Abstract:
Nucleic acids for detecting  Aspergillus  species and other filamentous fungi are provided. Unique internal transcribed space 2 coding regions permit the development of nucleic acid probes specific for five different species of  Aspergillus , three species of  Fusarium , four species of  Mucor , two species of  Penecillium , five species of  Rhizopus , one species of  Rhizomucor , as well as probes for  Absidia corymbifer, Cunninghamella elagans, Pseudallescheria boydii , and  Sporothrix schenkii . Methods are disclosed for the species-specific detection and diagnosis of infection by  Aspergillus, Fusarium, Mucor, Penecillium, Rhizomucor, absidia, Cunninghaemella, Pseudallescheria  or  Sporthrix  in a subject. Furthermore, genus-specific probes are also provided for  Aspergillus, Fusarium  and  Mucor , in addition to an all-fungus nucleic acid probe.

Description:
PRIORITY CLAIM 
     This is a divisional of U.S. patent application Ser. No. 09/423,233 filed Jun. 27, 2000, issued as U.S. Pat. No. 6,372,430. U.S. patent application Ser. No. 09/423,233 is a § 371 national stage of PCT/US98/08926 filed May 1, 1997, and claims the benefit of U.S. Provisional Application No. 60/045,400 filed May 2, 1997. 
    
    
     This invention was made in the Centers for Disease Control Mycotic Diseases Laboratories, an agency of the United States Government. 
    
    
     TECHNICAL FIELD 
     This application relates in general to the field of diagnostic microbiology. In particular, the invention relates to the species-specific detection of  Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus, Rhizomucor, Absidia, Cunninghamella, Pseudallescheria boydii  ( Scedosporium apiospermum ), and  Sporothrix species.    
     BACKGROUND OF THE INVENTION 
     In recent years, chemotherapy for hematological malignancies, and high-dose corticosteroid treatment for organ transplant recipients, along with the spread of AIDS, have greatly increased the number of immunocompromised patients (1, 12, 14, 43). Saprophytic filamentous fungi, such as  Aspergillus, Rhizopus , and  Mucor  species, found in the environment and considered to be of low virulence, are now responsible for an increasing number of infections in the immunocompromised host (17, 20, 43). In addition, these infections are often fulminant and rapidly fatal in immunocompromised patients (7, 11, 12, 20, 44). Morbidity and mortality is extremely high; for example, aspergillosis has a mortality rate of approximately 90% (8, 11). 
     To complicate matters, diagnosis is difficult and symptoms are often non-specific (18, 27, 29, 42, 44). Antibody-based tests can be unreliable due to the depressed or variable immune responses of immunocompromised patents (2, 9, 18, 46). Antigen detection tests developed to date have fallen short of the desired sensitivity (2, 9, 38). Radiographic evidence can be non-specific and inconclusive (5, 29, 36), although some progress in diagnosis has been made with the advent of computerized tomography (40). However, definitive diagnosis still requires either a positive blood or tissue culture or histopathological confirmation (3, 21). An added complication is that the invasive procedures necessary to obtain biopsy materials are often not recommended in thrombocytopenic patient populations (37, 41). 
     Even when cultures of blood, lung or rhinocerebral tissues are positive, morphological and biochemical identification of filamentous fungi can require several days for adequate growth and sporulation to occur, delaying targeted drug therapy. Some atypical isolates may never sporulate, making identification even more difficult (23). When histopathology is performed on tissue biopsy sections, the morphological similarities of the various filamentous fungi in tissue make differentiation difficult (16). Fluorescent antibody staining of histopathological tissue sections is not specific unless cross-reactive epitopes are absorbed out which can make the resultant antibody reactions weak (14, 19). Therapeutic choices vary (7, 41, 44) making a test to rapidly and specifically identify filamentous fungi urgently needed for the implementation of appropriately targeted therapy. Early and accurate diagnosis and treatment can decrease morbidity and increase the chances for patient survival (6, 27, 39). Furthermore, identification of filamentous fungi to at least the species level would be epidemiologically useful (24, 31, 43, 47). 
     PCR-based methods of detection, which show promise as rapid, sensitive means to diagnose infections, have been used in the identification of DNA from  Candida  species (13, 15, 30) and some other fungi, particularly  Aspergillus  species (31, 33, 45). However, most of these tests are only genus-specific (28, 38) or are directed to detect only single-copy genes (4, 35). Others have designed probes to detect multi-copy genes so as to increase test sensitivity (31, 33) but in doing so have lost test specificity because they have used highly conserved genes, which detect one or a few species but which are also plagued with cross-reactivities to human, fungal or even viral DNA (25, 31, 33). 
     Therefore, it is an object of the invention to provide improved materials and methods for detecting and differentiating  Aspergillus  and other filamentous fungal species in the clinical and laboratory settings. 
     SUMMARY OF THE INVENTION 
     The present invention relates to nucleic acids for detecting  Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus, Rhizomucor, Absidia, Cunninghamella, Pseudallescheria  ( Scedosporium ), and  Sporothrix  species. Unique internal transcribed spacer 2 coding regions permit the development of probes specific for five different  Aspergillus  species,  A. flavus, A. fumigatus, A. niger, A. terreus , and  A. nidulans . The invention thereby provides methods for the species-specific detection and diagnosis of  Aspergillus  infection in a subject. In addition, species probes have been developed for three  Fusarium , four  Mucor , two  Penicillium , five  Rhizopus  and one  Rhizomucor  species, as well as probes for  Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii  ( Scedosporium apiospermum ), and  Sporothrix schenckii . Generic probes for  Aspergillus, Fusarium , and  Mucor  species have also been developed. 
     These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention provides a simple, rapid, and useful method for differentiating filamentous fungal species from each other and from other medically important fungi. This invention enables a rapid, simple and useful method to isolate fungal DNA from host samples, and to apply the species- and genus-specific probes for the diagnosis of a disease. Ultimately, these probes can be used for in situ hybridization or in situ PCR diagnostics so that the morphology of host tissue, and microorganisms, remain intact. 
     The invention provides nucleic acids containing regions of specificity for five  Aspergillus , three  Fusarium , four  Mucor , two  Penicillium , five  Rhizopus  and one  Rhizomucor  species as well as probes for  Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii  ( Scedosporium apiospremum ), and  Sporothrix schenckii . These nucleic acids are from the internal transcribed spacer 2 (“ITS2”) region of ribosomal deoxyribonucleic acid (rDNA) of the genome of the aforementioned filamentous fungi. The ITS2 region is located between the 5.8S rDNA region and the 28S rDNA region. 
     In particular, the invention provides nucleic acids from  Aspergillus flavus  (SEQ ID NO:1),  Aspergillus fumigatus  (SEQ ID NO:2),  Aspergillus niger  (SEQ ID NO:3),  Aspergillus terreus  (SEQ ID NO:4),  Aspergillus nidulans  (SEQ ID NO:5),  Fusarium solani  (SEQ ID NO:6),  Fusarium moniliforme  (SEQ ID NO:7),  Mucor rouxii  (SEQ ID NO:8),  Mucor racemosus  (SEQ ID NO:9),  Mucor plumbeus  (SEQ ID NO:10),  Mucor indicus  (SEQ ID NO:11),  Mucor circinilloides f. circinelloides  (SEQ ID NO:12),  Rhizopus oryzae  (SEQ ID NO:13 and NO:14),  Rhizopus microsporus  (SEQ ID NO:15 and 16),  Rhizopus circinans  (SEQ ID NO:17 and 18).  Rhizopus stolonifer  (SEQ ID NO:19),  Rhizomucor pusillus  (SEQ ID NO:20),  Absidia corymbifera  (SEQ ID NO:21 and 22),  Cunninghamella elegans  (SEQ ID NO:23),  Pseudallescheria boydii  (teleomorph of  Scedosporium apiospermum ) (SEQ ID NO:24, 25, 26, and 27),  Penicillium notatum  (SEQ ID NO:28), and  Sporothrix schenkii  (SEQ ID NO:29). These sequences can be used to identify and distinguish the respective species of  Aspergillus, Fusarium, Mucor, Rhizopus , and  Penicillium , and identify and distinguish these species from each other and from  Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii  ( Scedosporium apiospermum ), and  Sporothrix schenkii.    
     Furthermore, the invention provides isolated nucleic acid probes derived from GenBank nucleic acid sequences (for  Penicillium marneffei  and  Fusarium oxysporum  only) or from the above nucleic acid sequences which may be used as species-specific identifiers of  Aspergillus flavus  (SEQ ID NO:30 and 31),  Aspergillus fumigatus  (SEQ ID NO:32),  Aspergillus niger  (SEQ ID NO:33),  Aspergillus terreus  (SEQ ID NO:34),  Aspergillus nidulans  (SEQ ID NO: 35),  Mucor rouxii  (SEQ ID NO:36),  Mucor plumbeus  (SEQ ID NO:37),  Mucor indicus  (SEQ ID NO:38),  Mucor circinilloides f. circinelloides  (SEQ ID NO:39),  Mucor racemosus  (SEQ ID NO:40),  Rhizopus oryzae  (SEQ ID NO:41),  Rhizopus circinans  (SEQ ID NO:42),  Rhizomucor pusillus  (SEQ ID NO:43),  Rhizopus stolonifer  (SEQ ID NO:44),  Pseudallescheria boydii  ( Scedosporium apiospermum ) (SEQ ID NO:45),  Penicillium notatum  (SEQ ID NO:46),  Penicillium marneffei  (SEQ ID NO:47 and 48),  Fusarium moniliforme  (SEQ ID NO:49),  Fusarium oxysporum  (SEQ ID NO:50),  Fusarium solani  (SEQ ID NO:51),  Cunninghamella elegans  (SEQ ID NO:52, 53, and 54),  Absidia corymbifera  (SEQ ID NO:55),  Sporothrix schenkii  (SEQ ID NO:56), and  Rhizopus microsporus  (SEQ ID NO:57). Such probes can be used to selectively hybridize with samples containing nucleic acids from species of  Aspergillus, Fusarium, Mucor, Rhizopus  (or  Rhizomucor ),  Penicillium , or from  Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii  ( Scedosporium apiospermum ), and  Sporothrix schenkii . These fungi can be detected after polymerase chain reaction or ligase chain reaction amplification of fungal DNA and specific probing of amplified DNA with DNA probes labeled with digoxigenin, reacted with anti-digoxigenin antibodies labeled with horseradish peroxidase and a colorimetric substrate, for example. Additional probes can routinely be derived from the sequences given in SEQ ID NOs:1–29, which are specific for the respective species. Therefore, the probes shown in SEQ ID NOs:30–57 are only provided as examples of the species-specific probes that can be derived from SEQ ID NOs:1–29. 
     Generic probes for  Aspergillus  (SEQ ID NO:58),  Fusarium , (SEQ ID NO:59) and  Mucor  (SEQ ID NO:60) species have also been developed to identify all members of their respective species which are listed above as well as an all fungus biotinylated probe (SEQ ID NO:61) to capture all species-specific and generic probes listed above for their detection. 
     By “isolated” is meant nucleic acid free from at least some of the components with which it naturally occurs. By “selective” or “selectively” is meant a sequence which does not hybridize with other nucleic acids to prevent adequate determination of an  Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus  or  Rhizomucor  genus or species or of  Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii  ( Scedosporium apiospermum ), or  Sporothrix schenckii  species. 
     The hybridizing nucleic acid should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes. As used herein to describe nucleic acids, the term “selectively hybridizes” excludes the occasional randomly hybridizing nucleic acids and thus has the same meaning as “specifically hybridizing”. The selectively hybridizing nucleic acids of the invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% complementarity with the segment of the sequence to which it hybridizes. 
     The invention contemplates sequences, probes and primers which selectively hybridize to the complementary, or opposite, strand of DNA as those specifically provided herein. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-specific or genus-specific hybridization capability is maintained. By “probe” is meant nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18 nucleotides. The invention provides isolated nucleic acids that selectively hybridize with the species-specific nucleic acids under stringent conditions and should have at least 5 nucleotides complementary to the sequence of interest. See generally,  Maniatis  (26). 
     If used as primers, the invention provides compositions including at least two nucleic acids which hybridize with different regions so as to amplify a desired region. Depending on the length of the probe or primer, target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of diagnosing the presence of the  Aspergillus , the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes (e.g.,  Aspergillus  DNA from a sample) is at least enough to distinguish hybridization with a nucleic acid from other yeasts and filamentous fungi. The invention provides examples of nucleic acids unique to each filamentous fungus in the listed sequences so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid. 
     Alternatively, the nucleic acid probes can be designed to have homology with nucleotide sequences present in more than one species of the fungi listed above. Such a nucleic acid probe can be used to selectively identify a group of species such as the generic probes listed for  Aspergillus  (SEQ ID NO:58),  Fusarium  (SEQ ID NO:59), and  Mucor  (SEQ ID NO:60) as well as all fungi listed (SEQ ID NO:61). Additionally, the invention provides that the nucleic acids can be used to differentiate the filamentous fungi listed in general from other filamentous fungi and yeasts, such as  Candida  species. Such a determination is clinically significant, since therapies for these infections differ. 
     The invention further provides methods of using the nucleic acids to detect and identify the presence of the filamentous fungi listed, or particular species thereof. The method involves the steps of obtaining a sample suspected of containing filamentous fungi. The sample may be taken from an individual, such as blood, saliva, lung lavage fluids, vaginal mucosa, tissues, etc., or taken from the environment. The filamentous fungal cells can then be lysed, and the DNA extracted and precipitated. The DNA is preferably amplified using universal primers derived from the internal transcribed spacer regions, 18S, 5.8S and 28S regions of the filamentous fungal rDNA. Examples of such universal primers are shown below as ITS1 (SEQ ID NO: 62), ITS3 (SEQ ID NO: 63), ITS4 (SEQ ID NO: 64). Detection of filamentous fungal DNA is achieved by hybridizing the amplified DNA with a species-specific probe that selectively hybridizes with the DNA. Detection of hybridization is indicative of the presence of the particular genus (for generic probes) or species (for species probes) of filamentous fungus. 
     Preferably, detection of nucleic acid (e.g. probes or primers) hybridization can be facilitated by the use of detectable moieties. For example, the species-specific or generic probes can be labeled with digoxigenin, and an all-fungus probe, such as described in SEQ ID NO:61, can be labeled with biotin and used in a streptavidin-coated microtiter plate assay. Other detectable moieties include radioactive labeling, enzyme labeling, and fluorescent labeling, for example. 
     The invention further contemplates a kit containing one or more species-specific probes, which can be used for the detection of particular filamentous fungal species and genera in a sample. Such a kit can also contain the appropriate reagents for hybridizing the probe to the sample and detecting bound probe. The invention may be further demonstrated by the following non-limiting examples. 
     EXAMPLES 
     In this example, PCR assay employing universal, fungus-specific primers and a simple, rapid EIA-based format for amplicon detection were used. 
     Extraction of Filamentous Fungal DNA 
     A mechanical disruption method was used to obtain DNA from filamentous fungal species and an enzymatic disruption method described previously (13) was used to obtain DNA from yeasts. Filamentous fungi were grown for 4 to 5 days on Sabouraud dextrose agar slants (BBL, division of Becton Dickinson, Cockeysville, Md.) at 35° C. Two slants were then washed by vigorously pipeting 5 mls of 0.01 M potassium phosphate buffered saline (PBS) onto the surface of each slant and the washes were transferred to 500 ml Erlenmeyer flasks containing 250 ml of Sabouraud dextrose broth (BBL). Flasks were then incubated for 4 to 5 days on a rotary shaker (140 rpm) at ambient temperature. Growth was then harvested by vacuum filtration through a sterile Whatman #1 filter paper which had been placed into a sterile Buchner funnel attached to a 2 L side-arm flask. The resultant cellular mat was washed on the filtration apparatus three times with sterile distilled water, removed from the filter paper by gentle scraping with a rubber policeman, and placed into a sterile Petri plate which was then sealed with parafilm and frozen at −20° C. until used. 
     Just prior to use, a portion of the frozen cellular mat, equal in size to a quarter, was removed and placed into a cold mortar (6″ diameter). Liquid nitrogen was added to cover the mat which was then ground into a powder with a pestle. Additional liquid nitrogen was added as needed to keep the mat frozen during grinding. 
     DNA was then purified using proteinase K and RNase treatment, multiple phenol extractions, and ethanol precipitation by conventional means (26). 
     PCR Amplification 
     The fungus-specific, universal primer pair ITS3 (5′- GCA TCG ATG AAG AAC GCA GC-3′) (SEQ ID NO: 63) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (SEQ ID NO: 64) was used to amplify a portion of the 5.8S rDNA region, the entire ITS2 region, and a portion of the 28S rDNA region for each species as previously described (13, 34). DNA sequencing used this primer pair and also the fungus-specific, universal primer pair ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) (SEQ ID NO: 62) and ITS4 to amplify a portion of the 18S rDNA region, the entire 5.8S region, the entire ITS1 and ITS2 regions, and a portion of the 28S rDNA region. 
     A DNA reagent kit (TaKaRa Biomedicals, Shiga, Japan) was used for PCR amplification of genomic DNA. PCR was performed using 2 μl of test sample in a total PCR reaction volume of 100 μl consisting of 10 μl of 10×Ex Taq buffer, 2.5 mM each of dATP, dGTP, dCTP, and dTTP, in 8 μl, 0.2 μl of each primer, and 0.5 U of TaKaRa Ex Taq DNA polymerase. Thirty cycles of amplification were performed in a Perkin-Elmer 9600 thermal cycler (Emeryville, Calif.) after initial denaturation of DNA at 95° C. for 5 minutes. Each cycle consisted of a denaturation step at 95° C. for 30 seconds, an annealing step at 58° C. for 30 seconds, and an extension step at 72° C. for 1 minute. A final extension at 72° C. for 5 minutes followed the last cycle. After amplification, samples were stored at −20° C. until used. 
                                   TABLE 1                   Synthetic Universal Oligonucleotides Used in PCR       and Hybridization Analyses            Primers   Nucleotide Sequence   Chemistry and       or Probes   (5′ to 3′)   Location               ITS3   GCA TCG ATG AAG AAC GCA GC   5.8S rDNA           (SEQ ID NO:63)   universal 5′               primer               ITS4   TCC TCC GCT TAT TGA TAT GC   28S rDNA           (SEQ ID NO:64)   universal 3′               primer               ITS1   TCC GTA GGT GAA CCT GCG G   18S rDNA           (SEQ ID NO:62)   universal 5′               primer                    
DNA Sequencing
 
     Primary DNA amplifications were conducted as described above. The aqueous phase of the primary PCR reaction was purified using QIAquick Spin Columns (Quiagen, Chatsworth, Calif.). DNA was eluted from each column with 50 μl of heat-sterilized Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0). 
     Purified DNA was labeled using a dye terminator cycle sequencing kit (ABI PRISM, Perkin Elmer, Foster City, Calif.). One mix was made for each of the primers so that sequencing could be performed in both the forward and reverse directions. The reaction volume (20 μl) contained 9.5 μl Terminator Premix, 2 μl (1 ng) DNA template, 1 μl primer (3.2 pmol) and 7.5 μl heat-sterilized distilled H 2 O. The mixture was then placed into a pre-heated (96° C.) Perkin Elmer 9600 thermal cycler for 25 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 4 minutes. The PCR product was then purified before sequencing using CentriSep spin columns (Princeton Separations, Adelphia, N.J.). DNA was then vacuum dried, resuspended in 6 μl of formamide-EDTA (5 μl deionized formamide plus 1 μl 50 mM EDTA, pH 8.0), and denatured for 2 min at 90° C. prior to sequencing using an automated capillary DNA sequencer (ABI Systems, Model 373, Bethesda, Md.). 
     The sequencing results were as follows: 
       Aspergillus flavus  5.8S ribosomal RNA gene, partial sequence, internal transcribed spacer 2, complete sequence, and 28S ribosomal RNA gene, partial sequence. 
                                       (SEQ ID NO:1)                    GCTGCCCATC AAGCACGGC TTGTGTGTTG GGTCGTCGTC                           CCCTCTCCGG GGGGGACGGG CCCCAAAGGC AGCGGCGGCA                       CCGCGTCCGA TCCTCGAGCG TATGGGGCTT TGTCACCCGC                       TCTGTAGGCC CGGCCGGCGC TTGCCGAACG CAAATCAATC                       TTTTTCCAGG TTGACCTCGG ATCAGGTAGG GATACCCGCT                       GAACTTCAA              Aspergillus fumigatus  5.8S ribosomal RNA gene, partial sequence, internal transcribed spacer 2, complete sequence, and 28S ribosomal RNA gene, partial sequence.
 
                                   (SEQ ID NO:2)                AAACTTTCAA CAATGGATCT CTTGGTTCCG GCATCGATGA                   AGAACGCAGC GAAATGCGAT AACTAATGTG AATTGCAGAA               TTCAGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCC               CTGGTATTCC GGGGGGCATG CCTGTCCGAG CGTCATTGCT               GCCCATCAAG CACGGCTTGT GTGTTGGGCC CCCGTCCCCC               TCTCCCGGGG GACGGGCCCG AAAGGCAGCG GCGGCACCGC               GTCCGGTCCT CGAGCGTATG GGGCTTGTCA CCTGCTCTGT               AGGCCCGGCC GGCGCCAGCC GACACCCAAC TTTATTTTTC               TAAGGTTGAC CTCGGATCAG GTAGGGATAC CCGCTGAACT TAAA              Aspergillus niger  5.8S ribosomal RNA gene, partial sequence, internal transcribed spacer 2, complete sequence, and 28S ribosomal RNA gene, partial sequence.
 
                                   (SEQ ID NO:3)                AAACTTTCAA CAATGGATCT CTTGGTTCCG GCATCGATGA                   AGAACGCAGC GAAATGCGAT AACTAATGTG AATTGCAGAA               TTCAGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCC               CTGGTATTCC GGGGGGCATG CCTGTCCGAG CGTCATTGCT               GCCCTCAAGC ACGGCTTGTG TGTTGGGTCG CCGTCCCCCT               CTCCCGGGGG ACGGGCCCGA AAGGCAGCGG CGGCACCGCG               TCCGATCCTC GAGCGTATGG GGCTTTGTCA CCTGCTCTGT               AGGCCCGGCC GGCGCCTGCC GACGTTATCC AACCATTTTT               TTCCAGGTTG ACCTCGGATC AGGTAGGGAT ACCCGCTGAA CTTAA              Aspergillus terreus  5.8S ribosomal RNA gene, partial sequence, internal transcribed spacer 2, complete sequence, and 28S ribosomal RNA gene, partial sequence.
 
                                       (SEQ ID NO:4)                    AAACTTTCAA CAATGGATCT CTTGGTTCCG GCATCGATGA                           AGAACGCAGC GAAATGCGAT AACTAATGTG AATTGCAGAA                       TTCAGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCC                       CTGGTATTCC GGGGGGGCAT GCCTGTCCGA GCGTCATTGC                       TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CTCGTCCCCC                       GGCTCCCGGG GGACGGGCCC GAAAGGCACC GGCGGCACCG                       CGTCCGGTCC TCGAGCGTAT GGGGCTTCGT CTTCCGCTCC                       GTAGGCCCGG CCGGCGCCCG CCGAACGCAT TTATTTGCAA                       CTTGTTTTTT TTTCCAGGTT GACCTCGGAT CAGGT              Aspergillus nidulans  5.8S ribosomal RNA gene, partial sequence, internal transcribed spacer 2, complete sequence, and 28S ribosomal RNA gene, partial sequence.
 
                                   (SEQ ID NO:5)                AAACTTTCAA CAATGGATCT CTTGGTTCCG GCATCGATGA                   AGAACGCAGC GAACTGCGAT AAGTAATGTG AATTGCAGAA               TTCAGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCC               CTGGCATTCC GGGGGGCATG CCTGTCCGAG CGTCATTGCT               GCCCTCAAGC CCGGCTTGTG TGTTGGGTCG TCGTCCCCCC               CCCCGGGGGA CGGGCCCGAA AGGCAGCGGC GGCACCGGTC               CGGTCCTCGA GCGTATGGGG CTTGGTCACC CGCTCGATTA               GGGCCGGCCG GGCGCCAGCC GGCGTCTCCA ACCTTATCTT               TCTCAGGTTG ACCTCGGATC AGGTAGGGAT ACCCGCTGAA CTTAA              Fusarium solani  (strain ATCC62877) internal transcribed spacer 2 and adjacent regions.
 
                                       (SEQ ID NO:6)                    GAAAATGCGA TAAGTAATGT GAATTGCAGA ATTCAGTGAA                           TCATCGAATC TTTGAACGCA CATTGCGCCC GCCAGTATTC                       TGGCGGGCAT GCCTGTTCGA GCGTCATTAC AACCCTCAGG                       CCCCCGGGCC TGGCGTTGGG GATCGGCGGA AGCCCCCTGC                       GGGCACAACG CCGTCCCCCA AATACAGTGG CGGTCCCGCC                       GCAGCTTCCA TTGCGTAGTA GCTAACACCT CGCAACTGGA                       GAGCGGCGCG GCCACGCCGT AAAACACCCA ACTTCTGAAT                       GTTGACCTCG AATCAGGTAG GAATACCCGC TGAACTTAA              Fusarium moniliforme  (strain ATCC38519) internal transcribed spacer 2 and adjacent regions.
 
                                       (SEQ ID NO:7)                    AAATGCGATA AGTAATGTGA ATTGCAAAAT TCAGTGAATC                           ATCGAATCTT TGAACGCACA TTGCGCCCGC CAGTATTCTG                       GCGGGCATGC CTGTTCGAGC GTCATTTCAA CCCTCAAGCC                       CCCGGGTTTG GTGTTGGGGA TCGGCAAGCC CTTGCGGCAA                       GCCGGCCCCG AAATCTAGTG GCGGTCTCGC TGCAGCTTCC                       ATTGCGTAGT AGTAAAACCC TCGCAACTGG TACGCGGCGC                       GGCCAAGCCG TTAAACCCCC AACTTCTGAA TGTTGACCTC                       GGATCAGGTA GGAATACCCG CTGAACTTAA              Mucor rouxii  (strain ATCC24905) internal transcribed spacer 2 and adjacent regions.
 
                                       (SEQ ID NO:8)                    AAAGTGCGAT AACTAGTGTG AATTGCATAT TCAGTGAATC                           ATCGAGTCTT TGAACGCAAC TTGCGCTCAT TGGTATTCCA                       ATGAGCACGC CTGTTTCAGT ATCAAAACAA ACCCTCTATC                       CAGCATTTTG TTGAATAGGA ATACTGAGAG TCTCTTGATC                       TATTCTGATC TCGAACCTCT TGAAATGTAC AAAGGCCTGA                       TCTTGTTTAA ATGCCTGAAC TTTTTTTTAA TATAAAGAGA                       AGCTCTTGCG GTAAACTGTG CTGGGGCCTC CCAAATAATA                       CTCTTTTTAA ATTTGATCTG AAATCAGGCG GGATTACCCG                       CTGAACTTAA              Mucor racemosus  (strain ATCC22365) internal transcribed spacer 2 and adjacent regions.
 
                                       (SEQ ID NO:9)                    AAAGTGCGAT AACTAGTGTG AATTGCATAT TCAGTGAATC                           ATCGAGTCTT TGAACGCAAC TTGCGCTCAT TGGTATTCCA                       ATGAGCACGC CTGTTTCAGT ATCAAAACAA ACCCTCTATC                       CAACTTTTGT TGTATAGGAT TATTGGGGGC CTCTCGATCT                       GTATAGATCT TGAAATCCCT GAAATTTACT AAGGCCTGAA                       CTTGTTTAAA TGCCTGAACT TTTTTTTAAT ATAAAGGAAA                       GCTCTTGTAA TTGACTTTGA TGGGGCCTCC CAAATAAATC                       TCTTTTAAAT TTGATCTGAA ATCAGGCGGG ATTACCCGCT                       GAACTTAA              Mucor plumbeus  (strain ATCC4740) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:10)                        AAAGTGCGAT AACTAGTGTG AATTGCATAT TCAGTGAATC                           ATCGAGTCTT TGAACGCAAC TTGCGCTCAT TGGTATTCCA                       ATGAGCACGC CTGTTTCAGT ATCAAAACAA ACCCTCTATC                       CAACTTTTGT TGTATAGGAT TATTGGGGGC CTCTCGATCT                       GTATAGATCT TGAAACCCTT GAAATTTACT AAGGCCTGAA                       CTTGTTTAAT GCCTGAACTT TTTTTTAATA TAAAGGAAAG                       CTCTTGTAAT TGACTTTGAT GGGGCCTCCC AAATAAATCT                       TTTTTAAATT TGATCTGAAA TCAGGTGGGA TTACCCGCTG                       AACTTAA              Mucor indicus  (strain ATCC4857) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:11)                        AAAGTGCGAT AACTAGTGTG AATTGCATAT TCAGTGAATC                           ATCGAGTCTT TGAACGCATC TTGCACTCAA TGGTATTCCA                       TTGAGTACGC CTGTTTCAGT ATCAAAAAC AACCCTTATT                       CAAAATTCTT TTTTTGAATA GATATGAGTG TAGCAACCTT                       ACAAGTTGAG ACATTTTAAA TAAAGTCAGG CCATATCGTG                       GATTGAGTGC CGATACTTTT TTAATTTTGA AAAGGTAAAG                       CATGTTGATG TCCGCTTTTT GGGCCTCCCA AATAACTTTT                       TAAACTTGAT CTGAAATCAG GTGGGATTAC CCGCTGAACT                       TAA              Mucor circinelloides f. circinelloides  (strain ATCC1209B) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:12)                        AAAGTGCGAT AACTAGTGTG AATTGCATAT TCAGTGAATC                           ATCGAGTCTT TGAACGCAAC TTGCGCTCAT TGGTATTCCA                       ATGAGCACGC CTGTTTCAGT ATCAAAACAA ACCCTCTATC                       CAACATTTTT GTTGAATAGG ATGACTGAGA GTCTCTTGAT                       CTATTCTGAT CTCGAAGCTC TTGAAATGTA CAAAGGCCTG                       ATCTTGTTTG AATGCCTGAA CTTTTTTTTA ATATAAAGAG                       AAGCTCTTGC GGTAAACTGT GCTGGGGCCT CCCAAATAAC                       ACATCTTTAA ATTTGATCTG AAATCAGGT GGGACTACCC                       GCTGAACTT AA              Rhizopus oryzae  (strain ATCC34965) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:13)                        AGTGCGATAA CTAGTGTGAA TTGCATATTC AGTGAATCAT                           CGAGTCTTTG AACGCAGCTT GCACTCTATG GTTTTTCTAT                       AGAGTACGCC TGCTTCAGTA TCATCACAAA CCCACACATA                       ACATTTGTTT ATGTGGTGAT GGGTCGCATC GCTGTTTTAT                       TACAGTGAGC ACCTAAAATG TGTGTGATTT TCTGTCTGGC                       TTGCTAGGCA GGAATATTAC GCTGGTCTCA GGATCTTTTT                       TTTTGGTTCG CCCAGGAAGT AAAGTACAAG AGTATAATCC                       AGTAACTTTC AAACTATGAT CTGAAGTCAG GTGGGATTAC                       CCGCTGAACT TAA              Rhizopus oryzae  (strain ATCC11886) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:14)                        AGTGCGATAA CTAGTGTGAA TTGCATATTC AGTGAATCAT                           CGAGTCTTTG AACGCAGCTT GCACTCTATG GTTTTTCTAT                       AGAGTACGCC TGCTTCAGTA TCATCACAAA CCCACACATA                       ACATTTGTTT ATGTGGTAAT GGGTCGCATC GCTGTTTTAT                       TACAGTGAGC ACCTAAAATG TGTGTGATTT TCTGTCTGGC                       TTGCTAGGCA GGAATATTAC GCTGGTCTCA GGATCTTTTT                       CTTTGGTTCG CCCAGGAAGT AAAGTACAAG AGTATAATCC                       AGCAACTTTC AAACTATGAT CTGAAGTCAG GTGGGATTAC                       CCGCTGAACT TAA              Rhizopus microsporus  (strain ATCC14056) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:15)                        AAAGTGCGAT AACTAGTGTG AATTGCATAT TCGTGAATCA                           TCGAGTCTTT GAACGCAGCT TGCACTCTAT GGATCTTCTA                       TAGAGTACGC TTGCTTCAGT ATCATAACCA ACCCACACAT                       AAAATTTATT TTATGTGGTG ATGGACAAGC TCGGTTAAAT                       TTAATTATTA TACCGATTGT CTAAAATACA GCCTCTTTGT                       AATTTTCATT AAATTACGAA CTACCTAGCC ATCGTGCTTT                       TTTGGTCCAA CCAAAAAACA TATAATCTAG GGGTTCTGCT                       AGCCAGCAGA TATTTTAATG ATCTTTAACT ATGATCTGAA                       GTCAAGTGGG ACTACCCGCT GAACTTAA              Rhizopus microsporus  (strain ATCC12276) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:16)                        AAAGTGCGAT AACTAGTGTG AATTGCATAT TCGTGAATCA                           TCGAGTCTTT GAACGCAGCT TGCACTCTAT GGATCTTCTA                       TAGAGTACGC TTGCTTCAGT ATCATAACCA ACCCACACAT                       AAAATTTATT TTATGTGGTG ATGGACAAGC TCGGTTAAAT                       TTAATTATTA TACCGATTGT CTAAAATACA GCCTCTTTGT                       AATTTTCATT AAATTACGAA CTACCTAGCC ATCGTGCTTT                       TTTGGTCCAA CCAAAAAACA TATAATCTAG GGGTTCTGCT                       AGCCAGCAAA TATTTTAATG ATCTTTAACC TATGATCTGA                       AGTCAAGTGG GACTACCCGC TGAACTTAA              Rhizopus circinans  (strain ATCC34106) internal transcribed spacer 2 and adjacent regions.
 
                                       (SEQ ID NO:17)                    AAATTGCGAT AACTAGTGTG AATTGCATTT TCAGTGAATC                           ATCGAGTCTT TGAACGCAT CTTGCGCTCT TGGGATTCTT                       CCCTAGAGCA CACTTGCTTC AGTATCATAA CAAAACCCTC                       ACCTAATATT TTTTTTTTTT AAAAAAAAAA TATTAGAGTG                       GTATTGGGGT CTCTTTGGTA ATTCTTTGTA ATTATAAAAG                       TACCCTTAAA TGTCATAAAC AGGTTAGCTT TAGCTTGCCT                       TTAAAGATCT TCTTAGGGTA TCATTACTTT TCGTAAATCT                       TTAATAGGCC TGTCACATAA TTCTACCCTT AAATTTCTTA                       AACCTTGATC TGAAGTCAAG TGGGAGTACC CGCTGAACTT AA              Rhizopus circinans  (strain ATCC34101) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:18)                        AAATTGCGAT AACTAGTGTG AATTGCATTT TCAGTGAATC                           ATCGAGTCTT TGAACGCATC TTGCGCTCTT GGGATTCTTC                       CCTAGAGCAC ACTTGCTTCA GTATCATAAC AAAACCCTCA                       CCTAATATTT TTTTTTAAAA AAAAAAAATA TTAGAGTGGT                       ATTGGGGTCT CTTTGGTAAT TCTTTGTAAT TATAAAAGTA                       CCCTTAAATG TCATAAACAG GTTAGCTTTA GCTTGCCTTT                       AAAGATCTTC TTAGGGTATC ATTACTTTTC GTAAATCTTT                       AATAGGCCTG TCACATAATT CTACCCTTAA ATTTCTTAAA                       CCTTGATCTG AAGTCAAGTG GGAGTACCCG CTGAACTTAA              Rhizous stolonifer  (strains ATCC14037 and 6227A) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:19)                        AAAGTGCGAT AACTAGTGTG AATTGCATAT TCAGTGAATC                           ATCGAGTCTT TGAACGCAAC TTGCACTCTA TGGTTTTCCG                       TAAAGTACGC TTGCTTCAGT ATCATAAAGA CCCCATCCTG                       ATTATTATTT TTTTATTAAA ATAATTAATT TTGGAGATAA                       TAAAAATGAG GCTCTTTCTT TTCTTTTTTT TTTTTTTAAA                       AAAAAGGGGG GGAAAGGGTC TTTTAAAATG GGCAAATTCT                       GGGTTTTTTA CTAAACCTGA ACTCCCCCCA AAAATTCAAA                       AAAAAAAAAA TGGGTTTTAC CAAATTTTTT TTTTTTTTCT                       CCTTTTTGTG TAGTTAATAC TCTATTAAAT TTATTTACTT                       GGTATTATAA CGATTATGCA AGAAGGGAGA GAACAAAGAA                       TAATGAAAGA GAGTTTTTAA ATAAATTCTT TTTTCATTTT                       TTCAATCAAT GATCTGAAGT CAAGTGGGAT TACCCGCTGA                       ACTTAA              Rhizomucor pusillus  (strain ATCC36606) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:20)                    AAATTGCGAA AAGTAATGCG ATCTGCAGCC TTTGCGAATC                       ATCGAATTCT CGAACGCACC TTGCACCCTT TGGTTCATCC                       ATTGGGTACG TCTAGTTCAG TATCTTTATT AACCCCTAAA                       GGTTTATTTT TTGATAAATC TTTGGATTTG CGGTGCTGAT                       GGATTTTCAT CCGTTCAAGC TACCCGAACA ATTTGTATGT                       TGTTGACCCT TGATATTTCC TTGAGGGCTT GCATTGGTAT                       CTAATTTTTT ACCAGTGTGC TTCGAGATGA TCAAGTATAA                       AGGTCAATCA ACCACAAATA AATTTCAACT ATGGATCTGA                       ACTTAGATGG GATTACCCGC TGAACTTAA              Absidia corymbifera  (strain ATCC46774) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:21)                    AAAGTGCGAT AATTATTGCG ACTTGCATTC ATAGCGAATC                       ATCGAGTTCT CGAACGCATC TTGCGCCTAG TAGTCAATCT                       ACTAGGCACA GTTGTTTCAG TATCTGCAAC TACCAATCAG                       TTCAACTTGG TTCTTTGAAC CTAAGCGAGC TGGAAATGGG                       CTTGTGTTGA TGGCATTCAG TTGCTGTCAT GGCCTTAAAT                       ACATTTAGTC CTAGGCAATT GGCTTTAGTC ATTTGCCGGA                       TGTAGACTCT AGAGTGCCTG AGGAGCAACG ACTTGGTTAG                       TGAGTTCATA ATTCCAAGTC AATCAGTCTC TTCTTGAACT                       AGGTCTTAAT CTTTATGGAC TAGTGAGAGG ATCTAACTTG                       GGTCTTCTCT TAAAACAAAC TCACATCTAG ATCTGAAATC                       AACTGAGATC ACCCGCTGAA CTTAA              Absidia corymbifera  (strain ATCC46773) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:22)                    AAAGTGCGAT AATTATTGCG ACTTGCATTC ATAGTGAATC                       ATCGAGTTCT TGAACGCATC TTGCGCCTAG TAGTCAATCT                       ACTAGGCACA GTTGTTTCAG TATCTGCATC CACCAATCAA                       CTTAACCTTT TGTGTTGAGT TGGAACTGGG CTTCTAGTTG                       ATGGCATTTA GTTGCTGTCA TGGCCTTAAA TCAATGTCCT                       AGGTGTTAGA ACATCTAACA CCGGATGGAA ACTTTAGAGC                       GCTTTAAGAG CAGCTTGGTT AGTGAGTTCA ATAATTCCAA                       GCATTAAGTC TTTTAATGAA CTAGCTTTTC TATCTATGGG                       ACACTACTTG GAGAAATCCA AGTAACCTTT AAACTCCCAT                       TTAGATCTGA AATCAACTGA GACCACCCGC TGAACTTAA              Cunninghamella elegans  (strain ATCC42113) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:23)                    AAATCGCGAT ATGTAATGTG ACTGCCTATA GTGAATCATC                       AAATCTTTGA AACGCATCTT GCACCTTATG GTATTCCATA                       AGGTACGTCT GTTTCAGTAC CACTAATAAA TCTCTCTCTA                       TCCTTGATGA TAGAAAAAAA AAAAATAATT TTTACTGGGC                       CCGGGGAATC CTTTTTTTTT TTTAATAAAA AGGACCAATT                       TTGGCCCAAA AAAAAGGGTT GAACTTTTTT TACCAGATCT                       TGCATCTAGT AAAAACCTAG TCGGCTTTAA TAGATTTTTA                       TTTTCTATTA AGTTTATAGC CATTCTTATA TTTTTTAAAA                       TCTTGGCCTG AAATCAGATG GGATACCCGC TGAACTTAA              Pseudallescheria boydii  (strain ATCC44328) internal transcribed spacer 2 and adjacent regions (teleomorph of  Scedosporium apiospermum ).
 
                                           (SEQ ID NO:24)                    AAATGCGATA AGTAATGTAA ATTGCAAAAT TCAGTGAATC                       ATCGAATCTT TGAAACGCAC ATTGCGCCCG GCAGTAATCT                       GCCGGGCATG CCTGTCCGAG CGTCATTTCA ACCCTCGAAC                       CTCCGTTTC CTTAGGGAAG CCTAGGGTCG GTGTTGGGGC                       GCTACGGCAA GTCCTCGCAA CCCCCGTAGG CCCTGAAATA                       CAGTGGCGGT CCCGCCGCGG TTGCCTTCTG CGTAGTAAGT                       CTCTTTTGCA AGCTCGCATT GGGTCCCGGC GGAGGCCTGC                       CGTCAAACCA CCTAACAACT CCAGATGGTT TGACCTCGGA                       TCAGGTAGGG TTACCCGCTG AACTTAA              Pseudallescheria boydii  (strain ATCC36282) internal transcribed spacer 2 and adjacent regions (teleomorph of  Scedosporium apiospermum ).
 
                                           (SEQ ID NO:25)                    GAAATGCGAT AAGTAATGTG AATTGCAGAA TTCAGTGAAT                       CATCGAATCT TTGAAACGCA CATTGCGCCC GGCAGTAATC                       TGCCGGGCAT GCCTGTCCGA GCGTCATTTC AACCCTCGAA                       CCTCCGTTTC CTCAGGGAAG CTCAGGGTCG GTGTTGGGGC                       GCTACGGCAA GTCTTCGCAA CCCTCCGTAG GCCCTGAAAT                       ACAGTGGCGG TCCCGCCGCG GTTGCCTTCT GCGTAGAAGT                       CTCTTTTGCA AGCTCGCATT GGGTCCCGGC GGAGGCCTGC                       CGTCAAACCA CCTATAACTC CAAATGGTTT GACCTCGGAT                       CAGGTAGGGT TACCCGCTGA ACTTAA              Scedosporium apiospermum  (strain ATCC64215) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:26)                    GAAATGCGAT AAGTAATGTG AATTGCAGAA TTCAGTGAATC                       ATCGAATCTT TGAACGCACA TTGCGCCCGG CAGTAATCTG                       CCGGGCATGC CTGTCCGAGC GTCATTTCAA CCCTCGAACC                       TCCGTTTCCT CAGGGAAGCT CAGGGTCGGT GTTGGGGCGC                       TACGGCGAGT CTTCGCGACC CTCCGTAGGC CCTGAAATAC                       AGTGGCGGTC CCGCCGCGGT TGCCTTCTGC GTAGTAAGTC                       TCTTTTGCAA GCTCGCATTG GGTCCCGGCG GAGGCCTGCC                       GTCAAACCAC CTATAACTCC AGATGGTTTG ACCTCGGATC                       AGGTAGGTAC CCGCTGAACT TAA              Scedosporium apiospermum  (strain ATCC46173) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:27)                    AAATGCGATA AGTAATGTGA ATTGCAGAAT TCAGTGAATC                       ATCGAATCTT TGAACGCACA TTGCGCCCGG CAGTAATCTG                       CCGGGCATGC CTGTCCGAGC GTCATTTCAA CCCTCGAACC                       TCCGTTTCCT CAGGGAAGCT CAGGGTCGGT GTTGGGGCGC                       TACGGCGAGT CTTCGCGACC CTCCGTAGGC CCTGAAATAC                       AGTGGCGGTC CCGCCGCGGT TGCCTTCTGC GTAGTAAGTC                       TCTTTTGCAA GCTCGCATTG GGTCCCGGCG GAGGCCTGCC                       GTCAAACCAC CTATAACTCC AGATGGTTTG ACCTCGGATC                       AGGTAGGTAC CCGCTGAACT TAA              Penicillium notatum  (strain ATCC10108) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:28)                    AAATGCGATA CGTAATGTGA ATTGCAAATT CAGTGAATCA                       TCGAGTCTT TGAACGCACA TTGCGCCCCC TGGTATTCCG                       GGGGGCATGC CTGTCCGAGC GTCATTGCTG CCCTCAAGCA                       CGGCTTGTGT GTTGGGCCCC GTCCTCCGAT CCCGGGGGAC                       GGGCCCGAAA GGCAGCGGCG GCACCGCGTC CGGTCCTCGA                       GCGTATGGGG CTTTGTCACC CGCTCTGTAG GCCCGGCCGG                       CGCTTGCCGA TCAACCCAAA TTTTTATCCA GGTTGACCTC                       GGATCAGGTA GGGATACCCG CTGAACTTAA              Sporothrix schenckii  (strain ATCC14284 ) internal transcribed spacer 2 and adjacent regions.
 
                                           (SEQ ID NO:29)                    GAAATGCGAT ACTAATGTGA ATTGCAGAAT TCAGCGAACC                       ATCGAATCTT TGAACGCACA TTGCGCCCGC CAGCATTCTG                       GCGGGCATGC CTGTCCGAGC GTCATTTCCC CCCTCACGCG                       CCCCGTTGCG CGCTGGTGTT GGGGCGCCCT CCGCCTGGCG                       GGGGGCCCCC GAAAGCGAGT GGCGGGCCCT GTGGAAGGCT                       CCGAGCGCAG TACCGAACGC ATGTTCTCCC CTCGCTCCGG                       AGGCCCCCCA GGCGCCCTGC CGGTGAAAAC GCGCATGACG                       CGCAGCTCTT TTTACAAGGT TGACCTCGGA TCAGGTGAGG                       ATACCCGCTG ACTTAA            
Contamination Precautions
 
     Precautions were taken to avoid possible contamination of PCR samples by following the guidelines of Fujita and Kwok (13, 22). All buffers and distilled water used for PCR assays were autoclaved and fresh PCR reagents were aliquoted prior to use. Physical separation of laboratory areas used to prepare PCR assays and to analyze PCR products, and the use of aerosol-resistant pipette tips, reduced possible cross-contamination of samples by aerosols. Appropriate negative controls were included in each test run, including controls omitting either the primer or the DNA template during PCR assays. 
     Agarose Gel Electrophoresis 
     Gel electrophoresis was conducted in TBE buffer (0.1 M Tris, 0.09 M boric acid, 1 mM EDTA, pH 8.4) at 80 V for 1 to 2 hours using gels composed of 1% (w/vol) agarose (International Technologies, New Haven, Conn.) and 1% (w/vol) NuSieve agar (FMC Bioproducts, Rockland, Me.). Gels were stained with 0.5 μg of ethidium bromide (EtBr) per ml of distilled H 2 O for 10 minutes followed by three serial washes for 10 minutes each with distilled H 2 O. 
     Microtitration Plate Enzyme Immunoassay for the Detection of PCR Products 
     Amplicons were detected using species-specific and genus probes labeled with digoxigenin andan all-filamentous fungal probe labeled with biotin in a streptavidin-coated microtiter plate format (13, 34). Ten μl of PCR product was added to each 1.5 ml Eppendorf tube. Single-stranded DNA was then prepared by heating the tubes at 95° C. for 5 minutes and cooling immediately on ice. Two-tenths of a ml of hybridization solution [4×SSC (saline sodium citrate buffer, 0.6 M NaCl, 0.06 M trisodium citrate, pH 7.0) containing 20 mM Hepes, 2 mM EDTA, and 0.15% (vol/vol) Tween 20] supplemented with 50 ng/ml each of the all- Aspergillus  biotinylated probe and a species-specific digoxigenin-labeled probe was added to each tube containing denatured PCR product. Tubes were mixed by inversion and placed in a water bath at 37° C. to allow probes to anneal to PCR product DNA. After 1 hour, 100 μl of each sample was added to duplicate wells of a commercially prepared streptavidin-coated microtitration plate (Boehringer Mannheim, Indianapolis, Ind.). The plate was incubated at ambient temperature for 1 hour with shaking, using a microtitration plate shaker (manufactured for Dynatech by CLTI, Middletown, N.Y.). Plates were washed 6 times with 0.01 M potassium phosphate buffered saline, pH 7.2, containing 0.05% Tween 20 (PBST). Each well then received 100 μl of horseradish peroxidase-conjugated, anti-digoxigenin Fab fragment (Boehringer Mannheim) diluted 1:1000 in hybridization buffer. After incubation at ambient temperature for 30 minutes with shaking, the plate was washed 6 times with PBST. One hundred μl of a mixture of one volume of 3, 3′, 5, 5′-tetramethyl benzidine peroxidase substrate (Kirkegaard and Perry Laboratories, Inc., Gaithersberg, Md.) and one volume of peroxidase solution (Kirkegaard and Perry Laboratories) was added to each well and the plate was placed at ambient temperature for 10 minutes for color development. The A 650nm  of each well was determined with a microtitration plate reader (UV Max, Molecular Devices, Inc., Menlo Park, Calif.). The absorbance value for the reagent blank, where DNA was absent but replaced with distilled H 2 O, was subtracted from each test sample. 
     Statistical Analysis 
     The Student&#39;s t test was used to determine differences between sample means. Means are expressed as the mean plus or minus the standard error from the mean. Differences were considered significant when P&lt;0.05. 
     The following probes were used to detect and distinguish each species. 
     
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Probe Sequences 
               
             
          
           
               
                   
                 5′ to 3′ 
               
               
                   
                 OLIGONUCLEOTIDE 
               
               
                 PROBES 
                 SEQUENCE 
               
               
                   
               
             
          
           
               
                 Generic Biotin Probe 
                 5′ end-labeled biotinylated probe 
                   
               
               
                   
                 5.8S region of rDNA 
               
               
                 B-58 
                 GAA TCA TCG A(AG)T CTT 
                 SEQ ID NO 61 
               
               
                   
                 TGA ACG 
               
               
                   
               
               
                 Digoxigenin-probe 
                 5′ end-labeled digoxigenin probe 
               
               
                   
                 ITS2 region of rDNA 
               
               
                 Aspergillus species 
               
               
                 
                   A. flavus 22 
                 
                 GCA AAT CAA TCT TTT TCC 
                 SEQ ID NO 30 
               
               
                 
                   A. flavus 23 
                 
                 GAA CGC AAA TCA ATC TTT 
                 SEQ ID NO 31 
               
               
                 
                   A. fumigatus 
                 
                 CCG ACA CCC ATC TTT ATT 
                 SEQ ID NO 32 
               
               
                 
                   A. niger 
                 
                 GAC GTT ATC CAA CCA TTT 
                 SEQ ID NO 33 
               
               
                 
                   A. nidulans 
                 
                 GGC GTC TCC AAC CTT ATC 
                 SEQ ID NO 35 
               
               
                 
                   A. terreus 
                 
                 GCA TTT ATT TGC AAC TTG 
                 SEQ ID NO 34 
               
               
                   
               
               
                 Fusarium species 
               
               
                 
                   F. moniliforme 
                 
                 TCT AGT GAC GGT CTC GCT 
                 SEQ ID NO 49 
               
               
                 
                   F. oxysporum 
                 
                 CGT TAA TTC GCG TTC CTC 
                 SEQ ID NO 50 
               
               
                 
                   F. solani 
                 
                 CTA ACA CCT CGC AAC TGG 
                 SEQ ID NO 51 
               
               
                   
                 AGA 
               
               
                   
               
               
                 Mucor species 
               
               
                 
                   M. circinelloides 
                 
                 AAC ATT TTT GTG AAT AGG 
                 SEQ ID NO 39 
               
               
                   
                 ATG 
               
               
                 
                   M. indicus 
                 
                 CGT GGA TTG AGT GCC GAT 
                 SEQ ID NO 38 
               
               
                 
                   M. plumbeus 
                 
                 GAA ACC CTT GAA ATT 
                 SEQ ID NO 37 
               
               
                 
                   M. rouxii 
                 
                 GAA TAG GAA TAC TGA GAG 
                 SEQ ID NO 36 
               
               
                 
                   M. racemosus 
                 
                 GAA ATC CCT GAA ATT 
                 SEQ ID NO 40 
               
               
                   
               
               
                 Penicillium species 
               
               
                   Penicillium marneffei  1 
                 GGG TTG GTC ACC ACC ATA 
                 SEQ ID NO 47 
               
               
                   Penicillium marneffei  2 
                 TGG TCA CCA CCA TAT TTA 
                 SEQ ID NO 48 
               
               
                 
                   Penicillium notatum 
                 
                 GAT CAA CCC AAA TTT TTA 
                 SEQ ID NO 46 
               
               
                   
               
               
                 Rhizopus species 
               
               
                 
                   R. circinans 
                 
                 CTT AGG GTA TCA TTA CTT 
                 SEQ ID NO 42 
               
               
                 
                   R. microsporus 
                 
                 CAT ATA ATC TAG GGG TTC 
                 SEQ ID NO 57 
               
               
                 
                   R. oryzae 
                 
                 GAG TAT AAT CCA G(CT)A  
                 SEQ ID NO 41 
               
               
                   
                 ACT 
               
               
                 
                   R. stolonifer 
                 
                 CTT GGT ATT ATA ACG ATT 
                 SEQ ID NO 44 
               
               
                 
                   Rhizomucor pusillus 
                 
                 TCC TTG AGG GCT TGC ATT 
                 SEQ ID NO 43 
               
               
                   
               
               
                 Other Genera 
               
               
                 
                   Absidia corymbifera 
                 
                 GTT GCT GTC ATG GCC TTA 
                 SEQ ID NO 55 
               
               
                   Cunninghamella elegans  4 
                 TAG TCG GCT TTA ATA GAT 
                 SEQ ID NO 52 
               
               
                   Cunninghamella elegans  5 
                 TAT TAA GTT TAT AGC CAT 
                 SEQ ID NO 53 
               
               
                   Cunninghamella elegans  6 
                 TAA GTT TAT AGC CAT TCT 
                 SEQ ID NO 54 
               
               
                 
                   Pseudallescheria boydii 
                 
                 AAG TCT CTT TTG CAA GCT 
                 SEQ ID NO 45 
               
               
                 
                   Sporothrix schoenckii 
                 
                 GAC GCG CAG CTC TTT TTA 
                 SEQ ID NO 56 
               
               
                   
               
               
                 Genus Probes 
               
               
                   G - ASPERGILLUS   
                 CCT CGA GCG TAT GGG GCT 
                 SEQ ID NO 58 
               
               
                   G - FUSARIUM   
                 CCC AAC TTC TGA ATG TTG 
                 SEQ ID NO 59 
               
               
                   G - MUCOR   
                 (AC)TG GGG CCT CCC AAA 
                 SEQ ID NO 60 
               
               
                   
                 TAA 
               
               
                   
               
             
          
         
       
     
     Species-specific probes to the ITS2 region of rDNA for  Aspergillus fumigatus  (SEQ ID NO:32),  A. flavus  (SEQ ID NO:31),  A. niger  SEQ ID NO:33),  A. terreus  (SEQ ID NO:34), and  A. nidulans  (SEQ ID NO:35) correctly identified each of the respective species (P&lt;0.001), and gave no false-positive reactions with  Rhizopus, Mucor, Fusarium, Penicillium , or  Candida  species. The  A. flavus  probe also recognized  A. oryzae , which belongs to the  A. flavus  group. Identification time was reduced from a mean of 5 days by conventional methods to 8 hours. 
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Aspergillus Probes 
               
             
          
           
               
                 Fungus 
                 
                   A. fumigatus 
                 
                 
                   A. nidulans 
                 
                 
                   A. niger 
                 
                 
                   A. terreus 
                 
                 
                   A. flavus 
                 
               
               
                   
               
               
                 
                   A. fumigatus 
                 
                 2.197 ± 0.187 
                 0.002 
                 0.000 
                 0.001 
                 0.001 
               
               
                 (n = 6) 
               
               
                 
                   A. nidulans 
                 
                 0.001 
                 1.315 ± 0.464 
                 0.002 
                 0.000 
                 0.001 
               
               
                 (n = 3) 
                   
               
               
                 
                   A. niger 
                 
                 0.000 
                 0.000 
                 1.242 ± 0.471 
                 0.001 
                 0.003 
               
               
                 (n = 5) 
                   
                   
               
               
                 
                   A. terreus 
                 
                 0.001 
                 0.000 
                 0.001 
                 1.603 ± 0.378 
                 0.001 
               
               
                 (n = 4) 
                   
                   
                   
               
               
                 
                   A. flavus 
                 
                 0.001 
                 0.001 
                 0.000 
                 0.001 
                 2.043 ± 0.390 
               
               
                 (n = 6) 
                   
                   
                   
                   
               
               
                 
                   A. oryzae 
                 
                 0.001 
                 0.002 
                 0.001 
                 0.001 
                 2.445 ± 0.106 
               
               
                 (n = 2) 
                   
                   
                   
                   
               
               
                 
                   A. parasitica 
                 
                 0.001 
                 0.002 
                 0.002 
                 0.002 
                 0.051 
               
               
                 (n = 1) 
               
               
                 
                   A. clavus 
                 
                 0.005 
                 0.005 
                 0.006 
                 0.005 
                 0.003 
               
               
                 (n = 1) 
               
               
                 
                   C. albicans 
                 
                 0.002 
                 0.001 
                 0.002 
                 0.000 
                 0.000 
               
               
                 (n = 1) 
               
               
                 
                   C. parasilosis 
                 
                 0.001 
                 0.002 
                 0.002 
                 0.002 
                 0.001 
               
               
                 (n = 1) 
               
               
                 
                   C. glabrata 
                 
                 0.001 
                 0.003 
                 0.001 
                 0.001 
                 0.005 
               
               
                 (n = 1) 
               
               
                 
                   C. krusei 
                 
                 0.002 
                 0.002 
                 0.002 
                 0.001 
                 0.001 
               
               
                 (n = 1) 
               
               
                 
                   C. tropicalis 
                 
                 0.002 
                 0.002 
                 0.001 
                 0.000 
                 0.001 
               
               
                 (n = 1) 
               
               
                 
                   F. moniliforme 
                 
                 0.003 
                 0.003 
                 0.001 
                 0.001 
                 0.001 
               
               
                 (n = 1) 
               
               
                 
                   F. solani 
                 
                 0.006 
                 0.002 
                 0.001 
                 0.000 
                 0.001 
               
               
                 (n = 1) 
               
               
                 
                   R. oryzae 
                 
                 0.001 
                 0.001 
                 0.001 
                 0.001 
                 0.001 
               
               
                 (n = 1) 
               
               
                 
                   M. racemosus 
                 
                 0.001 
                 0.002 
                 0.005 
                 0.002 
                 0.000 
               
               
                 (n = 1)  
               
               
                 
                   P. notatum 
                 
                 0.001 
                 0.002 
                 0.002 
                 0.002 
                 0.000 
               
               
                 (n = 1) 
               
               
                 Avg ± SD 
                 0.001 ± 0.002 
                 0.001 ± 0.001 
                 0.000 ± 0.002 
                 0.000 ± 0.002 
                 0.002 ± 0.010 
               
               
                 negative 
               
               
                 controls 
               
               
                   
               
             
          
         
       
     
     Species-specific probes to the ITS2 region of rDNA for  Fusarium oxysporum, F. solani , and  F. moniliforme , correctly identified each of the respective species (P&lt;0.001), and gave no false-positive reactions with  Blastomyces, Apophysomyces, Candida, Aspergillus, Mucor, Penecillium, Rhizopus, Rhizomucor, Absidia, Cunninghamella, Pseudallescheria, Sporothrix , or  Neosartorya . Empty boxes in Table 4 represent zero probe reactivity. 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Fusarium Probes 
               
             
          
           
               
                   
                   
                   
                   
                 Generic 
               
               
                 Fungus 
                 
                   F. oxysporum 
                 
                 
                   F. solani 
                 
                 
                   F. moniliforme 
                 
                 Fusarium  
               
               
                   
               
               
                 
                   F. oxysporum 
                 
                 1.40 ± 0.13 
                   
                   
                 1.76 ± 0.27 
               
               
                 (n = 3) 
               
               
                 
                   F. solani 
                 
                   
                 1.57 ± 0.07 
                   
                 1.35 ± 0.28 
               
               
                 (n = 5) 
               
               
                 
                   F. moniliforme 
                 
                   
                   
                 1.40 ± 0.01 
                 1.34 ± 0.91 
               
               
                 (n = 2) 
               
               
                 Negative control 
               
               
                 
                   A. fumigatus 
                 
               
               
                 
                   A. flavus 
                 
               
               
                 
                   A. niger 
                 
               
               
                 
                   A. nidulans 
                 
               
               
                 
                   A. terreus 
                 
               
               
                 
                   A. parasiticus 
                 
               
               
                 
                   A. clavatus 
                 
               
               
                 
                   P. marneffei 
                 
                   
                 0.01 
                 0.01 
               
               
                 
                   P. notatum 
                 
                 0.01 
                 0.01 
                 0.01 
               
               
                 
                   Rhizopus oryzae 
                 
                   
                 0.03 
                 0.01 
               
               
                 
                   Rhizopus microsporus 
                 
                   
                 0.01 
                 0.01 
               
               
                 
                   Rhizopus circinans 
                 
                   
                 0.01 
                 0.01 
               
               
                 
                   Rhizopus stolonifer 
                 
                   
                 0.01 
                 0.01 
               
               
                 
                   Rhizomucor pusillus 
                 
                   
                 0.03 
                 0.02 
               
               
                 
                   M. racemosus 
                 
               
               
                 
                   M. circinelloides 
                 
               
               
                 
                   M. rouxii 
                 
               
               
                 
                   M. plumbeus 
                 
               
               
                 
                   M. indicus 
                 
               
               
                 
                   Absidia corymbifera 
                 
                   
                 0.01 
                 0.01 
               
               
                 
                   Cunninghamella elegans 
                 
                   
                 0.01 
                 0.02 
               
               
                 
                   P. boydii 
                 
                   
                   
                 0.02 
               
               
                 
                   Sporothrix schenckii 
                 
                   
                 0.01 
                 0.01 
               
               
                 
                   C. albicans 
                 
               
               
                 
                   C. tropicalis 
                 
               
               
                 
                   C. krusei 
                 
               
               
                 
                   C. parasilosis 
                 
               
               
                 
                   C. glabrata 
                 
               
               
                 
                   Neosartorya fischeri 
                 
                   
                 0.01 
               
               
                 
                   Blastomyces dermatitidis 
                 
               
               
                 
                   Apophysomyces elegans 
                 
               
               
                 Average of negative controls 
                 0.001 ± 0.002 
                 0.005 ± 0.01 
                 0.004 ± 0.006 
               
               
                   
               
             
          
         
       
     
     Species-specific probes to various other zygomyces are presented in Table 5, showing correct identification of each species and no false positives. The exceptions are that the  M. circinelloides  probe hybridized with the  M. rouxii  DNA and the  M. plumbeus  probe hybridized with the  M. racemosus  DNA. However, the  M. rouxii  probe did not hybridize with  M. circinelloides  DNA, nor did the  M. racemosus  probe hybridize with  M. plumbeus  DNA. Therefore, by a process of elimination, each species can be correctly identified. Empty boxes in Table 5 represent zero probe reactivity. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Zygomyces Probes 
               
             
          
           
               
                   
                 D-probes 
               
             
          
           
               
                 FUNGUS 
                 RORY 
                 RMIC 
                 RCIR 
                 RSTOL 
                 RPUS 
                 MRACE 
                 MCIR 
                 MRX 
                 MPLUM 
                 MIND 
                 ABS 
                 CUN 
               
               
                   
               
               
                 
                   R. oryzae 
                 
                 1.50 ± 
                   
                   
                   
                 0.01 
                   
                   
                   
                   
                   
                   
                   
               
               
                 (n = 5) 
                 0.48 
               
               
                 
                   R. microsporus 
                 
                   
                 0.96 ± 
               
               
                 (n = 5) 
                   
                 0.61 
               
               
                 
                   R. circinans 
                 
                   
                   
                 1.56 ± 
               
               
                 (n = 3) 
                   
                   
                 0.19 
               
               
                 
                   R. stolonifer 
                 
                   
                   
                   
                 2.53 ± 
                   
                   
                 0.01 
               
               
                 (n = 5) 
                   
                   
                   
                 0.07 
               
               
                 
                   Rhizomucor  
                 
                   
                   
                   
                   
                 1.10 ± 
               
               
                 
                   pusillus 
                 
                   
                   
                   
                   
                 0.68 
               
               
                 (n = 2) 
               
               
                 
                   M. racemosus 
                 
                   
                   
                   
                 0.01 
                   
                 2.02 ± 
                   
                   
                 0.29 ± 
               
               
                 (n = 6) 
                   
                   
                   
                   
                   
                 0.34 
                   
                   
                 0.52 
               
               
                 
                   M. circinelloides 
                 
                   
                   
                   
                   
                   
                   
                 1.63 ± 
                 0.01 
                 0.02 
               
               
                 (n = 3) 
                   
                   
                   
                   
                   
                   
                 0.37 
               
               
                 
                   M. rouxii 
                 
                   
                   
                   
                   
                   
                   
                 1.77 
                 0.76 
               
               
                 (n = 1) 
               
               
                 
                   M. plumbeus 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                 2.14 ± 
               
               
                 (n = 2) 
                   
                   
                   
                   
                   
                   
                   
                   
                 0.25 
               
               
                 
                   M. indicus 
                 
                   
                 0.01 
                   
                   
                   
                   
                   
                   
                   
                 1.70 ± 
               
               
                 (n = 1) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 0.04 
               
               
                 
                   Absidia 
                 
                   
                   
                   
                   
                 0.01 
                   
                   
                   
                 0.01 
                   
                 1.61 ± 
               
               
                 
                   corymbifera 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 0.08 
               
               
                 (n = 2) 
               
               
                 
                   Cunninhamella 
                 
                   
                 0.01 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 2.26 ± 
               
               
                 
                   elegans 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 0.03 
               
               
                 (n = 2) 
               
               
                 Negative control 
               
               
                 
                   A. fumigatus 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                 0.01 
                 0.02 
               
               
                 
                   A. flavus 
                 
                   
                   
                   
                   
                 0.01 
                   
                   
                   
                   
                 0.05 
               
               
                 
                   A. niger 
                 
                   
                   
                   
                   
                   
                   
                   
                 0.01 
               
               
                 
                   A. nidulans 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                 0.01 
                 0.01 
               
               
                 
                   A. terreus 
                 
                 0.01 
               
               
                 
                   A. parasiticus 
                 
                   
                   
                   
                   
                 0.01 
                   
                   
                   
                   
                 0.03 
               
               
                 
                   A. clavatus 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 0.02 
               
               
                 
                   P. marneffei 
                 
                   
                   
                 0.01 
               
               
                 
                   P. notatum 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 0.03 
               
               
                 
                   F. oxysporum 
                 
                   
                   
                   
                   
                   
                   
                   
                 0.01 
               
               
                 
                   F. solani 
                 
               
               
                 
                   F. moniliforme 
                 
                 0.01 
                   
                   
                 0.01 
                   
                   
                   
                 0.01 
                   
                 0.01 
               
               
                 
                   P. boydii 
                 
                 0.02 
               
               
                 
                   Sporothrix 
                 
               
               
                 
                   schenckii 
                 
               
               
                 
                   C. albicans 
                 
               
               
                 
                   C. tropicalis 
                 
               
               
                 
                   C. krusei 
                 
               
               
                 
                   C. parasilosis 
                 
               
               
                 
                   C. glabrata 
                 
               
               
                 
                   Neosartorya 
                 
                   
                   
                 0.01 
               
               
                 
                   fischeri 
                 
               
               
                 
                   Blastomyces 
                 
               
               
                 
                   dermatitidis 
                 
               
               
                 
                   Apophysomyces 
                 
               
               
                 
                   elegans 
                 
               
               
                 Average 
                 0.001 ± 
                 0.001 ± 
                 0.000 ± 
                 0.000 ± 
                 0.001 ± 
                 0.001 ± 
                 0.001 ± 
                 0.001 ± 
                 0.003 ± 
                 0.005 ± 
                 0.001 ± 
               
               
                   
                 .004 
                 0.02 
                 0.002 
                 0.003 
                 0.003 
                 0.002 
                 0.002 
                 0.003 
                 0.005 
                 0.01 
                 0.001 
               
               
                   
               
             
          
         
       
     
     Species-specific probes to various other fungi are presented in Table 6, showing correct identification of each species and no false positives. Empty boxes in Table 6 represent zero probe reactivity. 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Pseudallescheria and Sporothrix Probes 
               
             
          
           
               
                   
                   
                   
                   
                 
                   Sporothrix 
                 
               
               
                 Fungus 
                 
                   P. boydii 
                 
                 
                   P.marneffei 
                 
                 
                   P.notatum 
                 
                 
                   schenckii 
                 
               
               
                   
               
               
                 
                   P. boydii 
                 
                 1.65 ± 0.48 
                   
                   
                   
               
               
                 (n = 4) 
               
               
                 
                   P. marneffei 
                 
                 0.01 
                 1.24 ± 0.12 
               
               
                 (n = 3) 
                   
               
               
                 
                   P. notatum 
                 
                   
                   
                 1.93 ± 0.25 
               
               
                 (n = 3) 
                   
                   
               
               
                 
                   Sporothrix schenckii 
                 
                 0.01 
                   
                   
                 1.94 ± 0.25 
               
               
                 (n = 3) 
                   
                   
                   
               
               
                 Negative control 
               
               
                 
                   A. fumigatus 
                 
                 0.01 
               
               
                 
                   A. flavus 
                 
               
               
                 
                   A. niger 
                 
               
               
                 
                   A. nidulans 
                 
               
               
                 
                   A. terreus 
                 
               
               
                 
                   A. parasiticus 
                 
               
               
                 
                   A. clavatus 
                 
                   
                   
                 0.11 
               
               
                 
                   F. oxysporum 
                 
                   
                 0.10 
               
               
                 
                   F. solani 
                 
                   
                 0.14 
               
               
                 
                   F. moniliforme 
                 
                   
                 0.08 
               
               
                 
                   R. oryzae 
                 
                 0.01 
               
               
                 
                   R. microsporus 
                 
                 0.01 
               
               
                 
                   R. circinans 
                 
                 0.01 
               
               
                 
                   R. stolonifer 
                 
                 0.01 
               
               
                 
                   Rhizomucor pusilus 
                 
               
               
                 
                   M. racemosus 
                 
                   
                 0.04 
               
               
                 
                   M. circinelloides 
                 
                 0.01 
                 0.09 
               
               
                 
                   M. rouxii 
                 
                 0.01 
               
               
                 
                   M. plumbeus 
                 
                   
                 0.05 
               
               
                 
                   M. indicus 
                 
               
               
                 
                   Absidia corymbifera 
                 
                 0.01 
               
               
                 
                   Cunninghamela bertholletiae 
                 
                 0.01 
               
               
                 
                   C. albicans 
                 
               
               
                 
                   C. tropicalis 
                 
                   
                 0.02 
               
               
                 
                   C. krusei 
                 
               
               
                 
                   C. parasilosis 
                 
               
               
                 
                   C. glabrata 
                 
               
               
                 
                   Neosatorya pseudofischeri 
                 
                   
                 0.03 
               
               
                 
                   Blastomyces dermatitidis 
                 
                 0.01 
               
               
                 
                   Apophysomyces elegans 
                 
                 0.01 
               
               
                 Average Negative Controls 
                 0.004 ± 0.002 
                 0.013 ± 0.03 
                 0.002 ± 0.019 
                 0.001 ± 0.002 
               
               
                   
               
             
          
         
       
     
     All of the references mentioned in this Specification are hereby incorporated by reference in their entirety. 
     REFERENCES 
     
         
         1. Ampel, N. M. 1996. Emerging disease issues and fungal pathogens associated with HIV infection. Emerg. Infec. Dis. 2:109–116. 
         2. Andriole, V. T. 1996 . Aspergillus  infections: problems in diagnosis and treatment. Infect. Agents and Dis. 5:47–54. 
         3. Andriole, V. T. 1993. Infections with  Aspergillus  species. Clin. Infec. Dis. 17 Suppl 2:S481–S486. 
         4. Bir, N., A. Paliwal, K. Muralidhar, P. Reddy, and P. U. Sarma. 1995. A rapid method for the isolation of genomic DNA from  Aspergillus fumigatus . Prep. Biochem. 25:171–181. 
         5. Blum, U., M. Windfuhr, C. Buitrago-Tellez, G. Sigmund, E. W. Herbst, and M. Langer. 1994. Invasive pulmonary aspergillosis. MRI, CT, and plain radiographic findings and their contribution for early diagnosis. Chest 106:1156–1161. 
         6. Caillot, D., O. Casasnovas, A. Bernard, J. F. Couaillier, C. Durand, B. Cuisenier, E. Solary, F. Piard, T. Petrella, A. Bonnin, G. Couaillault, M. Dumas, and H. Guy, 1997. Improved management of invasive pulmonary aspergillosis in neutropenic patients using early thoracic computed tomographic scan and surgery. J. Clin. Oncol. 15:139–147. 
         7. Denning, D. W. Therapeutic outcome in invasive aspergillosis. Clin. Infect. Dis. 23:608–615. 
         8. Denning, D. W. Diagnosis and management of invasive aspergillosis. Curr. Clin. Topics Inf. Dis. 16:277–299. 
         9. de Repentigny, L., L. Kaufman, G. T. Cole, D. Kruse, J. P. Latge, and R. C. Matthews. 1994. Immunodiagnosis of invasive fungal infections. J. Med. Vet. Mycol. 32 Suppl 1239–252. 
         10. Dupont, B., D. W. Denning, D. Marriott, A. Sugar, M. A. Viviani, and T. Sirisanthana. 1994. Mycoses in AIDS patients. J. Med. Vet. Mycol. 32 Suppl 1:221–239. 
         11. Fisher, B. D., D. Armstrong, B. Yu, and J. W. M. Gold. 1981. Invasive aspergillosis: progress in early diagnosis and treatment. Am. J. Med. 71:571–577. 
         12. Fridkin, S. K. and W. R. Jarvis. 1996. Epidemiology of nosocomial fungal infections. Clin. Microbiol. Rev. 9:499–511. 
         13. Fujita, S-I., B. A. Lasker, T. J. Lott, E. Reiss, and C. J. Morrison. 1995. Micro titration plate enzyme immunoassay to detect PCR-amplified DNA from  Candida  species in blood. J. Clin. Microbiol. 33:962–967. 
         14. Gordon, M. A., E. W. Lapa, and J. Kane. 1977. Modified indirect fluorescent antibody test for aspergillosis. J. Clin. Microbiol. 6:161–165. 
         15. Holmes, A. R., R. D. Cannon, M. G. Shepard, and H. F. Jenkinson. 1994. Detection of  Candida albicans  and other yeast in blood by PCR. J. Clin. Microbiol. 32:228–231. 
         16. Hung, C. C., S. C. Chang, P. C. Yang, W. C. Hseigh. 1994. Invasive pulmonary pseudallescheriasis with direct invasion of the thoracic spine in an immunocompromised patient. Eur. J. Clin. Microbiol. Infect. Dis. 13:749–751. 
         17. Kappe, R., and H. P. Seeliger. 1993. Serodiagnosis of deep-seated fungal infections. Curr. Topics Med. Mycol. 5:247–280. 
         18. Kappe, R., A. Schulze-Berge, H. G. Sonntag. 1996. Evaluation of eight antibody tests and one antigen test for the diagnosis of invasive aspergillosis. Mycoses 39:13–23. 
         19. Kaufman and Reiss, Manual of Clinical Microbiology. 
         20. Kremery, V., Jr., E. Kunova, Z. Jesenska, J. Trupl, S. Spanik, J. Mardiak, M. Studena, and E. Kukuckova. 1996. Invasive mold infections in cancer patients: 5 years&#39; experience with  Aspergillus, Mucor, Fusarium  and  Acremonium  infections. Supportive Care in Cancer 4:39–45. 
         21. Khoo, S. H., and D. W. Denning. 1994. Invasive aspergillosis in patients with AIDS. Clin. Infect. Dis 19 Suppl 1: S41–S48. 
         22. Kwok, S., and R. Higuichi. 1989. Avoiding false positives with PCR. Nature (London) 339:237–238. 
         23. Larone, D. H. Medically Important Fungi: A Guide to Identification. 3rd ed. ASM Press, Washington, D.C. 1995. 
         24. Leenders, A., A. van Belkum, S. Janssen, S. de Marie, J. Kluytmans, J. Wielenga, B. Lowenberg, and H. Verbrugh. Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward. J. Clin. Microbiol. 34:345–351. 
         25. Makimura, K., S. Y. Murayama, H. Yamaguchi. 1994. Specific detection of  Aspergillus  and  Penicillium  species from respiratory specimens by polymerase chain reaction (PCR). Jap. J. Med. Sci. Biol. 47:141–156. 
         26. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 
         27. Martino, P., and C. Girmenia. 1993. Diagnosis and treatment of invasive fungal infections in cancer patients. Supportive Care in Cancer. 1:240–244. 
         28. Melchers, W. J., P. E. Verweij, P. van den Hurk, A. van Belkum, B. E. De Pauw, J. A. Hoogkamp-Korstanje, and J. F. Meis. 1994. General primer-mediated PCR for detection of  Aspergillus  species. J. Clin. Microbiol. 32:1710–1717. 
         29. Miller, W. T. J., G. J. Sals, I. Frank, W. B. Gefter, M. Aronchick, W. T. Miller. 1994. Pulmonary aspergillosis patients with AIDS. Clinical and radiographic correlations. Chest 105:37–44. 
         30. Miyakawa, Y., T. Mabuchi, and Y. Fukazawa. 1993. New method for detection of  Candida albicans  in human blood by polymerase chain reaction. J. Clin. Microbiol. 31:3344–3347. 
         31. Montone, K. T., and L. A. Litzky. 1995. Rapid method for detection of  Aspergillus  5S ribosomal RNA using a genus-specific oligonucleotide probe. J. Clin. Microbiol. 103:48–51. 
         32. Rogers, T. R., K. A. Haynes, and R. A. Barnes. 1990. Value of antigen detection in predicting invasive aspergillosis. Lancet 336:1210–1213. 
         33. Sandhu, G. S., B. C. Kline, L. Stockman, and G. D. Roberts,. 1995. Molecular probes for diagnosis of fungal infections. J. Clin. Microbiol. 33:2913–2919. 
         34. Shin, J. H., F. S. Nolte, and C. J. Morrison. 1997. Rapid identification of  Candida  species in blood cultures using a clinically useful PCR method. J. Clin. Microbiol. in press. 
         35. Tang, C. M., D. W. Holden, A. Aufauvre-Brown, and J. Cohen. The detection of  Aspergillus  spp. by the polymerase chain reaction and its evaluation in bronchoalveolar lavage fluid. Amer. Rev. Respir. Dis 148:1313–1317. 
         36. Thompson, B. H., W. Stanford, J. R. Galvin, and Y. Kurlhara. 1995. Varied radiologic appearances of pulmonary aspergillosis. Radiographics 15:1273–1284. 
         37. Tierney, Jr. L. M. Aspergillosis. In Current Medical Diagnosis and Treatment. 33rd ed. Norwalk, Conn.: Appleton and Lange, 1994. 
         38. Verweij, P. E., J. P. Latge, A. J. Rijs, W. J. Melchers, B. E. De Pauw, J. A. Hoogkamp-Korstanje, and J. F. Mels. 1995. Comparison of antigen detection and PCR assay using bronchoalveolar lavage fluid for diagnosing invasive pulmonary aspergillosis in patients receiving treatment for hematological malignancies. J. Clin. Microbiol. 33:3150–3153. 
         39. von Eiff, M., N. Roos, R. Schulten, M. Hesse, M. Zuhisdorf, and J. van de Loo. 1995. Pulmonary aspergillosis: early diagnosis improves survival. Respiration 62:341–347. 
         40. von Eiff, M., N. Roos, W. Fegeler, C. von Eiff, R. Schulten, M. Hesse, M. Zuhisdorf, and J. van de Loo. 1996. Hospital acquired  Candida  and  Aspergillus  pneumonia—diagnostic approaches and clinical findings. J. Hosp. Infect. 32:17–28. 
         41. Walsh, T. J. 1993. Management of immunocompromised patients with evidence of an invasive mycosis. Hemat. Oncol. Clin. N .Amer. 7:1003–1026. 
         42. Walsh, T. J., C. Gonzalez, C. A. Lyman, S. J. Chanock, and P. A. Pizzo. 1996. Invasive fungal infections in children: recent advances in diagnosis and treatment. Adv. Ped. Inf. Dis. 11:187–290. 
         43. Walsh, T. J., B. De Pauw, E. Anaissle, and P. Martino. 1994. Recent advances in the epidemiology, prevention, and treatment of invasive fungal infections in neutropenic patients. J. Med. Vet. Mycol. 32 Supp 1:33–51. 
         44. Warnock, D. W. 1995. Fungal complications of transplantation: diagnosis, treatment, and prevention. J. Antimicrob. Chemother. 36 Suppl B:73–90. 
         45. Yamakami, Y., A. Hashimoto, I. Tokimatsu, and M. Nasu. 1996. PCR detection of DNA specific for  Aspergillus  species in serum of patients with invasive aspergillosis. J. Clin. Microbiol. 34:2464–2468. 
         46. Young, R. C., and J. E. Bennett. 1971. Invasive aspergillosis: absence of detectable antibody response. Am. Rev. Respir. Dis 104:710–716. 
         47. Zervos, M. J. and J. A. Vasquez. 1996. DNA analysis in the study of fungal infections in the immunocompromised host. Clin. Lab. Med. 16:73–88.