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 application claims priority to PCT/US98/08926, filed May 1, 1998, which 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 microsportis  (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, Fusariunm, 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 apiospermumn )(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 Tag buffer, 2.5 mM each of dATP, dGTP, dCTP, and dTTP, in 8 μl 0.2 μM of each primer, and 0.5 U of TaKaRa Ex Tag 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 
               
               
                 or Probes 
                 (5′ to 3′) 
                 and Location 
               
               
                   
               
               
                 ITS3 
                 GCA TCG ATG AAG 
                 5.85 rDNA universal 5′ 
               
               
                   
                 AAC GCA GC 
                 primer 
               
               
                   
                 (SEQ ID NO:63) 
               
               
                 ITS4 
                 TCC TCC GCT TAT 
                 28S rDNA universal 3′ 
               
               
                   
                 TGA TAT GC 
                 primer 
               
               
                   
                 (SEQ ID NO:64) 
               
               
                 ITSI 
                 TCC GTA GGT GAA 
                 185 rDNA universal 5′ 
               
               
                   
                 CCT GCG G 
                 primer 
               
               
                   
                 (SEQ ID NO:62) 
               
               
                   
               
             
          
         
       
     
     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 GAAAGGCAGC 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. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:12) 
               
             
          
         
       
     
       Rhizopus oryzae  (strain ATCC34965) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:13) 
               
             
          
         
       
     
       Rhizopus oryzae  (strain ATCC11886) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:14) 
               
             
          
         
       
     
       Rhizopus microsporus  (strain ATCC14056) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:15) 
               
             
          
         
       
     
       Rhizopus microsporus  (strain ATCC12276) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
             
           
               
                 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 (SEQ ID NO:16) 
               
             
          
         
       
     
       Rhizopus circinans  (strain ATCC34106) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO:17) 
               
             
          
         
       
     
       Rhizopus circinans  (strain ATCC34101) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO:18) 
               
             
          
         
       
     
       Rhizous stolonifer  (strains ATCC14037 and 6227A) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:19) 
               
             
          
         
       
     
       Rhizomucor pusillus  (strain ATCC36606) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
             
           
               
                 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 (SEQ ID NO:20) 
               
             
          
         
       
     
       Absidia corymbifera  (strain ATCC46774) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:21) 
               
             
          
         
       
     
       Absidia corymbifera  (strain ATCC46773) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO:22) 
               
             
          
         
       
     
       Cunninghamella elegans  (strain ATCC42113) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO:23) 
               
             
          
         
       
     
       Pseudallescheria boydii  (strain ATCC44328) internal transcribed spacer 2 and adjacent regions (teleomorph of  Scedosporium apiospennum ). 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:24) 
               
             
          
         
       
     
       Pseudallescheria boydii  (strain ATCC36282) internal transcribed spacer 2 and adjacent regions (teleomorph of  Scedosporium apiospermum ). 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:25) 
               
             
          
         
       
     
       Scedosporium apiospermum  (strain ATCC64215) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:26) 
               
             
          
         
       
     
       Scedosporium apiospermum  (strain ATCC46173) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 (SEQ ID NO:27) 
               
             
          
         
       
     
       Penicillium notatum  (strain ATCC10108) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
             
           
               
                 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 (SEQ ID NO:28) 
               
             
          
         
       
     
       Sporothrix schenckii  (strain ATCC14284) internal transcribed spacer 2 and adjacent regions. 
     
       
         
               
               
             
           
               
                   
                 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 
                 2  
               
               
                   
                   
               
               
                   
                 ATACCCGCTG ACTTAA (SEQ ID NO:29) 
               
             
          
         
       
     
     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 and an 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 650  nm 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 biontinylated 
                   
               
               
                   
                 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 ± 
                 0.002 
                 0.000 
                 0.001 
                 0.001 
               
               
                 (n = 6) 
                 0.187 
               
               
                 
                   A. nidulans 
                 
                 0.001 
                 1.315 ± 
                 0.002 
                 0.000 
                 0.001 
               
               
                 (n = 3) 
                   
                 0.464 
               
               
                 
                   A. niger 
                 
                 0.000 
                 0.000 
                 1.242 ± 
                 0.001 
                 0.003 
               
               
                 (n = 5) 
                   
                   
                 0.471 
               
               
                 
                   A. terreus 
                 
                 0.001 
                 0.000 
                 0.001 
                 1.603 ± 
                 0.001 
               
               
                 (n = 4) 
                   
                   
                   
                 0.378 
               
               
                 
                   A. flavus 
                 
                 0.001 
                 0.001 
                 0.000 
                 0.001 
                 2.043 ± 
               
               
                 (n = 6) 
                   
                   
                   
                   
                 0.390 
               
               
                 
                   A. oryzae 
                 
                 0.001 
                 0.002 
                 0.001 
                 0.001 
                 2.445 ± 
               
               
                 (n = 2) 
                   
                   
                   
                   
                 0.106 
               
               
                 
                   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 = I) 
               
               
                 
                   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 ± 
                 0.001 ± 
                 0.000 ± 
                 0.000 ± 
                 0.002 ± 
               
               
                 negative 
                 0.002 
                 0.001 
                 0.002 
                 0.002 
                 0.010 
               
               
                 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 ± 
                   
                   
                 1.76 ± 
               
               
                 (n = 3) 
                 0.13 
                   
                   
                 0.27 
               
               
                 
                   F. solani 
                 
                   
                 1.57 ± 
                   
                 1.35 ± 
               
               
                 (n = 5) 
                   
                 0.07 
                   
                 0.28 
               
               
                 
                   F. moniliforme 
                 
                   
                   
                 1.40 ± 
                 1.34 ± 
               
               
                 (n = 2) 
                 0.01 
                 0.91 
               
               
                 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 ± 
                 0.005 ± 
                 0.004 ± 
               
               
                   
                 0.002 
                 0.01 
                 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 pusillus 
                 
                   
                   
                   
                   
                 1.10 ± 
               
               
                 (n = 2) 
                   
                   
                   
                   
                 0.68 
               
               
                 
                   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 corymbifera 
                 
                   
                   
                   
                   
                 0.01 
                   
                   
                   
                 0.01 
                   
                 1.61 ± 
               
               
                 (n = 2) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 0.08 
               
               
                 
                   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 fischeri 
                 
                   
                   
                 0.01 
               
               
                 
                   Blastomyces 
                 
               
               
                 
                   dermatifidis 
                 
               
               
                 
                   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 ± 
                   
                   
                   
               
               
                 (n = 4) 
                 0.48 
               
               
                 
                   P. marneffei 
                 
                 0.01 
                 1.24 ± 
               
               
                 (n = 3) 
                   
                 0.12 
               
               
                 
                   P. notatum 
                 
                   
                   
                 1.93 ± 
               
               
                 (n = 3) 
                   
                   
                 0.25 
               
               
                 
                   Sporothrix schenckii 
                 
                 0.01 
                   
                   
                 1.94 ± 
               
               
                 (n = 3) 
                   
                   
                   
                 0.25 
               
               
                 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 bertholietiae 
                 
                 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 ± 
                 0.013 ± 
                 0.002 ± 
                 0.001 ± 
               
               
                   
                 0.002 
                 0.03 
                 0.019 
                 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. Trupi, 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 asperaillosis. 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. 
     
       
         
           
             61 
           
           
             1 
             208 
             DNA 
             Aspergillus flavus 
           
            1
gctgcccatc aagcacggct tgtgtgttgg gtcgtcgtcc cctctccggg ggggacgggc     60
cccaaaggca gcggcggcac cgcgtccgat cctcgagcgt atggggcttt gtcacccgct    120
ctgtaggccc ggccggcgct tgccgaacgc aaatcaatct ttttccaggt tgacctcgga    180
tcaggtaggg atacccgctg aacttcaa                                       208
 
           
             2 
             364 
             DNA 
             Aspergillus fumigatus 
           
            2
aaactttcaa caatggatct cttggttccg gcatcgatga agaacgcagc gaaatgcgat     60
aactaatgtg aattgcagaa ttcagtgaat catcgagtct ttgaacgcac attgcgcccc    120
ctggtattcc ggggggcatg cctgtccgag cgtcattgct gcccatcaag cacggcttgt    180
gtgttgggcc cccgtccccc tctcccgggg gacgggcccg aaaggcagcg gcggcaccgc    240
gtccggtcct cgagcgtatg gggcttgtca cctgctctgt aggcccggcc ggcgccagcc    300
gacacccaac tttatttttc taaggttgac ctcggatcag gtagggatac ccgctgaact    360
taaa                                                                 364
 
           
             3 
             365 
             DNA 
             Aspergillus niger 
           
            3
aaactttcaa caatggatct cttggttccg gcatcgatga agaacgcagc gaaatgcgat     60
aactaatgtg aattgcagaa ttcagtgaat catcgagtct ttgaacgcac attgcgcccc    120
ctggtattcc ggggggcatg cctgtccgag cgtcattgct gccctcaagc acggcttgtg    180
tgttgggtcg ccgtccccct ctcccggggg acgggcccga aaggcagcgg cggcaccgcg    240
tccgatcctc gagcgtatgg ggctttgtca cctgctctgt aggcccggcc ggcgcctgcc    300
gacgttatcc aaccattttt ttccaggttg acctcggatc aggtagggat acccgctgaa    360
cttaa                                                                365
 
           
             4 
             355 
             DNA 
             Aspergillus terreus 
           
            4
aaactttcaa caatggatct cttggttccg gcatcgatga agaacgcagc gaaatgcgat     60
aactaatgtg aattgcagaa ttcagtgaat catcgagtct ttgaacgcac attgcgcccc    120
ctggtattcc gggggggcat gcctgtccga gcgtcattgc tgccctcaag cccggcttgt    180
gtgttgggcc ctcgtccccc ggctcccggg ggacgggccc gaaaggcagc ggcggcaccg    240
cgtccggtcc tcgagcgtat ggggcttcgt cttccgctcc gtaggcccgg ccggcgcccg    300
ccgaacgcat ttatttgcaa cttgtttttt tttccaggtt gacctcggat caggt         355
 
           
             5 
             365 
             DNA 
             Aspergillus nidulans 
           
            5
aaactttcaa caatggatct cttggttccg gcatcgatga agaacgcagc gaactgcgat     60
aagtaatgtg aattgcagaa ttcagtgaat catcgagtct ttgaacgcac attgcgcccc    120
ctggcattcc ggggggcatg cctgtccgag cgtcattgct gccctcaagc ccggcttgtg    180
tgttgggtcg tcgtcccccc ccccggggga cgggcccgaa aggcagcggc ggcaccggtc    240
cggtcctcga gcgtatgggg cttggtcacc cgctcgatta gggccggccg ggcgccagcc    300
ggcgtctcca accttatctt tctcaggttg acctcggatc aggtagggat acccgctgaa    360
cttaa                                                                365
 
           
             6 
             319 
             DNA 
             Fusarium solani 
           
            6
gaaaatgcga taagtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca     60
cattgcgccc gccagtattc tggcgggcat gcctgttcga gcgtcattac aaccctcagg    120
cccccgggcc tggcgttggg gatcggcgga agccccctgc gggcacaacg ccgtccccca    180
aatacagtgg cggtcccgcc gcagcttcca ttgcgtagta gctaacacct cgcaactgga    240
gagcggcgcg gccacgccgt aaaacaccca acttctgaat gttgacctcg aatcaggtag    300
gaatacccgc tgaacttaa                                                 319
 
           
             7 
             310 
             DNA 
             Fusarium moniliforme 
           
            7
aaatgcgata agtaatgtga attgcaaaat tcagtgaatc atcgaatctt tgaacgcaca     60
ttgcgcccgc cagtattctg gcgggcatgc ctgttcgagc gtcatttcaa ccctcaagcc    120
cccgggtttg gtgttgggga tcggcaagcc cttgcggcaa gccggccccg aaatctagtg    180
gcggtctcgc tgcagcttcc attgcgtagt agtaaaaccc tcgcaactgg tacgcggcgc    240
ggccaagccg ttaaaccccc aacttctgaa tgttgacctc ggatcaggta ggaatacccg    300
ctgaacttaa                                                           310
 
           
             8 
             330 
             DNA 
             Mucor rouxii 
           
            8
aaagtgcgat aactagtgtg aattgcatat tcagtgaatc atcgagtctt tgaacgcaac     60
ttgcgctcat tggtattcca atgagcacgc ctgtttcagt atcaaaacaa accctctatc    120
cagcattttg ttgaatagga atactgagag tctcttgatc tattctgatc tcgaacctct    180
tgaaatgtac aaaggcctga tcttgtttaa atgcctgaac ttttttttaa tataaagaga    240
agctcttgcg gtaaactgtg ctggggcctc ccaaataata ctctttttaa atttgatctg    300
aaatcaggcg ggattacccg ctgaacttaa                                     330
 
           
             9 
             328 
             DNA 
             Mucor racemosus 
           
            9
aaagtgcgat aactagtgtg aattgcatat tcagtgaatc atcgagtctt tgaacgcaac     60
ttgcgctcat tggtattcca atgagcacgc ctgtttcagt atcaaaacaa accctctatc    120
caacttttgt tgtataggat tattgggggc ctctcgatct gtatagatct tgaaatccct    180
gaaatttact aaggcctgaa cttgtttaaa tgcctgaact tttttttaat ataaaggaaa    240
gctcttgtaa ttgactttga tggggcctcc caaataaatc tcttttaaat ttgatctgaa    300
atcaggcggg attacccgct gaacttaa                                       328
 
           
             10 
             327 
             DNA 
             Mucor plumbeus 
           
            10
aaagtgcgat aactagtgtg aattgcatat tcagtgaatc atcgagtctt tgaacgcaac     60
ttgcgctcat tggtattcca atgagcacgc ctgtttcagt atcaaaacaa accctctatc    120
caacttttgt tgtataggat tattgggggc ctctcgatct gtatagatct tgaaaccctt    180
gaaatttact aaggcctgaa cttgtttaat gcctgaactt ttttttaata taaaggaaag    240
ctcttgtaat tgactttgat ggggcctccc aaataaatct tttttaaatt tgatctgaaa    300
tcaggtggga ttacccgctg aacttaa                                        327
 
           
             11 
             322 
             DNA 
             Mucor indicus 
           
            11
aaagtgcgat aactagtgtg aattgcatat tcagtgaatc atcgagtctt tgaacgcatc     60
ttgcactcaa tggtattcca ttgagtacgc ctgtttcagt atcaaaaaca acccttattc    120
aaaattcttt ttttgaatag atatgagtgt agcaacctta caagttgaga cattttaaat    180
aaagtcaggc catatcgtgg attgagtgcc gatacttttt taattttgaa aaggtaaagc    240
atgttgatgt ccgctttttg ggcctcccaa ataacttttt aaacttgatc tgaaatcagg    300
tgggattacc cgctgaactt aa                                             322
 
           
             12 
             330 
             DNA 
             Mucor circinelloides f. 
           
            12
aaagtgcgat aactagtgtg aattgcatat tcagtgaatc atcgagtctt tgaacgcaac     60
ttgcgctcat tggtattcca atgagcacgc ctgtttcagt atcaaaacaa accctctatc    120
caacattttt gttgaatagg atgactgaga gtctcttgat ctattctgat ctcgaagctc    180
ttgaaatgta caaaggcctg atcttgtttg aatgcctgaa ctttttttta atataaagag    240
aagctcttgc ggtaaactgt gctggggcct cccaaataac acatctttaa atttgatctg    300
aaatcaggtg ggactacccg ctgaacttaa                                     330
 
           
             13 
             333 
             DNA 
             Rhizopus oryzae 
           
            13
agtgcgataa ctagtgtgaa ttgcatattc agtgaatcat cgagtctttg aacgcagctt     60
gcactctatg gtttttctat agagtacgcc tgcttcagta tcatcacaaa cccacacata    120
acatttgttt atgtggtgat gggtcgcatc gctgttttat tacagtgagc acctaaaatg    180
tgtgtgattt tctgtctggc ttgctaggca ggaatattac gctggtctca ggatcttttt    240
ttttggttcg cccaggaagt aaagtacaag agtataatcc agtaactttc aaactatgat    300
ctgaagtcag gtgggattac ccgctgaact taa                                 333
 
           
             14 
             333 
             DNA 
             Rhizopus oryzae 
           
            14
agtgcgataa ctagtgtgaa ttgcatattc agtgaatcat cgagtctttg aacgcagctt     60
gcactctatg gtttttctat agagtacgcc tgcttcagta tcatcacaaa cccacacata    120
acatttgttt atgtggtaat gggtcgcatc gctgttttat tacagtgagc acctaaaatg    180
tgtgtgattt tctgtctggc ttgctaggca ggaatattac gctggtctca ggatcttttt    240
ctttggttcg cccaggaagt aaagtacaag agtataatcc agcaactttc aaactatgat    300
ctgaagtcag gtgggattac ccgctgaact taa                                 333
 
           
             15 
             348 
             DNA 
             Rhizopus microsporus 
           
            15
aaagtgcgat aactagtgtg aattgcatat tcgtgaatca tcgagtcttt gaacgcagct     60
tgcactctat ggatcttcta tagagtacgc ttgcttcagt atcataacca acccacacat    120
aaaatttatt ttatgtggtg atggacaagc tcggttaaat ttaattatta taccgattgt    180
ctaaaataca gcctctttgt aattttcatt aaattacgaa ctacctagcc atcgtgcttt    240
tttggtccaa ccaaaaaaca tataatctag gggttctgct agccagcaga tattttaatg    300
atctttaact atgatctgaa gtcaagtggg actacccgct gaacttaa                 348
 
           
             16 
             349 
             DNA 
             Rhizopus microsporus 
           
            16
aaagtgcgat aactagtgtg aattgcatat tcgtgaatca tcgagtcttt gaacgcagct     60
tgcactctat ggatcttcta tagagtacgc ttgcttcagt atcataacca acccacacat    120
aaaatttatt ttatgtggtg atggacaagc tcggttaaat ttaattatta taccgattgt    180
ctaaaataca gcctctttgt aattttcatt aaattacgaa ctacctagcc atcgtgcttt    240
tttggtccaa ccaaaaaaca tataatctag gggttctgct agccagcaaa tattttaatg    300
atctttaacc tatgatctga agtcaagtgg gactacccgc tgaacttaa                349
 
           
             17 
             361 
             DNA 
             Rhizopus circinans 
           
            17
aaattgcgat aactagtgtg aattgcattt tcagtgaatc atcgagtctt tgaacgcatc     60
ttgcgctctt gggattcttc cctagagcac acttgcttca gtatcataac aaaaccctca    120
cctaatattt ttttttttta aaaaaaaaat attagagtgg tattggggtc tctttggtaa    180
ttctttgtaa ttataaaagt acccttaaat gtcataaaca ggttagcttt agcttgcctt    240
taaagatctt cttagggtat cattactttt cgtaaatctt taataggcct gtcacataat    300
tctaccctta aatttcttaa accttgatct gaagtcaagt gggagtaccc gctgaactta    360
a                                                                    361
 
           
             18 
             360 
             DNA 
             Rhizopus circinans 
           
            18
aaattgcgat aactagtgtg aattgcattt tcagtgaatc atcgagtctt tgaacgcatc     60
ttgcgctctt gggattcttc cctagagcac acttgcttca gtatcataac aaaaccctca    120
cctaatattt ttttttaaaa aaaaaaaata ttagagtggt attggggtct ctttggtaat    180
tctttgtaat tataaaagta cccttaaatg tcataaacag gttagcttta gcttgccttt    240
aaagatcttc ttagggtatc attacttttc gtaaatcttt aataggcctg tcacataatt    300
ctacccttaa atttcttaaa ccttgatctg aagtcaagtg ggagtacccg ctgaacttaa    360
 
           
             19 
             486 
             DNA 
             Rhizopus stolonifer 
           
            19
aaagtgcgat aactagtgtg aattgcatat tcagtgaatc atcgagtctt tgaacgcaac     60
ttgcactcta tggttttccg taaagtacgc ttgcttcagt atcataaaga ccccatcctg    120
attattattt ttttattaaa ataattaatt ttggagataa taaaaatgag gctctttctt    180
ttcttttttt tttttttaaa aaaaaggggg ggaaagggtc ttttaaaatg ggcaaattct    240
gggtttttta ctaaacctga actcccccca aaaattcaaa aaaaaaaaaa tgggttttac    300
caaatttttt ttttttttct cctttttgtg tagttaatac tctattaaat ttatttactt    360
ggtattataa cgattatgca agaagggaga gaacaaagaa taatgaaaga gagtttttaa    420
ataaattctt ttttcatttt ttcaatcaat gatctgaagt caagtgggat tacccgctga    480
acttaa                                                               486
 
           
             20 
             349 
             DNA 
             Rhizomucor pusillus 
           
            20
aaattgcgaa aagtaatgcg atctgcagcc tttgcgaatc atcgaattct cgaacgcacc     60
ttgcaccctt tggttcatcc attgggtacg tctagttcag tatctttatt aacccctaaa    120
ggtttatttt ttgataaatc tttggatttg cggtgctgat ggattttcat ccgttcaagc    180
tacccgaaca atttgtatgt tgttgaccct tgatatttcc ttgagggctt gcattggtat    240
ctaatttttt accagtgtgc ttcgagatga tcaagtataa aggtcaatca accacaaata    300
aatttcaact atggatctga acttagatgg gattacccgc tgaacttaa                349
 
           
             21 
             425 
             DNA 
             Absidia corymbifera 
           
            21
aaagtgcgat aattattgcg acttgcattc atagcgaatc atcgagttct cgaacgcatc     60
ttgcgcctag tagtcaatct actaggcaca gttgtttcag tatctgcaac taccaatcag    120
ttcaacttgg ttctttgaac ctaagcgagc tggaaatggg cttgtgttga tggcattcag    180
ttgctgtcat ggccttaaat acatttagtc ctaggcaatt ggctttagtc atttgccgga    240
tgtagactct agagtgcctg aggagcaacg acttggttag tgagttcata attccaagtc    300
aatcagtctc ttcttgaact aggtcttaat ctttatggac tagtgagagg atctaacttg    360
ggtcttctct taaaacaaac tcacatctag atctgaaatc aactgagatc acccgctgaa    420
cttaa                                                                425
 
           
             22 
             399 
             DNA 
             Absidia corymbifera 
           
            22
aaagtgcgat aattattgcg acttgcattc atagtgaatc atcgagttct tgaacgcatc     60
ttgcgcctag tagtcaatct actaggcaca gttgtttcag tatctgcatc caccaatcaa    120
cttaaccttt tgtgttgagt tggaactggg cttctagttg atggcattta gttgctgtca    180
tggccttaaa tcaatgtcct aggtgttaga acatctaaca ccggatggaa actttagagc    240
gctttaagag cagcttggtt agtgagttca ataattccaa gcattaagtc ttttaatgaa    300
ctagcttttc tatctatggg acactacttg gagaaatcca agtaaccttt aaactcccat    360
ttagatctga aatcaactga gaccacccgc tgaacttaa                           399
 
           
             23 
             359 
             DNA 
             Cunninghamella elegans 
           
            23
aaatcgcgat atgtaatgtg actgcctata gtgaatcatc aaatctttga aacgcatctt     60
gcaccttatg gtattccata aggtacgtct gtttcagtac cactaataaa tctctctcta    120
tccttgatga tagaaaaaaa aaaaataatt tttactgggc ccggggaatc cttttttttt    180
tttaataaaa aggaccaatt ttggcccaaa aaaaagggtt gaactttttt taccagatct    240
tgcatctagt aaaaacctag tcggctttaa tagattttta ttttctatta agtttatagc    300
cattcttata ttttttaaaa tcttggcctg aaatcagatg ggatacccgc tgaacttaa     359
 
           
             24 
             346 
             DNA 
             Pseudallescheria boydii 
           
            24
aaatgcgata agtaatgtaa attgcaaaat tcagtgaatc atcgaatctt tgaaacgcac     60
attgcgcccg gcagtaatct gccgggcatg cctgtccgag cgtcatttca accctcgaac    120
ctccgtttcc ttagggaagc ctagggtcgg tgttggggcg ctacggcaag tcctcgcaac    180
ccccgtaggc cctgaaatac agtggcggtc ccgccgcggt tgccttctgc gtagtaagtc    240
tcttttgcaa gctcgcattg ggtcccggcg gaggcctgcc gtcaaaccac ctaacaactc    300
cagatggttt gacctcggat caggtagggt tacccgctga acttaa                   346
 
           
             25 
             346 
             DNA 
             Pseudallescheria boydii 
           
            25
gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaaacgca     60
cattgcgccc ggcagtaatc tgccgggcat gcctgtccga gcgtcatttc aaccctcgaa    120
cctccgtttc ctcagggaag ctcagggtcg gtgttggggc gctacggcaa gtcttcgcaa    180
ccctccgtag gccctgaaat acagtggcgg tcccgccgcg gttgccttct gcgtagaagt    240
ctcttttgca agctcgcatt gggtcccggc ggaggcctgc cgtcaaacca cctataactc    300
caaatggttt gacctcggat caggtagggt tacccgctga acttaa                   346
 
           
             26 
             344 
             DNA 
             Scedosporium apiospermum 
           
            26
gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac     60
attgcgcccg gcagtaatct gccgggcatg cctgtccgag cgtcatttca accctcgaac    120
ctccgtttcc tcagggaagc tcagggtcgg tgttggggcg ctacggcgag tcttcgcgac    180
cctccgtagg ccctgaaata cagtggcggt cccgccgcgg ttgccttctg cgtagtaagt    240
ctcttttgca agctcgcatt gggtcccggc ggaggcctgc cgtcaaacca cctataactc    300
cagatggttt gacctcggat caggtaggta cccgctgaac ttaa                     344
 
           
             27 
             343 
             DNA 
             Scedosporium apiospermum 
           
            27
aaatgcgata agtaatgtga attgcagaat tcagtgaatc atcgaatctt tgaacgcaca     60
ttgcgcccgg cagtaatctg ccgggcatgc ctgtccgagc gtcatttcaa ccctcgaacc    120
tccgtttcct cagggaagct cagggtcggt gttggggcgc tacggcgagt cttcgcgacc    180
ctccgtaggc cctgaaatac agtggcggtc ccgccgcggt tgccttctgc gtagtaagtc    240
tcttttgcaa gctcgcattg ggtcccggcg gaggcctgcc gtcaaaccac ctataactcc    300
agatggtttg acctcggatc aggtaggtac ccgctgaact taa                      343
 
           
             28 
             309 
             DNA 
             Penicillium notatum 
           
            28
aaatgcgata cgtaatgtga attgcaaatt cagtgaatca tcgagtcttt gaacgcacat     60
tgcgccccct ggtattccgg ggggcatgcc tgtccgagcg tcattgctgc cctcaagcac    120
ggcttgtgtg ttgggccccg tcctccgatc ccgggggacg ggcccgaaag gcagcggcgg    180
caccgcgtcc ggtcctcgag cgtatggggc tttgtcaccc gctctgtagg cccggccggc    240
gcttgccgat caacccaaat ttttatccag gttgacctcg gatcaggtag ggatacccgc    300
tgaacttaa                                                            309
 
           
             29 
             336 
             DNA 
             Sporothrix schenckii 
           
            29
gaaatgcgat actaatgtga attgcagaat tcagcgaacc atcgaatctt tgaacgcaca     60
ttgcgcccgc cagcattctg gcgggcatgc ctgtccgagc gtcatttccc ccctcacgcg    120
ccccgttgcg cgctggtgtt ggggcgccct ccgcctggcg gggggccccc gaaagcgagt    180
ggcgggccct gtggaaggct ccgagcgcag taccgaacgc atgttctccc ctcgctccgg    240
aggcccccca ggcgccctgc cggtgaaaac gcgcatgacg cgcagctctt tttacaaggt    300
tgacctcgga tcaggtgagg atacccgctg acttaa                              336
 
           
             30 
             18 
             DNA 
             Aspergillus flavus 
           
            30
gcaaatcaat ctttttcc                                                   18
 
           
             31 
             18 
             DNA 
             Aspergillus fumigatus 
           
            31
gaacgcaaat caatcttt                                                   18
 
           
             32 
             18 
             DNA 
             Aspergillus fumigatus 
           
            32
ccgacaccca tctttatt                                                   18
 
           
             33 
             18 
             DNA 
             Aspergillus niger 
           
            33
gacgttatcc aaccattt                                                   18
 
           
             34 
             18 
             DNA 
             Aspergillus terreus 
           
            34
gcatttattt gcaacttg                                                   18
 
           
             35 
             18 
             DNA 
             Aspergillus nidulans 
           
            35
ggcgtctcca accttatc                                                   18
 
           
             36 
             18 
             DNA 
             Mucor rouxii 
           
            36
gaataggaat actgagag                                                   18
 
           
             37 
             15 
             DNA 
             Mucor indicus 
           
            37
gaaacccttg aaatt                                                      15
 
           
             38 
             18 
             DNA 
             Mucor indicus 
           
            38
cgtggattga gtgccgat                                                   18
 
           
             39 
             21 
             DNA 
             Mucor circinelloides f. 
           
            39
aacatttttg tgaataggat g                                               21
 
           
             40 
             15 
             DNA 
             Mucor racemosus 
           
            40
gaaatccctg aaatt                                                      15
 
           
             41 
             18 
             DNA 
             Rhizopus oryzae 
           
            41
gagtataatc cagyaact                                                   18
 
           
             42 
             18 
             DNA 
             Rhizopus circinans 
           
            42
cttagggtat cattactt                                                   18
 
           
             43 
             18 
             DNA 
             Rhizomucor pusillus 
           
            43
tccttgaggg cttgcatt                                                   18
 
           
             44 
             18 
             DNA 
             Rhizopus stolonifer 
           
            44
cttggtatta taacgatt                                                   18
 
           
             45 
             18 
             DNA 
             Pseudallescheria boydii 
           
            45
aagtctcttt tgcaagct                                                   18
 
           
             46 
             18 
             DNA 
             Penicillium notatum 
           
            46
gatcaaccca aattttta                                                   18
 
           
             47 
             18 
             DNA 
             Penicillium marneffei 
           
            47
gggttggtca ccaccata                                                   18
 
           
             48 
             18 
             DNA 
             Penicillium marneffei 
           
            48
tggtcaccac catattta                                                   18
 
           
             49 
             18 
             DNA 
             Fusarium moniliforme 
           
            49
tctagtgacg gtctcgct                                                   18
 
           
             50 
             18 
             DNA 
             Fusarium oxysporum 
           
            50
cgttaattcg cgttcctc                                                   18
 
           
             51 
             21 
             DNA 
             Fusarium solani 
           
            51
ctaacacctc gcaactggag a                                               21
 
           
             52 
             18 
             DNA 
             Cunninghamella elegans 
           
            52
tagtcggctt taatagat                                                   18
 
           
             53 
             18 
             DNA 
             Cunninghamella elegans 
           
            53
tattaagttt atagccat                                                   18
 
           
             54 
             18 
             DNA 
             Cunninghamella elegans 
           
            54
taagtttata gccattct                                                   18
 
           
             55 
             18 
             DNA 
             Absidia corymbifera 
           
            55
gttgctgtca tggcctta                                                   18
 
           
             56 
             18 
             DNA 
             Sporothrix schenckii 
           
            56
gacgcgcagc tcttttta                                                   18
 
           
             57 
             18 
             DNA 
             Rhizopus microsporus 
           
            57
catataatct aggggttc                                                   18
 
           
             58 
             18 
             DNA 
             Aspergillus sp. 
           
            58
cctcgagcgt atggggct                                                   18
 
           
             59 
             18 
             DNA 
             Fusarium sp. 
           
            59
cccaacttct gaatgttg                                                   18
 
           
             60 
             18 
             DNA 
             Mucor sp. 
           
            60
mtggggcctc ccaaataa                                                   18
 
           
             61 
             21 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence B-58 biotin
      probe 
             
           
            61
gaatcatcga rtctttgaac g                                               21