTATGAGCTCGAGTGAG

SEQUENCE 1462 - 1483

GGGAAATTAGTGTTATGTTGTT

Cryptococcus neoformans var. gattii

GENOTYPE 5 SEQUENCE - 41 - 62

TGGATACAAGAGGCTTTGCTTT

SEQUENCE - 57 96

TGCTTTTTGACGATGAACTTGTCAAAGTGTGG

SEQUENCE - 255 - 279 TGACGGGTACTAGAGACACTTGC

SEQUENCE - 305 - 328

TCGTCATACACATCCTATTGATTT

SEQUENCE - 350 - 370

TACTGGTGCTTGTGTTGCCTA SEQUENCE - 458 - 475

TTATAGTATTGG

SEQUENCE - 465 - 486 TAGTATTGGTTACTTGCC

SEQUENCE - 523 - 538

ATGAAAAACAGGTAAA

SEQUENCE - 529 - 548 AACAGGTAAATGTGGTATGG

SEQUENCE - 637 - 672

GTTGACCCAACCTGACAGTG

SEQUENCE - 747 - 790

ATCACTAAGAATGACTCCAGTCGCGGGGGACTTGGAACTTCTCT SEQUENCE 909 - 950

GTCTTTGGAGGATGTAAGATTTCATCGTGTCATAGCTCAAGT

SEQUENCE 981 - 1046

CATGGATCGGGTGCCACCCCTGGTATGAGCTCGAGTGAGACTGTGCAAGGTCAA

TTTGGCCTTGGG SEQUENCE 1462 - 1483

GGGAAATTAGTGTCATGTTCTT

The region from about 390 to about 550 may also provide useful discriminating probes, but the artisan is cautioned to consider misalignments due to multiple repeats.

Designing Probes and Primers

Region-specific primers or probes derived from the nucleotide sequences provided can be used to prime DNA synthesis and PCR amplification, as well as to directly identify samples containing pathogenic yeast, using hybridization methodologies. Innis et al., PCR Protocols, Academic Press. San Diego, CA (1990). The primers are preferably at least about 15 bases, and generally less than about 35 bases, and more preferably at least about 18 bases to about 27 bases in length.

When selecting pairs of primer sequence, it is preferred that the primer pairs have approximately the same G + C content, so that melting temperatures are approximately the same. As a general guide, the formula 3(G+C) + 2(A+T) = oC, is useful. In general, sequences having high degrees of GC or AT content should be avoided.

^3 For example, some typical probes will have from about 20 percent GC to about 80 percent GC content , while preferred probes have from about 40 to about 60 percent CG content.

Probes are designed based on nucleotide differences as viewed in sequence alignments, like Figure 1. Probes are typically less than about 50 bases in length, preferably at least about 15 bases, and generally less than about 35 bases, and more preferably at least about 18 bases to about 27 bases in length.

With reference to the cladogram of Figure 2 and the Figure 1 sequences, it is evident that the individual clusters, and each genotype within a cluster, have specific sequence regions that can be used to construct hybridization (discriminating) probes for resolution from other members of the cluster and other genotypes in the database, described below, and, thus, for highly specific identification. Discriminating probes are based on nucleotide differences as viewed in sequence alignments.

Identification of the genotype-specific sequence regions useful as probes is first done visually, using alignments like those in Figure 1, and confirmed by computer search within the genotype sequence database, followed by verification of probe specificity by a search of GenBank. If the sequence fails any of these tests, other variable regions of the database in use are searched for a new probe sequence. The quality of the probe (Tm, potential of stem loops, etc.) is then determined using a sequence analysis program (Oligo, National Biosciences, Inc.) This process is set out in more detail below in the Examples.2 Probe sequences are usually chosen to give a Tm of 60 to 70oC, using the percent GC algorithm. Further probe design is provided below in the Examples.

Methods

The methods of the invention typically utilize nucleic acid-based probes to detect the presence of a pathogenic yeast and to ascertain its genotype. In these methods a sample suspected of containing a pathogenic yeast is provided. This sample may be provided from a human subject or any other source thought to harbor the yeast, such as plants and animals, especially food products.

The sample is subjected to standard DNA extraction protocols. For example, DNA can be extracted from a blood sample as described by Haynes, et al.

(1995). In this procedure, erythrocytes are lysed and lymphocytes, as well as any resident yeast cells are pelleted by centrifugation. The supernatant is carefully removed and any

ft yeast cells present in the pellet are converted to spheroplasts, as follows. The pellet is washed twice with distilled water, then resident yeast cells are converted to spheroplasts by incubating for 2 hrs at 37 oC in 10 mM citrate buffer, pH 5.8, 1M sorbitol and 10 mg/ml lysing enzymes from Trichoderma harzianum (Sigma), which is freshly prepared for each procedure. The spheroplasts are washed in the spheroplasting buffer and then lysed in distilled water and centrifuged in a microfuge to pellet cell debris. The supernatant can be used for PCR without further purification.

The isolated nucleic acids are either detected directly by hybridization using a discriminating probe or subjected to an optional amplification, like polymerase chain reaction (PCR) or ligation chain reaction (LCR).

The polymerase chain reaction (PCR) procedure amplifies specific nucleic acid sequences through a series of manipulations including denaturation. annealing of oligonucleotide primers, and extension of the primers with DNA polymerase (Mullis. K.

B. et al., U.S. Pat. Nos. 4,683,202. 4,683,195; Mullis, K. B., EP 201.184; Erlich, H., EP 50,424, EP 84,796, EP 258,017, EP 237,362; Erlich, H., U.S. Pat. No. 4,582,788; Saiki,

R. et al., U.S. Pat. No. 4,683,202; Mullis, K. B. et al. Cold Spring Harbor Symp. Quant.

Biol. 51:263 (1986); Saiki, R. et al. Science 230:1350 (1985); Saiki, R. et al. Science

231:487 (1988); Loh, E. Y. et al. Science 243:217 (1988)). These steps can be repeated many times, potentially resulting in large amplification of the number of copies of the original specific sequence. It has been shown that even a single copy of a DNA sequence can be amplified to produce hundreds of nanograms of product (Li. H. et al. Nature

335:414 (1988)).

"Ligase Chain Reaction" (LCR) schemes based on ligation of two (or more) oligonucleotides in the presence of a nucleic acid target having the sequence of the resulting "di-oligonucleotide, " thereby amplifying the di-oligonucleotide, are also well known. Wuet al., Genomics 4: 560 (1989); Backman et al., EP 320,308; Wallace, B., EP

336,731; and Orgel, L., WO 89/09835.

In one aspect of the invention, the amplification serves directly as a detection method. In other words, in such an aspect, whole amplification products are detected. For example, primers may be designed such that they anneal at their 3' ends only in the presence of certain genotypes or serotypes. In the context of PCR, amplification is successful only in the presence of nucleic acid from the target organisms. Likewise, in LCR, such a primer will ligate to the adjacent probe only in the presence of nucleic acid from certain target organisms. The products may be detected visually (e.g., ethidium bromide-stained gel under uv light) or by other means, like autoradiography, using labeled probes, or using avidin-biotin technology, and the like. Beneficially, however, an amplified product, like a PCR product, is subjected to a treatment imparting further specificity on the process. For example, a capture step may be further employed using a discriminating (capture) probe. The amplified product is captured by hybridization to the specific capture probes immobilized on a solid substrate, for example, a microplate, and the captured material is detected. Detection may be achieved, for example, by generating biotinylated amplification products, and bound amplification product then is detected with streptavidin-horse radish peroxidase conjugate (SAHRP). The assay may be formatted in a microplate, using removable 8 or 12-well strips.

By this procedure, universal PCR primers can be used and PCR conditions are uniform for all yeasts; the need to match primer duplex stabilities is eliminated, and specificity lies in the sequence of the capture hybridization probe. In a further refinement of this procedure, capture hybridization probe sequences and hybridization conditions can be modified so that all hybridizations, regardless of probe sequence, may be executed at a single stringency. When amplified product is detected by hybridization to plates bearing specific capture probes, the hybridization protocol presented below in Examples is followed. Total time for execution of this hybridization protocol, including hands-on time, is about 90 minutes.

In a preferred embodiment, a capture-based assay is used where the capture probe is immobilized via a spacer, rather than being directly attached to the substrate. The inventors observed that amplified product capture by probes covalently coupled to a spacer arm was superior to capture by probes coated directly on the plate surface. Generally, nanogram levels of amplified product per well are detectable in plates with surface adsorbed probes. Plates bearing spacer arms, in contrast, allowed detection at 125 pg amplified product (108 copies of a 1 kb dsDNA amplified product) per well.

Techniques for coupling oligonucleotides to substrates are well known in the art. High quality plates coated with a spacer arm of 600 carbons may be obtained from Genetic Vectors, Inc. (5201 NW 72 Avenue, Suite 100, Miami, Florida 33166). To covalently immobilize DNA probes in the wells to serve as capture probes, wells coated with spacer arms were incubated at 50°C with a probe coating mix consisting of 25μl of lOmM carbodiimide (EDC) and 75/xl of probe (1.33ng/μl). The solutions were prepared in freshly made lOmM 1-methyl-imidazole. After 6 hrs incubation at 50°C, the wells were washed with 0.4M NaOH and 0.25% Tween 20, pre-warmed to 50°C, and incubated in the same solution for 15 min. The described washing procedure was repeated, followed by a final wash with distilled water at 25 °C. The coated and washed plates (or strips in a strip holder) were stored dry at 4°C in sealed plastic bags. Kits

The assay kits of the invention typically are self-contained, with the exception of PCR reagents. All buffers, primers and coated wells are supplied in the kits. The assay can be formatted on any solid matrix, like 8-well or 12-well strips, depending upon the number of yeasts targeted by the kit. The strips may be color coded or labeled with a probe code to allow easy identification of the specificity of the probes in the wells. Kits also usually contain a positive control consisting of a probe specific for a conserved sequence present in all amplified products.

One embodiment is an assay kit for the nine genotypes of C. neoformans. In this embodiment, the discriminating genotype capture probes are arranged in 8-well strips; one strip for each genotype probe, from strip 1 through 9. Strip 10 contains the conserved sequence positive control probe, 11 contains an unrelated probe as negative control and 12 is devoid of capture probe, providing a reagent blank. For each patient, twelve lOμl aliquots of PCR reaction mixture containing unknown amplified products are dispensed into duplicate rows of wells (patient 1 in wells Al through B12, patient 2 in wells Cl through D12, etc.) to start the hybridization assay. In this way, samples from up to four patients can be run in duplicate on one plate.

Database Applications

Because nucleotide sequences are useful to discriminate among species and genotypes, the inventive molecules disclosed in Figure 1 are useful as members of a database, which may also include the sequences of other intergenic spacer regions of pathogenic yeast, especially Cryptococcus strains. Such a database may be used, for example, in designing probes as described above, in testing the novelty and non- obviousness of newly sequenced materials, or in conjunction with the described methods in identifying a yeast species or genotype, and thus in determining a course of therapeutic treatment. Accordingly, one aspect of the invention contemplates a database of DNA sequences of Cryptococcus intergenic spacer regions. A preferred embodiment contains at least one of the inventive sequences stored on a computer readable medium. For example, the individual sequences may be grouped with regard to the individual functional and structural groups mentioned above. While the individual sequences of a database may exist in printed form, they are preferably in electronic form, as in an ascii or a text file. They may also exist as word processing files or they may be stored in database applications like DB2, Sybase, Oracle, GCG and GenBank. One skilled in the art will understand the range of applications suitable for using and storing the electronic embodiments of the invention.

"Computer readable media" refers to any medium that can be read and accessed by a computer. These include: magnetic storage media, like floppy discs, hard drives and magnetic tape; optical storage media, like CD-ROM; electrical storage media, like RAM and ROM; and hybrids of these categories, like magnetic/optical storage media. One skilled in the art will readily understand the scope of computer readable media and how to implement them.

References

Boekhout, T.. Kamp, M.. & Gueho. E. 1998. Med Mycol. 36(6):365-72.

Boekhout. T., & van Belkum, A. 1997. Curr Genet. 32(3):203-8.

Boekhout, T., van Belkum, A., Leenders, A.C., Verbrugh, H.A., Mukamurangwa. P., Swinne, D., & Scheffers. WA. 1997. Int J Syst Bacteriol. 47(2):432-42. Brummer, E.. Kamei, K., & Miyaji, M. 1998. Mycopathologia. 142(l):3-7 Buchanan, K.L., & Murphy, J.W. 1998. Emerg Infect D is. 4(l):71-83. Casadevall, A. & Perfect. J.R. 1998. Cryptococcus neoformans. ASM Press, Washington, D.C. Darwazah, A., Berg, G.. & Faris, B. 1999. J Infect. 38(2): 130-1

Diaz, M.R. & Fell, J.W. 2000 . Antonie Van Leeuwenhoek 77. In Press.

I Espinel-Ingroff, A., Stockman, L., Roberts, G., Pincus, D., Pollack, J., & Marler, J.

1998. J Clin Microbiol. 36(4):883-6.

Faergemann, J. 1997. Mycoses. 40, Suppl. 1: 29-32.

Fan, M., Chen, L.C., Ragan, M.A., Gutell, R.R., Warner, J.R., Currie, B.P. & Casadevall, A. 1995. J. Med. Vet. Mycol. 33(4): 215-21.

Fell, J.W. 1993. Mol. Mar.Biol. Biotechnol. 3: 174-180.

Fell, J.W. 1995. J Ind Microbiol. 14(6): 475-7.

Fell, J.W. & Blatt, G.M. 1999. Journal of Industrial Microbiology & Biotechnology. 21:

677-681. Fell, J.W., Boekhout, T. , Fonseca, A., Scorzetti, G. & Statzell-Tallman. A. 2000.

Internat. J. Syst. Evol. Microbiol. In Press.

Gozdasoglu, S., Ertem, M., Buyukkececi, Z., Yavuzdemir, S.. Bengisun, S.. Ozenci. H.,

Tacyildiz. N., Unal, E.. Yavuz, G.. Deda, G. , & Aysev, D. 1999. Med Pediatr Oncol.

32(5): 344-8. Gueho, E., Boekhout, T., Ashbee, H.R., Guillot, J., Van Belkum, A., & Faergemann, J.

1998. Med Mycol. 36, Suppl 1:220-9

Haynes, K.A., Westerneng, T.J., Fell, J.W., & Moens, W. 1995. J Med Vet Mycol.

33(5)319-25.

Hamilton, J. 1998 Med. Mycol. 36(6): 351-64. Hoppe, J.E. & Frey, P. 1999. Eur. J. Clin. Microbiol. Infect. Dis. 18(3): 188-91.

Hsu. C.F., Wang, C.C., Hung, C.S., Cheng, S.N., Chen, Y.H., & Chu, M.L. 1998.

Chung Hua Min Kuo Hsiao Erh Ko I Hsueh Hui Tsa Chih. 39(3): 191-4.

Huang, Y.C., Lin, T.Y., Leu. H.S., Peng, H.L., Wu, J. H. & Chang, H.Y. 1999.

Infection. 27(2):97-102. Jordan, J.A. & Durso, M.B. 1996. Mol. Diagn. 1(1): 51-8.

Kawakami, S., Ono, Y., Miyazawa, Y., & Yamaguchi, H. 1998. Kansenshogaku Zasshi.

72(2): 105-13.

Kohno. S., Var a, A., Kwon-Chung, K.J., & Hara, K. 1994. Kansenshogaku Zasshi.

68(12):1512-7. Krcmery, V., Krupova, I., & Denning D.W. 1999a, J Hosp Infect. 41(3): 181-94.

Krcmery, V. Jr., Mateicka, F., Kunova, A., Spanik S., Gyarfas, J., Sycova, Z., & Trupl,

J. 1999b. Support Care Cancer. 7(l):39-43.

tf Kurtzman, C.P. & Fell, J.W. 1998. The Yeasts. A Taxonomic Study, 4th Edition.

Alsevier, Amsterdam.

Kwon-Chung, K.J. 1998. In: Kurtzman, C.P. & J.W. Fell. The Yeasts. A Taxonomic

Study, 4th Edition. Elsevier, Amsterdam. Lascaux, A.S., Bouscarat, F., Descamps, V. , Casalino, E., Picard-Dahan, C, Crickx, B.,

& Belaich, S. 1998. Ann Bermatol Venereol. 125(2):lll-3.

Lopez-Ribot, J.L., McAtee, R.K. Perea, S. Kirkpatrick, W.R. Rinaldi, M.G. & Patterson

T.F.. 1999. Antimicrob. Agents Chemother. 43(7): 1621-30.

Mannarelli, B.M. & Kurtzman, C.P. 1998. J. Clin. Microbiol. 36(6): 1634-41. Milan, E.P., Malheiros, E.S., Fischman, O., & Colombo, A.L. 1997. Mycopathologia.

137(3):153-7.

Morace. G., Pagano, L.. Sanguinetti. M., Posteraro, B., Mele. L.. Equitani, F.. D'Amore,

G., Leone, G., & Fadda. G. 1999. J Clin Microbiol. 37(6): 1871-5.

Muller, F.M., Groll A.H., & Walsh, TJ. 1999. Eur J Pediatr. 158(3): 187-99. Nagai, H., Yamakami, Y., Hashimoto. A., Tokimatsu, I. & Nasu, M. 1999. J Clin

Microbiol.37(3):694-9.

Olesen, G., Yilmaz, M.K., Palshof, T., Moller, J.K., & Peterslund, N.A. 1997. Ugeskr

Laeger. 159(10): 1438-42.

Paugam, A., Benchetrit, M., Fiacre, A., Tourte-Schaefer, C, & Dupouy-Camet, J. 1999. Med Mycol. 37(1): 11-7.

Pernice, I., Lo Passo, C, Criseo. G.. Pernice, A., & Todaro-Luck, F. 1998. Mycoses.

41(3-4): 117-24.

Pfaller. M.A., Messer, S.A., Hollis, R.J., Jones R.N. , Doern G.V., Brandt, M.E., &

Hajjeh. R.A. 1999. Diagn Microbiol Infect Dis. 33(4):217-22. Prariyachatigul, C, Chaiprasert, A., Meevootisom, V., & Pattanakitsakul, S. 1996. J

Med Vet Mycol. 34(4):251-8.

Reiss, E., Tanaka, K., Bruker, G., Chazalet, V., Coleman, D.,, Debeaupuis, J.P.,

Hanazawa, R., Latge, J.P., Lortholary, J., Makimura, K., Morrison, C.J., Murayama,

S.Y., Saoe, S., Paris, S., Sarfati, J., Shibuya, K., Sullivan. D., Uchida, K., & Yamaguchi, H. 1998. Med Mycol. 1:249-57.

Slavoski, L.A., & Tunkel, A. R. 1995. Clin Neuropharmacol. 18(2):95-112. Sorrel, T.C., Brownlee, A.G., Ruma. P., Malik, R., Pfeiffer, T.J., & Ellis, D.H. 1996a. J Clin Microbiol. 34(5):1261-3.

Sorrell, T.C., Chen, S.C., Ruma, P., Meyer, W., Pfeiffer, T.J., Ellis, D.H., & Brownlee, A.G. 1996b. J Clin Microbiol. 34(5): 1253-60. Van Belkum, A., Boekhout, T., & Bosboom, R. 1994. J Clin Microbiol. 32(10:2528-32. Viscoli, C. & Castagnola, E. 1999. Int. J. Infect. Dis. 3(2): 109-18 Viviani, M.A., Wen, H., Roverselli, A., Caldarelli-Stefano, R., Cogliati, M., Ferrante, P., & Tortorano, A.M. 1997. J Med Vet Mycol. 35(5):355-60.

Wadlin, J.K., Hanko, G., Stewart. R., Pape, J., & Nachamkin, I. 1999. J Clin Microbial. 37(6): 1967-70.

Warnock, D.W. 1998. J Antimicrob Chemother. 41, Suppl D:95-105.

* * *

The foregoing detailed description and the following working examples are provided to illustrate the invention and are not meant to be limiting. The artisan will readily appreciate different additional embodiments within the scope of the invention with reference to common knowledge in the art.

Examples

Example 1 : DNA Purification and Sequencing Yeast isolates were obtained from type collections at the University of

Miami, the United States Department of Agriculture (USDA) and Centraalbureau voor Schimmelcultures (CBS) in the Netherlands. DNA purified with the following protocol was used to obtain the IGS sequence data underlying the phylogenetic tree of Figure 2. Cells were grown as pure cultures for 12-14 hrs in GYP (2% glucose, 0.1 % yeast extract, and 0.5% peptone). Cells were pelleted by centrifugation and washed with distilled water. Washed cells were converted to spheroplasts by incubation for 2 hrs at 37°C in 10 mM sodium citrate buffer, pH 5.8, 1M sorbitol and 10 mg/ml Lysing Enzymes from Trichoderma harzianum (containing cellulase, protease and chitinase activities; Sigma), freshly prepared for each extraction procedure. DNA was extracted and purified from the spheroplasts using the QIAamp Tissue Kit (QIAGEN, Inc.; Santa Clarita, California), following the standard kit protocol. The DNA was amplified with umversal primers (see below) using MJ Research Thermal Cycler Model PTC100 (MJ Research, Inc.; Waltham,

Massachusetts). The resulting amplified product was purified with the QIAquick PCR Kit.

The IGS region examined was between the LrDNA gene and the 5S rRNA gene. Amplification of this IGS region used two primers, LrDNA primer LRU (5' TTA CCA CAG GGA TAA CTG GC) and 5S region primer 5SR (5' GGA TCG GAC GGG

GCA GGG TGC). Primer 5SR represents the antisense complement of nucleotides 24 to 44 from the 5' end of the secondary structure of the Cryptococcus neoformans 5S region

(Fan. et al. 1995). The individual strands were cycle sequenced with 5SR for the reverse strand and LR12 (5' CTG AAC GCC TCT AAG TCA GAA) for the forward strand. LR12 is closer to the 3 ' end of the LrDNA than LR11.

Example 2: Genotype Identification

To develop a molecular method for differentiating strains, we examined the IGS regions of serotypes of the pathogen C. neoformans (discussed below), the industrially important species Phaffia rhodozyma (Fell & Blatt 1999; Fell, et al., 2000), and the low temperature Antarctic yeasts in the genus Mrakia (Diaz & Fell 2000 ).

Figure 3 shows a portion of the IGS region for five different strains of Phaffia rhodozyma: (a) ATCC24228; (b) ATCC24230; (c) CBS6938; (d) VKMY2786; (e) CBS5905. These strains can be resolved based upon deletions and insertions in the IGS region.

In Mrakia (not shown) and C. neoformans (Figure 1), most differences in the IGS region are at a level of base sequences rather than deletions and insertions, although a region of deletions and insertions lies at the 3' end of IGS1, from about 1202 to about 1445. The IGS region lying between the LrDNA and the 5S rRNA genes of 91 C. neoformans strains has been examined (Figure 1). Our results for C. neoformans, correlated with serotype data , are summarized in the phylogentic tree in Figure 2.

Sequence analyses and probe hybridizations are powerful tools for accurate identification. The extent of the sequence divergence underlying the phylogenetic tree in Figure 2 provides a variety of regions for design of hybridization probes and primers for rapid and concise identification of genotypes within this group of highly pathogenic yeasts. The C. neoformans genotypes segregate into two phylogenetically distinct clades (Figure 2). The upper clade, representing genotypes la, lb, lc, 2a, 2b, and 2c, which are

M considered to be the mating types of C. neoformans var. neoformans (Kwon-Chung 1998), are worldwide pathogens that cause systemic and central nervous system disease in immune compromised patients. The lower clad, representing genotypes 3, 4 and 5, comprises Cryptococcus neoformans var. gattii, which is pathogenic in immune competent patients in the tropics. The distribution of serotypes A and D among C. neoformans var. neoformans genotypes of the upper clade (Fig. 2) suggests hybridization occurred between A and D serotypes. The presence of serotype hybrids and multiple genotypes has been independently confirmed by amplified fragment length polymorphism (AFLP) analysis (Boekhout, personal communication). Since hybridization of serotypes can take place within and between varieties of C. neoformans, identification of pathogens based on serotypes can be misleading. Consequently, genotyping via IGS sequence analysis provides an accurate representation of the presence of specific strains.

Example 3: Designing PCR (Universal) probes Biotinylated Universal PCR primers are synthesized and HPLC purified using conventional techniques. Their purity is confirmed by gel electrophoresis. The quality of biotinylation is tested by retention assay on Streptavidin coated agarose beads. Primer concentrations are determined by absorbance at 260 nm. An amount of primer equivalent to 1 A.U. at 260 nm is added to 50 μl of Streptavidin-coated agarose beads (Pierce). This suspension is adjusted to 1.0 ml with phosphate buffered saline (PBS) and mixed by briefly vortexing. The suspension is incubated for 10 minutes at room temperature then centrifuged to pellet the beads. The supernatant is harvested and the absorbance read at 260 nm. Primers should show at least 85% retention on the beads for best performance. Universal primers are tested for efficacy using a standard yeast DNA sample, titrated in 10-fold serial dilutions in distilled water to provide 0 (no DNA), 1, 10, 100, 1,000, 10,000, 100,000 and 1 ,000,000 copies of the target sequence per PCR reaction. For kits, primers typically are acceptable when they can detect 1,000 DNA copies per PCR reaction. Each amplified product should yield a single major product band on an agarose gel when lμg of amplified product is applied. Each amplified product also should give a signal of 1.0 to 2.0 A.U. on a standard plate assay against its complement capture probe. Example 4: Designing Capture Probes

Initial probe design uses sequence databases of intergenic spacer regions

(like the sequences of Figure 1) for sequence alignment. Probes are designed to incorporate polymorphisms close to the middle of the sequence whenever possible. Melting temperatures, and probe secondary structure are determined using the program "Oligo"

(National Biosciences, Inc.).

The selected capture probe sequences are coated on plates and tested to evaluate performance in the hybridization assay. The assay is carried out as described in Example 5. All probes are evaluated under the same assay conditions. Probes are challenged with their complementary target amplified product, with a negative amplified product varying 25% or more from the target sequence, and with one or more cross- reactive amplified products bearing a low number of mismatches (1 to 3 bases). Generally acceptable performance criteria include: horseradish peroxidase (HRP) signal of 1.0 to 2.0 A.U. at 450nm in response to the positive amplified product, negative and cross-reactive signals no higher than 10% of the positive signal, and a background signal, whichis generated in the absence of amplified product DNA, that is no higher than 10% of the gross positive signal.

Probes that yield high nonspecific signals, as defined by a high signal from negative amplified products, a high cross-reactive signal, or both, are modified by sequentially removing nucleotides from the 5' and 3' ends of the probe oligonucleotide. This panel of shortened probes, derived from the original probe, is tested for performance under the standard assay conditions. Strips from plates coated with each of the modifications are tested simultaneously. If a probe does not meet performance criteria after this series of modifications, another probe is selected and the process is repeated.

Example 5 : Developing a PCR-based Kit

In designing a PCR-based assay, cells from pure cultures are grown for 12- 14 hrs in GYP broth. Cells are washed twice with distilled water, then converted to spheroplasts by incubating for 2 hrs at 37 oC in 10 mM citrate buffer, pH 5.8, 1M sorbitol and 10 mg/ml lysing enzymes from Trichoderma harzianum (Sigma), which is freshly prepared for each procedure. The spheroplasts are washed in the spheroplasting buffer and

M then lysed in distilled water and centrifuged in a microfuge to pellet cell debris. The supernatant is used for PCR without further purification.

The selected capture probe sequences are coated on plates and tested to evaluate performance in the hybridization assay. The assay is described below. All probes are evaluated under the same assay conditions. Probes are challenged with their complementary target amplified product, with a negative amplified product varying 25 % or more from the target sequence, and with one or more cross-reactive amplified products bearing a low number of mismatches (1 to 3 bases). Generally acceptable performance criteria include: horseradish peroxidase (HRP) signal of 1.0 to 2.0 A.U. at 450nm in response to the positive amplified product, negative and cross-reactive signals no higher than 10% of the positive signal, and a background signal, whichis generated in the absence of amplified product DNA. that is no higher than 10% of the gross positive signal.

PCR reactions are carried out using 5 '-biotinylated umversal primers. The reaction solution contains: target DNA; lOmM Tris HC1 (pH9); 50mM KC1; 0.1 % Triton X-100; 2mM MgC12; 50pmoles of each biotinylated primer; 2.5 U of AmpliTaq DNA polymerase; dNTPs containing 200 nmoles each of dGTP, dCTP, dATP and TTP. Amplified products are 5' labeled with the 5 '-biotinylated PCR primers. PCR reaction mixtures are incubated in an MJ Research PTC 100 thermal cycler using the following program: 94oC for 2 min., followed by 30 cycles at 94oC for 30 sec, 64oC for 90 sec and 72oC for 30 sec, followed by 72oC for 8 min. Purification is by the QIAquick PCR Purification Kit (QIAGEN), following the manufacturer's protocol. Amplified product concentrations are determined by absorbance at 260nm. and the synthesis of approximately 1 kb amplified products is confirmed by agarose gel electrophoresis.

Amplified product detection is by hybridization to plates bearing specific capture probes, according to the following hybridization protocol. Probe-coated plates, and solutions and buffers needed for the assay are obtained from Genetic Vectors, Inc. (Miami, Florida) Capture probe oligonucleotides are synthesized, purified and coupled to plates by Genetic Vectors, Inc.. Total time for execution of the hybridization protocol is about 90 minutes. 1. Transfer 10 μl aliquots of biotinylated amplified product to the probe-coated wells.

2. Add 5μl of denaturation reagent.

3. Incubate at room temperature for 2 minutes.

4. Add 85 μl of hybridization buffer.

5. Incubate at 60oC for 30 minutes.

6. Wash twice with wash buffer.

7. Add 200μl 3M TMAC. 8. Incubate at 60oC for 10 minutes

9. Wash twice with wash buffer.

10. Add lOOμl of diluted (1 :5 ,000) Streptavidin-HRP conjugate.

11. Incubate at 37oC for 10 minutes.

12. Wash three times with wash buffer. 13. Add lOOμl of HRP substrate.

14. Incubate at 37oC for 10 minutes.

15. Add 25μl of stop solution.

16. Read at 450nm.

Example 6: Standard LCR Conditions

The following is adapted from the instruction manual for LCR available from Stratagene (Cat. # 200520, Revision # 057001). Reactions are carried out in a conventional thermocyler, as described above for use in PCR.

Reactions are assembled in 500 01 microfuge tubes. Add to each tube: 1 1 template (100 pg)

1 1 oligonucleotide mixture (10 ng/Dl; about 25 bases long each)

2 1 10X buffer (200 mM Tris-HCl (pH 7.5); 200 mM KC1; 100 mM MgC12; 1 % NP-40 detergent; 0.1 mM rATP; 10 mM DTT)

15 1 sterile deionized water

1 1 Pfu ligase (4 U)

Denature for 4 minutes at 94°C and anneal for 3 minutes at 60°C. Cycle 20- 25 times using the following steps: 92°C for 20 seconds and 60°C for 20 seconds.

3⁽* Products of LCR are resolved by agarose gel electrophoresis and visualized with ethidium bromide staining. Cryptococcus neoformans genotypes can be identified in the LCR product by the hybridization assay, performed as described in Example 5.

Claims

WE CLAIM:

1. An assemblage, comprising two universal primers adapted for a nucleic acid amplification protocol, wherein each primer has a sequence selected from the group consisting of suitable portions in suitable orientations of the sequences depicted in Figure 1. and minor variants of said suitable portions.

2. An assemblage according to claim 1, further comprising a discriminating primer adapted to discriminate among Cryptococcus neoformans genotypes, wherein the disciminating primer has a sequence selected from the group consisting of suitable portions in suitable orientations of the sequences depicted in Figure 1 , and minor variants of said suitable portions.

3. An assemblage according to claim 2, wherein the discriminating primer is immobilized on a solid matrix.

4. A universal primer adapted from a suitable portion of a sequence selected from the group consisting of, with reference to Figure 1, positions 1-65, 105-190, 172-191, 184-241, 337-369, 423-440, 423-449, 586-603, 586-600, 683-716, 875-892, 960- 988, 1056-1088, 1082-1122, 1108-1140, 1178-1264, 1284-1449 and minor variants of said primer and complements of said primer.

5. A universal primer according to claim 4, said sequence being selected from the group consisting of: GGTCTCGGGGGGCTTCCTCT, GGCTTCCTCTAGAGACTTGG, GGTCAAGCAAAGTCTAGAAAAG, GGTGAGTATGTGATGTGA, ACAAGACAAGTAGGGAA. their complements, and minor variants thereof.

6. A discriminating primer adapted from a suitable portion of a sequence selected from the group consisting of:

CTCTAACATGTTGGGTCTCGGGGGGCTTCCTCTAGAGACTTGGATGTAAGGGGC TTTACGCATTC;

CATCAGTCTTCAGCTTGGGT; TGGGGGACTTGGGAGCTGGTGCTTGTGTCGCAT; GGGGGACTTGGGGTAAGACGCCTTGCA; TATGTATATAGTTGA;

GGTGAGTATGTGATGTGAGAAGGATGGTACCCCATCCTG;

CTTATGAAGTGTGGATGACTCTAGGAGGGGTCTGAGAATGTGCTGTGCCAGCCA GGCTGATAATAGTTTAATTGTTAGCTTGGACTTGTACACAGTCTCATCAGTCTTC AGCTTGGGTCATTGATATTCTGTAAGTCAAAACTTATCCATTCACTGTAGTAGGT TGCGGAGGACTTGAAAGTTCTCTCTGCAATTGAGACACTTACCAGGCCATCGCA

AGTTGGCAGT;

GAGAATGTGCTGTGCCAGCCAGGCTGATAATAGTTTAATTGTTAGCTTGGACTT

GTACACAGTCTCATCAGTCTTCAGCT; TAGGTTGCGGAGGACTT;

GTTGCGGAGGACTTGAAAG;

CAGCTTTCTATGCATGCAGCCTCGGCGCCGACAGTTGAAAAAAATGTATAGTCT

CTGGACCATGAGAGA; TTGAATGTGTAATAGCTCAAGTGCCAGGAG;

GTCAATAGCCACCTGGGTGAGTATGTG;

AACCGACTTGAGGGAGTGTCAGGATGCATG;

GCAGACTCAGCCTTTTATCTTCGGCCTCACTGGCACACGCTTGAAGATCA;

TGGAAGCGGCGCTAGGGCCAAGCGGCTTGTCGCTGTC;

ATCTTATCGACAGGCTGGTGTTTCCTTCGTTCCCTTTCGTCTTTCAACTCGTCTTA

G; AAGGAAACAAGATAAAGTAGGACAAGACAAGTAGGGAGGTTAGTG;

GTGTGCTGTGCGAGCCCGGCTGATAATTGTTTTCATTGTTAGCTTGGATTGCACA

CACAGTCTCATCAGTCTTCAGCTTGGGTTATCGATATTCTGTAAGTCCAAACTTA

TCCATTCACATTCACTGTAGTGTAGGTTGCAGAGAGGACTTGGAAAGTTCTCTCT

CTGCACTGG;

TGGGTTATCGATATTCTTCTGTAAGTCCAAACTTATCCATTCACATTCACTGTAG

TGTAGGTTGCAGAGAGGACTTGGAAAGTTCTCTCTCTGCACTGG;

GGTTGCAGAGAGGACTTGG; GCATACTACTGATTT;

GCATACAGCCTTGGCGCCGGCAGCCGAAAAAAATGTATGGTCTTTAGAC;

TGAGAGATTTCGATGTGTTATAGCTCAAGTACCAGGAGCAGTAGTCATGATCTG

AAATCATGGATCTGTTTCCACGCCTGGTGTGAGCAGGAGTAAGACTTTGAAGTT

GACCTTGGGTGACAAAAAAATGGGGTGGTGTCAGGAACCACCCAGGTGAGTATG

TGAGAGATTTCGATGTGTTATAGCTCAAGTACCAGGAGCAGTAGTCATGATCTG

AAATCATGGATCTGTTTCCACGCCTGGTGTGAGCAGGAGTAAGACTTTGAAGTT

GACCTTGGGTGACAAAAAAATGGGGTGGTGTCAGGAACCACCCAGGTGAGTATG

; GGTGGTGTCAGGAACCACCCAGGTGAGTATGTG;

GCTGGCGAAGATGCAGTCAGCAACCAAAAAATTCTGGTAAGCACTCTGAAACGA

CTTGGGCGAGTCTCAGGATGCATGAGAGGCTTGCCGATCACAATTTTATCGAGC

AGGCGATGTTTCCTTCATTCCATTTCATCTTTCAGCTGGCCTTAGGATACGGCAG AACAAGACAAGTAGGGAAGTTAGTGTTATAATCTT

AGCCAGGCTGATAATAGTTTAA; GTACGCTACTGATTT;

GTGACTTCTTTTAGTCGTGGG;

GTAATCGTGAACCTGGGGGTCATGGATCTGTTTCTACACCTGATGTGAGCTGGA GCAAGACTTTGGAGTTGACCTTGGGTGACAAAAAAT;

GGGTGGTGTCAATAGCCACCTGGG; GTACACTACTGATTTTACGGT;

GGAGCAGTAATTGTGAAACCTGAGGTCATGGATCTGTTTCCACACCTGATGTGA GCTGGAGCAAGAGTTTGGAGTTGACTTTGGGCGACAAAAAATGGGATGGTGTCA ; CCAACAAAAAAACCCGGTAAGCAC;

TGAAGATCATCAGAGGCACTCATTTAACCCTGTTCCCATTATCAG; TTGTCAGCTTGGACTTGT; CTCTGGACCATGAGAGAGTTGAATGTGAATAGC; TGGACCATGAGAGAGTTGAATGTGTAATAGCTCAAG;

GTGAACCTGAGGTCATGG; TGGGGTGATGTCAATAGCCACCTG

GATGC ATGGGC AGACTC AGCC ; ATTT AACCCTGTCCCC ATTATC AG

ATCGATATTCTTCTGT AAGTC ; ATTCTTCTGT AAGTCC AAACT

TACGCATTCACATAATATTAGTTGGGGACTGAGG; GCAAGTAGAGTCAAACAG AGGAGAGAGGTCGGGCTGGTAATTAGTAATATCAGTCAGTCATTTCAGCT; GCTGGCGCCATCGATACTTTATAAGTCAAACTTAACCATTTCACTGTGTAGTA; GTTATGCGGCAAGCAAG; TGTGCTTATAGCATAGTC;

GCTGGTCAAGCAAACGTTTAAGTTGAGTCAAGCT: CAGTCTCGATGTCGCTGG; AGT AGCTGTGATCTGAGGCC ATGG ATC ; CAAAAAGTTGGGGCGGTGTCAGCAGCGGACACGGTGAGT; TGGTACCCGATCTTGTTGGTGAAGGTGCAGTCA:

TTGGAAGAGTCTCGGGATGCAGGAGCGGATTCAGCGTTTTCTTTGGCCTTACTTA TCGAGGGA;

TTCCCTTTTCGTTTTTCAACCGGTCATAGAAACAAGATTCTTAAGACGTGATTTG ATTAGA; TGCTTTTTTTTATGACGATGACTTGTCAAAGTGTGG;

AGTAGTAGGCTCTGAATTACTAGAGACACTTGC;

CAAGTTGGCAGGCAGGCAGG; GCAGGACACACATACTATTGATTT;

TGCTGGTGCTTGAGTTGCATA;

GAGAGAGTATATGCATGTCGCGGGGGGGGGACTTGGCT ; TTAAAGTATTTAG; GTTTGACCCGACCTGACGGTG;

4° ATCGCTAAGAATTACTCCGGTCGCGGGGGGCTTGCAACTTGTCT;

GTCTTTGGACGATGTGAGATTTCATTGTGTAATAGC;

GTTTCCATACCTGGTATAAGCTCGAGTGAGACTGTGCAAGTT;

GAGGGACTGGGTGTCCCCTTCGTTCCC ;

GGTCATAAAAACAAGATTCTAAGACGT; GGGAAATTAGTGTTATATTCTT

TGCTTTTTTACGATGACTTGTCAAAGTGTGG; TCGTACACATACTATTGATTT

GGCT ACTGGTACTGTGTTGC AT ; TACTGGTACTGTGTTGC ATA

TTATATAGTATT; GTTGACCCGACCTGATGGTG

ATCACTAAGAATTACTCCAGTCGCGGGGGGCTTGTAACTTCTCT;

GACG ATGT AAGATTTCATCGCGT AAT AGC ;

GGATCTGGTTCCACACCTGGTATGAGCTCGAGTGAG;

GGG AAATTAGTGTTATGTTGTT ; TGGAT AC AAGAGGCTTTGCTTT ;

TGCTTTTTGACGATGAACTTGTC AAAGTGTGG ;

TGACGGGTACTAGAGAC ACTTGC ; TCGTC ATAC AC ATCCT ATTGATTT

TACTGGTGCTTGTGTTGCCTA; TTATAGTATTGG; TAGTATTGGTTACTTGCC

ATGAAAAACAGGTAAA; AACAGGTAAATGTGGTATGG

GTTGACCCAACCTGACAGTG;

ATCACTAAGAATGACTCCAGTCGCGGGGGACTTGGAACTTCTCT;

GTCTTTGGAGGATGTAAGATTTCATCGTGTCATAGCTCAAGT;

CATGGATCGGGTGCCACCCCTGGTATGAGCTCGAGTGAGACTGTGCAAGGTCAA

TTTGGCCTTGGG; GGGAAATTAGTGTCATGTTCTT: their complements; and minor variants thereof.

7. An isolated nucleic acid having a sequence selected from the group consisting of those disclosed in Figure 1.

8. A nucleic acid sequence in computer readable form having a sequence selected from the group disclosed in Figure 1.

9. A method of discriminating among pathogenic yeasts, comprising amplifying a suspected yeast-containing sample using two universal primers adapted for a nucleic acid amplification protocol, wherein each primer has a sequence selected from the group consisting of suitable portions in suitable orientations of the sequences depicted in Figure 1, and minor variants of said suitable portions.

/

10. A method according to claim 9, further comprising bringing the resultant amplified product into contact with a discriminating primer adapted to discriminate among Cryptococcusneoformans genotypes, wherein the discriminating primer has a sequence selected from the group consisting of suitable portions in suitable orientations of the sequences depicted in Figure 1, and minor variants of said suitable portions.

11. A method according to claim 10, wherein said discriminating primer is elongated in a polymerase chain reaction.

12. A method according to claim 11. wherein said discriminating primer is immobilized.

13. A method of discriminating among pathogenic yeasts, comprising amplifying the intergenic spacer region of a suspected pathogenic yeast-containing sample and bringing the resultant amplified product into contact with a discriminating primer adapted to discriminate among Cryptococcus neoformans genotypes, wherein the discriminating primer has a sequence selected from the group consisting of suitable portions in suitable orientations of the sequences depicted in Figure 1 , and minor variants of said suitable portions.

14. A method of distinguishing among genotypes of pathogenic yeasts, comprising amplifying a sample with a pair of primers, at least one of which is a discriminating primer, and detecting the resultant amplification product.

15. A method of distinguishing among genotypes of pathogenic yeasts, comprising amplifying the intergenic spacer region of a yeast and contacting the resulting amplification product with a discriminating primer.

4i