Abstract:
Fungi and bacteria can be detected and rapidly quantified by using the nucleotide sequences taught here that are specific to the particular species or group of species of fungi or bacteria. Use of the sequences can be made with fluorescent labeled probes, such as in the TaqMan™ system which produces real time detection of polymerase chain reaction (PCR) products. Other methods of detection and quantification based on these sequences include hybridization, conventional PCR or other molecular techniques.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application is a continuation in part of Ser. No. 09/290,990, filed Apr. 14, 1999 now abanonded, which claims priority from provisional application Ser. No. 60/081,773, filed Apr. 15, 1998, the entire contents of both of which are hereby incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a method of identifying and quantifying specific fungi and bacteria using specific DNA sequences, as described and taught herein. These sequences can be used with real time detection of PCR products with a fluorogenic probe system or other molecular approaches like hybridizations. 
     BACKGROUND OF THE INVENTION 
     Fungi and bacteria are the source of or contribute to many health problems including infections, gastroenteritis, ulcers, asthma, allergies and sinusitis. The rapid identification of these microorganisms is critical for diagnosis and treatment. In addition, detecting and/or quantifying these microorganism in the environment may help to prevent adverse health effects. 
     Limitations of Current Technology 
     In order to determine the risk fungi and bacteria pose to human health, it is necessary to know what fungi and bacteria are present and in what numbers. Fungi and bacteria can be ingested, inhaled, or might enter the body through abrasions or punctures. It is important to identify these microorganisms as specifically and as rapidly as possible. Some species of a particular genus are harmless whereas others of the same genus may cause significant health effects. So without knowing precisely what microorganisms are present and in what numbers, it is impossible to evaluate the potential for negative health effects or the establishment of a risk assessment. 
     In the past, the detection and quantitative measurement of fungi and bacteria in samples has been performed either by direct microscopic examination of the collected cells or by growing cells on a suitable medium and identification and enumeration of the resultant colonies. The first method is highly labor intensive and is subject to potential errors in the recognition and positive identification. The second method is both time consuming and subject to significant quantitative inaccuracy. Both methods require extensive experience on the part of the analyst. 
     Some molecular approaches, such as the conventional polymerase chain reaction (PCR) procedure, are subject to inaccuracies due to the difficulty of quantifying the product. This procedure is also relatively slow and requires expertise in molecular biology. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to overcome the aforesaid deficiencies in the prior art. 
     It is an object of the present invention to provide a simple, reliable method for detecting and quantifying some fungi and bacteria by using the nucleotide sequences specific to each species or group of species of fungi and bacteria, as described herein. 
     According to the present invention, fungi and bacteria can be identified and quantified by using a nucleotide sequence specific to the particular species or, in the case of some fungi, group of species. Many methods including using real time, probe-based detection of polymerase chain reaction (PCR) products (e.g. TaqMan™ system) or other methods of detection and quantification including hybridization or conventional PCR could be used with these sequences. 
     Theory 
     Each microorganism is unique because of the sequence of some of the nucleotides in its DNA. However, there are many sequences which are common to more than one organism. There is thus a hierarchy or classification into which all microorganisms can be arranged. The “species” are typically the finest level of distinction that is recognized for separation of different members of a given genus. In the past, species were separated on the basis of morphological or biochemical differences. In order to identify or separate different species on the basis of its DNA sequence, one finds sequences that are unique to a given species but at the same time common to all isolates of a given species. 
     For this invention the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA) of the different fungi were used. For the bacteria, the sequences of unique enzymes were chosen. 
     To apply this invention, a number of possible detection methods are possible. For example, the TaqMan™, 3′-5′ exonuclease assay signals the formation of PCR amplicons by a process involving the nucleolytic degradation of a double-labeled fluorogenic probe that hybridizes to the target template at a site between the two primer recognition sequences (cf. U.S. Pat. No. 5,876,930). The model 7700 automates the detection and quantitative measurement of these signals, which are stoichiometrically related to the quantities of amplicons produced, during each cycle of amplification In addition to providing substantial reductions in the time and labor requirements for PCR analyses, this technology permits simplified and potentially highly accurate quantification of target sequences in the reactions. 
     There are additional systems and other molecular approaches that operate upon essentially the same principal. What is common to all of these technologies is the need for the identification of specific sequences that are unique to the targeted organism but common to all members of the species. The present invention teaches these identifying sequences and gives a description of the practical application of the sequences in the identification and quantification of specific fungi and bacteria. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates the sensitivity of the assay of the present invention. 
     FIG. 2 shows the actual vs. the expected amounts of conidia detected. 
     FIG. 3 shows TaqMan Threshold Responses from ten-fold dilutions of a single DNA extract. 
     FIG. 4 shows  H. pylori  counts per assay plotted against cycle threshold values. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     DNA Extraction 
     Genomic DNA is extracted using standard methods, e.g., the glass bead milling and glass milk adsorption method or any similar procedure of extracting genomic DNA. 
     Reactions are prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Applied Biosystems, Foster City Calif.) by addition of the following components; 12.5 μl of TaqMan Universal Master Mix (a 2×-concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs with UTP, passive reference dye and optimized buffer components, PE Applied Biosystems, Foster City Calif.); 2.5 μl of a mixture of forward and reverse primers (10 nM each); 2.5 μl of 400 nM TaqMan probe; 2.5 μl of 2 mg/ml bovine serum albumin, fraction V (Sigma Chemical, St. Louis, Mo.) and 5 μl of DNA template. 
     For each targeted fungus or bacterium, the appropriate forward primer, reverse primer and probe (Tables 1 and 2) are to be obtained. The probe is labeled with an appropriate set of dyes or other markers for the particular system of measurement being used. 
     For each target species or group of species, a calibrator sample with a known number of conidia or cells is used as a standard. To ensure that the sample matrix does not affect the PCR reaction and, thus the quantitative results, an internal standard is used. Addition of these conidia or cells to both the test and calibrator samples normalize the target species or group for potential sample to sample variability in DNA extraction efficiencies. 
     
       
         
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 List of Fungal Primers and Probes 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   Absidia coerulea / glauca   
               
             
          
           
               
                 Forward Primer NS92F: 
                 5′-CACCGCCCGTCGCTAC (SEQ ID NO:1) 
               
               
                   
               
               
                 Reverse Primer AcoerR1: 
                 5′-TCTAGTTTGCCATAGTTCTCTTCCAG (SEQ ID NO:2) 
               
               
                   
               
               
                 Probe 
                 MucP1: 
                 5′-CCGATTGAATGGTTATAGTGAGCATATGGGATC (SEQ ID NO:3) 
               
               
                   
               
             
          
           
               
                 
                   Absidia corymbifera 
                 
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:4) 
               
               
                   
               
               
                 Reverse Primer 
                 AcoryR1: 5′-GCAAAGCGTTCCGAAGGACA (SEQ ID NO:5) 
               
               
                   
               
               
                 Probe 
                 AcoryP1: 5′-ATGGCACGAGCAAGCATTAGGGACG (SEQ ID NO:6) 
               
               
                   
               
             
          
           
               
                 
                   Acremonium strictum 
                 
               
             
          
           
               
                 Forward Primer 
                 AstrcF1: 5′-CAACCCATTGTGAACTTACCAAAC (SEQ ID NO:7) 
               
               
                   
               
               
                 Reverse Primer 
                 AstrcR1: 5′-CGCCCCTCAGAGAAATACGATT (SEQ ID NO:8) 
               
               
                   
               
               
                 Probe 
                 AstrcP1: 5′-TCAGCGCGCGGTGGCCTC (SEQ ID NO: 9) 
               
               
                   
               
             
          
           
               
                 
                   Alternaria alternata 
                 
               
             
          
           
               
                 Forward Primer 
                 AaltrF1: 5′-GGCGGGCTGGAACCTC (SEQ ID NO:10) 
               
               
                   
               
               
                 Reverse Primer 
                 AltrR1-1: 5′GCAATTACAAAAGGTTTATGTTTGTCGTA (SEQ ID NO:11) 
               
               
                   
               
               
                 Reverse Primer 
                 AaltrR1-2: 5′-TGCAATTACTAAAGGTTTATGTTTGTCGTA (SEQ ID NO:12) 
               
               
                   
               
               
                 Probe 
                 AaltrP1: 5′-TTACAGCCTTGCTGAATTATTCACCCTTGTCTTT (SEQ ID NO:13) 
               
               
                   
               
             
          
           
               
                   Apophysomyces elegans  and  Saksenea vasiformis   
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:14) 
               
               
                   
               
               
                 Reverse Primer 
                 AelegR1: 5′-GACTCGAATGAGTTCTCGCTTC 
                 (SEQ ID NO:15) 
               
               
                   
               
               
                 Probe 
                 AelegP1: 5′-TGGCCAAGACCAGAATATGGGATTGC (SEQ ID NO:16) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus flavus/oryzae 
                 
               
             
          
           
               
                 Forward Primer 
                 AflavF1: 5′-CGAGTGTAGGGTTCCTAGCGA (SEQ ID NO:17) 
               
               
                   
               
               
                 Reverse Primer 
                 AflavR1: 5′-CCGGCGGCCATGAAT 
                 (SEQ ID NO:18) 
               
               
                   
               
               
                 Probe 
                 AtlavP1: 5′-TCCCACCCGTGTTTACTGTACCTTAGTTGCT (SEQ ID NO:19) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus fumigatus, Neosartorya fischeri 
                 
               
             
          
           
               
                 Forward Primer 
                 AfumiF1: 5′-GCCCGCCGTTTCGAC (SEQ ID NO:20) 
               
               
                   
               
               
                 Reverse Primer 
                 AfumiR1: 5′-CCGTTGTTGAAAGTTTTAACTGATTAC (SEQ ID NO:21) 
               
               
                   
               
               
                 Probe 
                 AfumiP1: 5′-CCCGCCGAAGACCCCAACATG (SEQ ID NO:22) 
               
               
                   
               
             
          
           
               
                   Aspergillus niger / foetidus / phoenicus   
               
             
          
           
               
                 Forward Primer 
                 AnigrF1: 5′-GCCGGAGACCCCAACAC-3′ (SEQ ID NO:23) 
               
               
                   
               
               
                 Reverse Primer 
                 AnigrR1: 5′-TGTTGAAAGTTTTAACTGATTGCATT-3′ (SEQ ID NO:24) 
               
               
                   
               
               
                 Probe 
                 AnigrP1: 5′-AATCAACTCAGACTGCACGCTTTCAGACAG (SEQ ID NO:25) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus nomius 
                 
               
             
          
           
               
                 Forward Primer 
                 AflavF1: 5′-CGAGTGTAGGGTTCCTAGCGA-3′ (SEQ ID NO:26) 
               
               
                   
               
               
                 Reverse Primer 
                 AnomiR1: 5′-CCGGCGGCCTTGC-3′ (SEQ ID NO:27) 
               
               
                   
               
               
                 Probe 
                 AflavP1: 5′-TCCCACCCGTGTTTACTGTACCTTAGTTGCT (SEQ ID NO:28) 
               
               
                   
               
             
          
           
               
                   Aspergillus ochraceus / ostianus / auricomus   
               
             
          
           
               
                 Forward Primer 
                 AochrF1: 5′-AACCTCCCACCCGTGTATACC-3′ (SEQ ID NO:29) 
               
               
                   
               
               
                 Reverse Primer 
                 AochrR1: 5′-CCGGCGAGCGCTGTG-3′ 
                 (SEQ ID NO:30) 
               
               
                   
               
               
                 Probe 
                 AochrP1: 5′-ACCTTGTTGCTTCGGCGAGCCC 
                 (SEQ ID NO:31) 
               
               
                   
               
             
          
           
               
                   Aspergillus parasiticus / sojae   
               
             
          
           
               
                 Forward Primer 
                 AflavF1: 5′-CGAGTGTAGGGTTCCTAGCGA-3′ (SEQ ID NO:32) 
               
               
                   
               
               
                 Reverse Primer 
                 AparaR3: 5′-GCCCGGGGCTGACG 
                 (SEQ ID NO:33) 
               
               
                   
               
               
                 Probe 
                 AflavP1: 5′-TCCCACCCGTGTTTACTGTACCTTAGTTGCT (SEQ ID NO:34) 
               
               
                   
               
             
          
           
               
                   Aspergillus restrictus / caesillus / conicus   
               
             
          
           
               
                 Forward Primer 
                 ArestF2: 5′-CGGGCCCGCCTTCAT-3′ (SEQ ID NO:35) 
               
               
                   
               
               
                 Reverse Primer 
                 ArestR1: 5′-GTTGTTGAAAGTTTTAACGATTTTTCT (SEQ ID NO:36) 
               
               
                   
               
               
                 Probe 
                 ArestP1: 5′-CCCGCCGGAGACTCCAACATTG (SEQ ID NO:37) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus sydowii 
                 
               
             
          
           
               
                 Forward Primer 
                 AsydoE1: 5′-CAACCTCCCACCCGTGAA (SEQ ID NO:38) 
               
               
                   
               
               
                 Reverse Primer 
                 versR1: 5′-CCATTGTTGAAAGTTTTGACTGATTTTA (SEQ ID NO:39) 
               
               
                   
               
               
                 Probe 
                 versP1:  5′-AGACTGCATCACTCTCAGGCATGAAGTTCAG (SEQ ID NO:40) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus tamarii 
                 
               
             
          
           
               
                 Forward Primer 
                 AflavF1: 5′-CGAGTGTAGGGTTCCTAGCGA (SEQ ID NO: 41) 
               
               
                   
               
               
                 Reverse Primer 
                 AtamaR1: 5′-CCCGGCGGCCTTAA (SEQ ID NO:42) 
               
               
                   
               
               
                 Probe 
                 AflavP1: 5′-TCCCACCCGTGTTTACTGTACCTTAGTTGCT (SEQ ID NO:43) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus terreus 
                 
               
             
          
           
               
                 Forward Primer 
                 AterrFl: 5′-TTACCGAGTGCGGGTCTTTA (SEQ ID NO:44) 
               
               
                   
               
               
                 Reverse Primer 
                 AterrR1: 5′-CGGCGGCCAGCAAC (SEQ ID NO:45) 
               
               
                   
               
               
                 Probe 
                 AterrP1: 5′-AACCTCCCACCCGTGACTATTGTACCTTG (SEQ ID NO:46) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus ustus 
                 
               
             
          
           
               
                 Forward Primer 
                 AustsF1: 5′-GATCATTACCGAGTGCAGGTCT (SEQ ID NO:47) 
               
               
                   
               
               
                 Reverse Primer 
                 AustsR1: 5′-GCCGAAGCAACGTTGGTC (SEQ ID NO:48) 
               
               
                   
               
               
                 Probe 
                 AustsP1: 5′-CCCCCGGGCAGGCCTAACC (SEQ ID NO:49) 
               
               
                   
               
             
          
           
               
                 
                   Aspergillus versicolor 
                 
               
             
          
           
               
                 Forward Primer 
                 AversF2: 5′-CGGCGGGGAGCCCT (SEQ ID NO:50) 
               
               
                   
               
               
                 Reverse Primer 
                 versR1:  5′-CCATTGTTGAAAGTTTTGACTGATTTTA (SEQ ID NO:51) 
               
               
                   
               
               
                 Probe 
                 versP1:  5′-AGACTGCATCACTCTCAGGCATGAAGTTCAG (SEQ ID NO:52) 
               
               
                   
               
             
          
           
               
                 
                   Chaetomium globosum 
                 
               
             
          
           
               
                 Forward Primer 
                 CglobF1: 5′-CCGCAGGCCCTGAAAAG (SEQ ID NO:53) 
               
               
                   
               
               
                 Reverse Primer 
                 CglobR1: 5′-CGCGGCGCGACCA (SEQ ID NO:54) 
               
               
                   
               
               
                 Probe 
                 CglobP1: 5′-AGATGTATGCTACTACGCTCGGTGCGACAG (SEQ ID NO:55) 
               
               
                   
               
             
          
           
               
                 
                   Cladosporium cladosporioides 
                 
               
             
          
           
               
                 Type 1 
                   
               
               
                   
               
               
                 Forward Primer 
                 Cclad1F1: 5′-CATTACAAGTGACCCCGGTCTAAC (SEQ ID NO:56) 
               
               
                   
               
               
                 Reverse Primer 
                 CcladR1:  5′-CGCGGCGCGACCA (SEQ ID NO:57) 
               
               
                   
               
               
                 Probe 
                 CcladP1:  5′-CCGGGATGTTCATAACCCTTTGTTGTCC (SEQ ID NO:58) 
               
               
                   
               
               
                 Type 2 
               
               
                 Forward Primer 
                 Cclad2F1: 5′-TACAAGTGACCCCGGCTACG (SEQ ID NO:59) 
               
               
                   
               
               
                 Reverse Primer 
                 CcladR1:  5′-CCCCGGAGGCAACAGAG (SEQ ID NO:60) 
               
               
                   
               
               
                 Probe 
                 CcladP1: 5′-CCGGGATGTTCATAACCCTTTGTTGTCC (SEQ ID NO:61) 
               
               
                   
               
             
          
           
               
                 
                   Cladosporium herbarum 
                 
               
             
          
           
               
                 Forward Primer 
                 CherbF1: 5′-AAGAACGCCCGGGCTT (SEQ ID NO:62) 
               
               
                   
               
               
                 Reverse Primer 
                 CherbR1:  5′-CGCAAGAGTTTGAAGTGTCCAC (SEQ ID NO:63) 
               
               
                   
               
               
                 Probe 
                 CherbP1:  5′-CTGGTTATTCATAACCCTTTGTTGTCCGACTCTG (SEQ ID NO:64) 
               
               
                   
               
             
          
           
               
                 
                   Cladosporium sphaerospermum 
                 
               
             
          
           
               
                 Forward Primer 
                 CsphaF1:  5′-ACCGGCTGGGTCTTTCG (SEQ ID NO:65) 
               
               
                   
               
               
                 Reverse Primer 
                 CsphaR1:  5′-GGGGTTGTTTTACGGCGTG (SEQ ID NO:66) 
               
               
                   
               
               
                 Probe 
                 CsphaP1:  5′-CCCGCGGCACCCTTTAGCGA (SEQ ID NO:67) 
               
               
                   
               
             
          
           
               
                   Conidiobolus coronatus / incongruus   
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:68) 
               
               
                   
               
               
                 Reverse Primer 
                 ConiR1:   5′-TGACCAAGTTTGACCAATTTCTCTA (SEQ ID NO:69) 
               
               
                   
               
               
                 Probe 
                 ConiP1:   5′-ATGGTTTAGTGAGGCCTCTGGATTTGAAGCTT (SEQ ID NO:70) 
               
               
                   
               
             
          
           
               
                 
                   Cunninghamella elegans 
                 
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:71) 
               
               
                   
               
               
                 Reverse Primer 
                 CunR1: 5′-AATCTAGTTTGCCATAGTTCTCCTCA (SEQ ID NO:72) 
               
               
                   
               
               
                 Probe 
                 CunP1: 5′-TGAATGGTCATAGTGAGCATGTGGGATCTTT (SEQ ID NO:73) 
               
               
                   
               
             
          
           
               
                   Emericella nidulans / rugulosa / quadrilineata   
               
             
          
           
               
                 Forward Primer 
                 AversF1: 5′-CAACCTCCCACCCGTGAC (SEQ ID NO:74) 
               
               
                   
               
               
                 Reverse Primer 
                 AniduR1:  5′-CATTGTTGAAAGTTTTGACTGATTTGT (SEQ ID NO:75) 
               
               
                   
               
               
                 Probe 
                 versP1:   5′-AGACTGCATCACTCTCAGGCATGAAGTTCAG (SEQ ID NO:76) 
               
               
                   
               
             
          
           
               
                   Eurotium amstelodami / chevalieri / herbariorum / rubrum / repens   
               
             
          
           
               
                 Forward Primer 
                 EamstF1:  5′-GTGGCGGCACCATQTCT (SEQ ID NO:77) 
               
               
                   
               
               
                 Reverse Primer 
                 EamstR1:  5′-CTGGTTAAAAAGATTGGTTGCGA (SEQ ID NO:78) 
               
               
                   
               
               
                 Probe 
                 EamstP1:  5′-CAGCTGGACCTACGGGAGCGGG (SEQ ID NO:79) 
               
               
                   
               
             
          
           
               
                 
                   Epicoccum nigrum 
                 
               
             
          
           
               
                 Forward Primer 
                 EnigrF1: 5′-TTGTAGACTTCGGTCTGCTACCTCTT (SEQ ID NO:80) 
               
               
                   
               
               
                 Reverse Primer 
                 EnigrR1:  5′-TGCAACTGCAAAGGGTTTGAAT (SEQ ID NO:81) 
               
               
                   
               
               
                 Probe 
                 EnigrP1:  5′-CATGTCTTTTGAGTACCTTCGTTTCCTCGGC (SEQ ID NO:82) 
               
               
                   
               
             
          
           
               
                   Geotrichum candidum  strain UAMH 7863 
               
             
          
           
               
                 Forward Primer 
                 GeoF1: 5′-GATATTTCTTGTGAATTGCAGAAGTGA (SEQ ID NO:83) 
               
               
                   
               
               
                 Reverse Primer 
                 GeoR1:    5′-TTGATTCGAAATTTTAGAAGAGCAAA (SEQ ID NO:84) 
               
               
                   
               
               
                 Probe 
                 GeoP1:    5′-CAATTCCAAGAGAGAAACAACGCTCAAACAAG (SEQ ID NO:85) 
               
               
                   
               
             
          
           
               
                 Geotrichum candidum 
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:86) 
               
               
                   
               
               
                 Reverse Primer 
                 GcandR1:  5′-AGAAAAGTTGCCCTCTCCAGTT (SEQ ID NO:87) 
               
               
                   
               
               
                 Probe 
                 GeoP2:    5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:88) 
               
               
                   
               
             
          
           
               
                 
                   Geotrichum klebahnii 
                 
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:89) 
               
               
                   
               
               
                 Reverse Primer 
                 GklebR1:  5′-AAAAGTCGCCCTCTCCTGC (SEQ ID NO:90) 
               
               
                   
               
               
                 Probe 
                 GeoP2:    5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:91) 
               
               
                   
               
             
          
           
               
                 
                   Memnoniella echinata 
                 
               
             
          
           
               
                 Forward Primer 
                 StacF4 5′-TCCCAAACCCTTATGTGAACC (SEQ ID NO:92) 
               
               
                   
               
               
                 Reverse Primer 
                 MemR1: 5′-TGTTTATACCACTCAGACGATACTCAAGT (SEQ ID NO:93) 
               
               
                   
               
               
                 Probe 
                 MemP1:    5′-CTCGGGCCCGGAGTCAGGC (SEQ ID NO:94) 
               
               
                   
               
             
          
           
               
                 
                   Mortierella polycephala/wolfii 
                 
               
             
          
           
               
                 Forward Primer 
                 NS92F:    5′-CACCGCCCGTCGCTAC (SEQ ID NO:95) 
               
               
                   
               
               
                 Reverse Primer 
                 MortRl:   5′-TGACCAAGTTTGGATAACTTTTCAG (SEQ ID NO:96) 
               
               
                   
               
               
                 Probe 
                 MortP1:   5′-CTTAGTGAGGCTTTCGGATTGGATCTAGGCA (SEQ ID NO:97) 
               
               
                   
               
             
          
           
               
                 
                   Mucor mucedo 
                 
               
             
          
           
               
                 Forward primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:98) 
               
               
                   
               
               
                 Reverse Primer 
                 MmuceR1:  5′-CTAAATAATCTAGTTTGCCATAGTTTTCG (SEQ ID NO:99) 
               
               
                   
               
               
                 Probe 
                 MucP1:    5′-CCGATTGAATGGTTATAGTGAGCATATGGGATC (SEQ ID NO:100) 
               
               
                   
               
             
          
           
               
                 
                   Mucor 
                 
               
               
                   amphibiorum / circinelloides / heimalis / indicus / mucedo / racemosus / 
               
               
                   ramosissimus  and  Rhizopus   
               
               
                   azygosporus / homothalicus / microsporus / oligosporus / oryzae   
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:101) 
               
               
                   
               
               
                 Reverse Primer 
                 MucR1-1:  5′-CCTAGTTTGCCATAGTTCTCAGCAG (SEQ ID NO:102) 
               
               
                   
               
               
                 Probe 
                 MucP1:    5′-CCGATTGAATGGTTATAGTGAGCATATGGGATC (SEQ ID NO:103) 
               
               
                   
               
             
          
           
               
                 
                   Myrothecium verrucaria/roridum 
                 
               
             
          
           
               
                 Forward Primer 
                 MyroF1: 5′-AGTTTACAAACTCCCAAACCCTTT (SEQ ID NO:104) 
               
               
                   
               
               
                 Reverse Primer 
                 MyroR1:   5′-GTGTCACTCAGAGGAGAAAACCA (SEQ ID NO:105) 
               
               
                   
               
               
                 Probe 
                 MyroP1:   5′-CGCCTGGTTCCGGGCCC (SEQ ID NO:106) 
               
               
                   
               
             
          
           
               
                 
                   Paecilomyces lilacinus 
                 
               
             
          
           
               
                 Forward Primer 
                 PlilaF1:  5′-CCCACTGTGAACCTTACCTCAG (SEQ ID NO:107) 
               
               
                   
               
               
                 Reverse Primer 
                 PlilaR1:  5′-GCTTGTGCAACTCAGAGAAGAAAT (SEQ ID NO:108) 
               
               
                   
               
               
                 Probe 
                 PlilaP1:  5′-CCGCCCGCTGGGCGTAATG (SEQ ID NO:109) 
               
               
                   
               
             
          
           
               
                 
                   Paecilomyces variotii 
                 
               
             
          
           
               
                 Forward Primer 
                 PvariF1:  5′-CCCGCCGTGGTTCAC (SEQ ID NO:110) 
               
               
                   
               
               
                 Forward Primer 
                 PvariF2:  5′-CGAAGACCCCTGGAACG (SEQ ID NO:111) 
               
               
                   
               
               
                 Reverse Primer 
                 PvariR1:  5′-GTTGTTGAAAGTTTTAATTGATTGATTGT (SEQ ID NO:112) 
               
               
                   
               
               
                 Probe 
                 PvariP1:  5′-CTCAGACGGCAACCTTCCAGGCA (SEQ ID NO:113) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium 
                 
               
               
                   aurantiogriseum / polonicum / viridicatum / freii / verrucosum */ hirsutum   
               
             
          
           
               
                 Forward Primer 
                 PauraF1:  5′-CGGGCCCGCCTTTAC (SEQ ID NO:114) 
               
               
                   
               
               
                 Reverse Primer 
                 PauraR1-1: 5′-GAAAGTTTTAAATAATTTATATTTTCACTCAGAGTT (SEQ ID NO:115) 
               
               
                   
               
               
                 Probe 
                 PenP2:    5′-CGCGCCCGCCGAAGACA (SEQ ID NO:116) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium aurantiogriseum/polonicum/viridicatum/freii 
                 
               
             
          
           
               
                 Forward Primer 
                 PauraF2:  5′-ACCGAGTGAGGGCCCTT (SEQ ID NO:117) 
               
               
                   
               
               
                 Reverse Primer 
                 PauraR6:  5′-CCCGGCGGCCAGTA 
                 (SEQ ID NO:118) 
               
               
                   
               
               
                 Probe 
                 PenP3:    5′-TCCAACCTCCCACCCGTGTTTATTT (SEQ ID NO:119) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium brevicompactum*/alberechii 
                 
               
             
          
           
               
                 Forward Primer 
                 PbrevF1:  5′-CCTTGTTGCTTCGGCGA (SEQ ID NO:120) 
               
               
                   
               
               
                 Reverse Primer 
                 PbrevR2:  5′-TCAGACTACAATCTTCAGACAGAGTTCTAA (SEQ ID NO:121) 
               
               
                   
               
               
                 Probe 
                 PbrevP1:  5′-CCTGCCTTTTGGCTGCCGGG (SEQ ID NO:122) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium 
                 
               
               
                   chrysogenum / griseofulvum / glandicola / coprophilum / expansum   
               
               
                 and  Eupenicillium crustaceum / egyptiacum   
               
             
          
           
               
                 Forward Primer 
                 PchryF1:  5′-CGGGCCCGCCTTAAC (SEQ ID NO:123) 
               
               
                   
               
               
                 Reverse Primer 
                 PchryR1-1: 5′-GAAAGTTTTAAATAATTTATATTTTCACTCAGAGTA (SEQ ID NO:124) 
               
               
                   
               
               
                 Reverse Primer 
                 PchryR2-1: 5′-GAAAGTTTTAAATAATTTATATTTTCACTCAGACCA (SEQ ID NO:125) 
               
               
                   
               
               
                 Probe 
                 PenP2: 5′-CGCGCCCGCCGAAGACA (SEQ ID NO:126) 
               
               
                   
               
             
          
           
               
                   Penicillium citrinum / sartoryi / westlingi   
               
             
          
           
               
                 Forward Primer 
                 PcitrF1: 5′-CCGTGTTGCCCGAACCTA (SEQ ID NO:127) 
               
               
                   
               
               
                 Reverse Primer 
                 PcitrR1:   5′-TTGTTGAAAGTTTTAACTAATTTCGTTATAG (SEQ ID NO:128) 
               
               
                   
               
               
                 Probe 
                 PcitrP2: 5′-CCCCTGAACGCTGTCTGAAGTTGCA (SEQ ID NO:129) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium corylophilum 
                 
               
             
          
           
               
                 Forward Primer 
                 PcoryF1: 5′-GTCCAACCTCCCACCCA (SEQ ID NO:130) 
               
               
                   
               
               
                 Reverse Primer 
                 PcoryR3-1: 5′-GCTCAGACTGCAATCTTCAGACTGT (SEQ ID NO:131) 
               
               
                   
               
               
                 Probe 
                 PcoryP1:   5′-CTGCCCTCTGGCCCGCG (SEQ ID NO:132) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium decumbens 
                 
               
             
          
           
               
                 Forward Primer 
                 PdecuF3:   5′-GGCCTCCGTCCTCCTTTG  (SEQ ID NO:133) 
               
               
                   
               
               
                 Reverse Primer 
                 PdecuR3:   5′-AAAAGATTGATGTGTTCGGCAG (SEQ ID NO:134) 
               
               
                   
               
               
                 Probe 
                 PdecuP2:   5′-CGCCGGCCGGACCTACAGAG (SEQ ID NO:135) 
               
               
                   
               
             
          
           
               
                   Penicillium echinulatum / solitum / camembertii / commune / crustosum   
               
             
          
           
               
                 Forward Primer 
                 PchryF1:   5′-CGGGCCCGCCTTAAC (SEQ ID NO:136) 
               
               
                   
               
               
                 Reverse Primer 
                 PauraR1-1: 5′-GAAAGTTTTAAATAATTTATATTTTCACTCAGAGTT (SEQ ID NO:137) 
               
               
                   
               
               
                 Probe 
                 PenP2:     5′-CGCGCCCGCCGAAGACA (SEQ ID NO:138) 
               
               
                   
               
             
          
           
               
                   Penicillium expansum / coprophilum   
               
             
          
           
               
                 Forward Primer 
                 PauraF2:   5′-ACCGAGTGAGGGCCCTT (SEQ ID NO:139) 
               
               
                   
               
               
                 Reverse Primer 
                 PchryR6:   5′-CCCGGCGGCCAGTT (SEQ ID NO:140) 
               
               
                   
               
               
                 Probe 
                 PenP3:     5′-TCCAACCTCCCACCCGTGTTTATTT (SEQ ID NO:141) 
               
               
                   
               
             
          
           
               
                   Penicillium fellutanum / charlesii   
               
             
          
           
               
                 Forward Primer 
                 PfellF1: 5′-AACCTCCCACCCGTGTATACTTA (SEQ ID NO:142) 
               
               
                   
               
               
                 Reverse Primer 
                 PfellR1:   5′-CTTATCGCTCAGACTGCAAGGTA (SEQ ID NO:143) 
               
               
                   
               
               
                 Probe 
                 PfellP1:  CGGTTGCCCCCCGGCG (SEQ ID NO:144) 
               
               
                   
               
             
          
           
               
                   Penicillium janthinellum / raperi   
               
             
          
           
               
                 Forward Primer 
                 PjantF2: 5′-CCCACCCGTGTTTATCATACCTA (SEQ ID NO:145) 
               
               
                   
               
               
                 Reverse Primer 
                 PjantR2:   5′-TTGAAAGTTTTAACTGATTTAGCTAATCG (SEQ ID NO:146) 
               
               
                   
               
               
                 Probe 
                 PjantP2:   5′-TGCAATCTTCAGACAGCGTTCAGGG (SEQ ID NO:147) 
               
               
                   
               
             
          
           
               
                   Penicillium madriti / gladioli   
               
             
          
           
               
                 Forward Primer 
                 PauraF1:   5′-CGGGCCCGCCTTTAC (SEQ ID NO:148) 
               
               
                   
               
               
                 Reverse Primer 
                 PchryR1-1: 5′-GAAAGTTTTAAATAATTTATATTTTCACTCAGAGTA (SEQ ID NO:149) 
               
               
                   
               
               
                 Reverse Primer 
                 PchryR2-1: 5′-GAAAGTTTTAAATAATTTATATTTTCACTCAGACCA (SEQ ID NO:150) 
               
               
                   
               
               
                 Probe 
                 PenP2:     5′-CGCGCCCGCCGAAGACA (SEQ ID NO:151) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium Oxalicum 
                 
               
             
          
           
               
                 Forward Primer 
                 PoxalF1: 5′-GGGCCCGCCTCACG (SEQ ID NO:152) 
               
               
                   
               
               
                 Reverse Primer 
                 PoxalR1: 5′-GTTGTTGAAAGTTTTAACTGATTTAGTCAAGTA (SEQ ID NO:153) 
               
               
                   
               
               
                 Probe 
                 PoxalP1: 5′-ACAAGAGTTCGTTTGTGTGTCTTCGGCG (SEQ ID NO:154) 
               
               
                   
               
             
          
           
               
                 
                   Penicillium roquefortii 
                 
               
             
          
           
               
                 Forward Primer 
                 PchryF1: 5′-CGGGCCCGCCTTAAC (SEQ ID NO:155) 
               
               
                   
               
               
                 Reverse Primer 
                 ProquR2: 5′-TTAAATAATTTATATTTGTTCTCAGACTGCAT (SEQ ID NO:156) 
               
               
                   
               
               
                 Probe 
                 PenP2: 5′-CGCGCCCGCCGAAGACA (SEQ ID NO:157) 
               
               
                   
               
             
          
           
               
                   Penicillium simplicissimum / ochrochloron   
               
             
          
           
               
                 Forward Primer 
                 PsimpF1-1: 5′-AACCTCCCACCCGTGTTGATT (SEQ ID NO:158) 
               
               
                   
               
               
                 Reverse Primer 
                 PsimpR2-1: 5′-GAGATCCGTTGTTGAAAGTTTTATCTG (SEQ ID NO:159) 
               
               
                   
               
               
                 Reverse Primer 
                 PsimpR3-1: 5′-GAGATCCGTTGTTGAAAGTTTTAACAG (SEQ ID NO:160) 
               
               
                   
               
               
                 Probe 
                 PsimpP1: 5′-CCGCCTCACGGCCGCC (SEQ ID NO:161) 
               
               
                   
               
             
          
           
               
                   Penicillium spinulosum / glabrum / thomii / pupurescens   
               
               
                 and  Eupenicillium lapidosum   
               
             
          
           
               
                 Forward Primer 
                 PspinF1: 5′-GTACCTTGTTGCTTCGGTGC (SEQ ID NO:162) 
               
               
                   
               
               
                 Reverse Primer 
                 PspinR1: 5′-CGTTGTTGAAAGTTTTAACTTATTTAGTTTAT (SEQ ID NO:163) 
               
               
                   
               
               
                 Probe 
                 PspinP1: 5′-TCCGCGCGCACCGGAG (SEQ ID NO:164) 
               
               
                   
               
             
          
           
               
                   Rhizomucor miehei / pusillus / variabilis   
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:165) 
               
               
                   
               
               
                 Reverse Primer 
                 RmucR1: 5′-GTAGTTTGCCATAGTTCGGCTA (SEQ ID NO:166) 
               
               
                   
               
               
                 Probe 
                 RmucP1: 5′-TTGAATGGCTATAGTGAGCATATGGGAGGCT (SEQ ID NO:167) 
               
               
                   
               
             
          
           
               
                 
                   Rhizopus stolonifer 
                 
               
             
          
           
               
                 Forward Primer 
                 NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:168) 
               
               
                   
               
               
                 Reverse Primer 
                 RstolR1: 5′-GCTTAGTTTGCCATAGTTCTCTAACAA (SEQ ID NO:169) 
               
               
                   
               
               
                 Probe 
                 MucP1: 5′-CCGATTGAATGGTTATAGTGAGCATATGGGATC (SEQ ID NO:170) 
               
               
                   
               
             
          
           
               
                 
                   Scopulariopsis asperula 
                 
               
             
          
           
               
                 Forward Primer 
                 SCbrvF1: 5′-CCCCTGCGTAGTAGATCCTACAT (SEQ ID NO:171) 
               
               
                   
               
               
                 Reverse Primer 
                 SCasprR1: 5′-TCCGAGGTCAAACCATGAGTAA (SEQ ID NO:172) 
               
               
                   
               
               
                 Probe 
                 ScopP1: 5′-TCGCATCGGGTCCCGGCG (SEQ ID NO:173) 
               
               
                   
               
             
          
           
               
                   Scopulariopsis brevicaulis / fusca   
               
             
          
           
               
                 Forward Primer 
                 SCbrvF1: 5′-CCCCTGCGTAGTAGATCCTACAT (SEQ ID NO:174) 
               
               
                   
               
               
                 Reverse Primer 
                 SCbrvR1: 5′-TCCGAGGTCAAACCATGAAATA (SEQ ID NO:175) 
               
               
                   
               
               
                 Probe 
                 ScopP1: 5′-TCGCATCGGGTCCCGGCG (SEQ ID NO:176) 
               
               
                   
               
             
          
           
               
                 
                   Scopulariopsis brumptii 
                 
               
             
          
           
               
                 Forward Primer 
                 SCbrmF1: 5′-CCCCTGCGTAGTAGTAAAACCA (SEQ ID NO:177 
               
               
                   
               
               
                 Reverse Primer 
                 SCbrmR1: 5′-CCGAGGTCAAACATCTTTGG (SEQ ID NO:178) 
               
               
                   
               
               
                 Probe 
                 ScopP1: 5′-TCGCATCGGGTCCCGGCG (SEQ ID NO:179) 
               
               
                   
               
             
          
           
               
                 
                   Scopulariopsis chartarum 
                 
               
             
          
           
               
                 Forward Primer 
                 SCchrF1: 5′-CCCCCTGCGTAGTAGTAAAGC (SEQ ID NO: 180) 
               
               
                   
               
               
                 Reverse Primer 
                 SCchrR1: 5′-TCCGAGGTCAAACCATCAAG (SEQ ID NO:181) 
               
               
                   
               
               
                 Probe 
                 ScopP1: 5′-TCGCATCGGGTCCCGGCG (SEQ ID NO:182) 
               
               
                   
               
             
          
           
               
                 
                   Scopulariopsis sphaerospora 
                 
               
             
          
           
               
                 Forward Primer 
                 SCsphF1: 5′-CCCCCTGCGTAGTAGTTTACAA (SEQ ID NO:183) 
               
               
                   
               
               
                 Reverse Primer 
                 SCsphR1: 5′-CCGAGGTCAAACCATCAAAAG (SEQ ID NO:184) 
               
               
                   
               
               
                 Probe 
                 ScopP1: 5′-TCGCATCGGGTCCCGGCG (SEQ ID NO:185) 
               
               
                   
               
             
          
           
               
                 
                   Stachybotrys chartarum 
                 
               
             
          
           
               
                 Forward Primer 
                 StacF4 TCCCAAACCCTTATGTGAACC (SEQ ID NO:186) 
               
               
                   
               
               
                 Reverse Primer 
                 StacRS GTTTGCCACTCAGAGAATACTGAAA (SEQ ID NO:187) 
               
               
                   
               
               
                 Probe 
                 StacP2 CTGCGCCCGGATCCAGGC (SEQ ID NO:188) 
               
               
                   
               
             
          
           
               
                   Trichoderma asperellum / hamatum   
               
             
          
           
               
                 Forward Primer 
                 TasprF1: 5′-CCCAAACCCAATGTGAACGT (SEQ ID NO:189) 
               
               
                   
               
               
                 Reverse Primer 
                 TasprR2-1: 5′-GGACTACAGAAAGAGTTTGGTTGCTT (SEQ ID NO:190) 
               
               
                   
               
               
                 Probe 
                 TridP1: 5′-CCAAACTGTTGCCTCGGCGGG (SEQ ID NO:191) 
               
               
                   
               
             
          
           
               
                   Trichoderma asperellum / hamatuim / viride * 
               
             
          
           
               
                 Forward Primer 
                 TasprF1: 5′-CCCAAACCCAATGTGAACGT (SEQ ID NO:192) 
               
               
                   
               
               
                 Reverse Primer 
                 TasprR1: 5′-TTTGCTCAGAGCTGTAAGAAATACG (SEQ ID NO:193) 
               
               
                   
               
               
                 Probe 
                 TridP1: 5′-CCAAACTGTTGCCTCGGCGGG (SEQ ID NO:194) 
               
               
                   
               
             
          
           
               
                 
                   Trichoderma harzianum 
                 
               
             
          
           
               
                 Forward Primer 
                 TharzF1: 5′-TTGCCTCGGCGGGAT (SEQ ID NO:195) 
               
               
                   
               
               
                 Reverse Primer 
                 TharzR1:   5′-ATTTTCGAAACGCCTACGAGA (SEQ ID NO:196) 
               
               
                   
               
               
                 Probe 
                 TharzP1:   5′-CTGCCCCGGGTGCGTCG (SEQ ID NO:197) 
               
               
                   
               
             
          
           
               
                   Trichoderma longibrachiatum / citroviride   
               
             
          
           
               
                 Forward Primer 
                 TlongF1: 5′-TGCCTCGGCGGGATTC (SEQ ID NO:198) 
               
               
                   
               
               
                 Reverse Primer 
                 TlongR1: 5′-CGAGAAAGGCTCAGAGCAAAAAT (SEQ ID NO:199) 
               
               
                   
               
               
                 Probe 
                 TlongP1: 5′-TCGCAGCCCCGGATCCCA (SEQ ID NO:200) 
               
               
                   
               
             
          
           
               
                   Trichoderma viride */ atroviride / koningii   
               
             
          
           
               
                 Forward Primer 
                 TviriF1: 5′-CCCAAACCCAATGTGAACCA (SEQ ID NO:201) 
               
               
                   
               
               
                 Reverse Primer 
                 TviriR1: 5′-TCCGCGAGGGGACTACAG (SEQ ID NO:202) 
               
               
                   
               
               
                 Probe 
                 TridP1: 5′-CCAAACTGTTGCCTCGGCGGG (SEQ ID NO:203) 
               
               
                   
               
             
          
           
               
                   Ulocladium atrum / chartarum   
               
             
          
           
               
                 Forward Primer 
                 UatrmF1: 5′-GCGGGCTGGCATCCTT (SEQ ID NO:204) 
               
               
                   
               
               
                 Reverse Primer 
                 UatrmR1: 5′-TTGTCCTATGGTGGGCGAA (SEQ ID NO:205) 
               
               
                   
               
               
                 Probe 
                 UloP1: 5′-TGAATTATTCACCCGTGTCTTTTGCGTACTTCT (SEQ ID NO:206) 
               
               
                   
               
             
          
           
               
                 
                   Ulocladium botrytis 
                 
               
             
          
           
               
                 Forward Primer 
                 UbotrF1: 5′-CCCCCAGCAGTGCGTT (SEQ ID NO:207) 
               
               
                   
               
               
                 Reverse Primer 
                 UbotrR1: 5′-CTGATTGCAATTACAAAAGGTTTATG (SEQ ID NO:208) 
               
               
                   
               
               
                 Probe 
                 UloP1: 5′-TGAATTATTCACCCGTGTCTTTTGCGTACTTCT (SEQ ID NO:209) 
               
               
                   
               
             
          
           
               
                 
                   Wallemia sebi 
                 
               
             
          
           
               
                 WsebiF1: 
                 5′-GGCTTAGTGAATCCTTCGGAG (SEQ ID NO:210) 
               
               
                   
               
               
                 WsebiR1: 
                 5′-GTTTACCCAACTTTGCAGTCCA (SEQ ID NO:211) 
               
               
                   
               
               
                 WsebiP1: 
                 5′-TGTGCCGTTGCCGGCTCAAATAG (SEQ ID NO:212) 
               
               
                   
               
             
          
           
               
                 Universal Fungal 
               
               
                 ASSAY 1 
               
               
                   
               
             
          
           
               
                 Forward Primer 
                 5.8F1:  5′-AACTTTCAACAACGGATCTCTTGG (SEQ ID NO:213) 
               
               
                   
               
               
                 Reverse Primer 
                 5.8R1: 5′-GCGTTCAAAGACTCGATGATTCAC (SEQ ID NO:214) 
               
               
                   
               
               
                 Probe 
                 5.8P1:  5′-CATCGATGAAGAACGCAGCGAAATGC (SEQ ID NO:215) 
               
               
                   
               
               
                 ASSAY 2 
               
               
                   
               
               
                 Forward Prime 
                 NS92F:  5′-CACCGCCCGTCGCTAC (SEQ ID NO:216) 
               
               
                   
               
               
                 Reverse Primer 
                 ZygR1:  5′-TAATGATCCTTCCGCAGGTTC (SEQ ID NO:217) 
               
               
                   
               
               
                 Probe 
                 ZygP1:  5′-CCTACGGAAACCTTGTTACGACTTTTACTTCCTCTAAA (SEQ ID NO:218) 
               
               
                   
               
               
                 *Assay does not detect all strains of the indicated species 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 List of Bacterial Primers and Probes 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   Escherichia coli 
                 
                   
               
               
                 Forward Primer 
                 uidAF1: 5′-GGGCAGGCCAGCGTATC (SEQ ID NO:219) 
               
               
                   
               
               
                 Reverse Primer 
                 uidAR1: 5′-CCCACACTTTGCCGTAATGA (SEQ ID NO:220) 
               
               
                   
               
               
                 Reverse Primer 
                 uidAR2: 5′-CGTACACTTTTCCCGGCAAT (SEQ ID NO:221) 
               
               
                   
               
               
                 Probe 
                 uidAP1: 5′-TGCTGCGTTTCGATGCGGTCA (SEQ ID NO:222) 
               
               
                   
               
             
          
           
               
                 
                   Helicobacter pylorii 
                 
               
             
          
           
               
                 Forward Primer 
                 HpylF1: 5′-GGGTATTGAAGCGATGTTTCCT (SEQ ID NO:223) 
               
               
                   
               
               
                 Reverse Primer 
                 HpylR1:  5′-GCTTTTTTGCCTTCGTTGATAGT (SEQ ID NO:224) 
               
               
                   
               
               
                 Probe 
                 HpylP1:  5′-CTCGTAACCGTGCATACCCCTATTGAG (SEQ ID NO:225) 
               
               
                   
               
             
          
         
       
     
     One skilled in the art will appreciate that primers and/or probes can be used which are not identical to the ones described above, as long as there is substantial similarity between the sequences. For purposes of the present invention, “substantial similarity” means that more than 90-110% of the sequence is the same as the sequences enumerated above. 
     Performance of Assay 
     Standard procedures for the operation of the model 7700 or similar detection system are used. This includes, for example with the model 7700, use of all default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisting of two min at 50° C. 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Cycle threshold (C T ) determinations, i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were automatically performed by the instrument for each reaction using default parameters. Assays for fungal target sequences and  G. candidum  (reference) sequences in the same DNA samples are performed in separate reaction tubes. 
     Quantification of Fungal or Bacterial Target 
     Quantification is performed by first subtracting mean reference sequence C T  values from mean target sequence C T  values for both test samples and a pre-specified calibrator sample to obtain ΔC T  values. Calibrator sample ΔC T  values are then subtracted from ΔC T  values of the test samples to obtain ΔΔC T  values. Assuming an amplification efficiency of one (i.e. a doubling of the target sequence for each cycle), the ratio of target sequences in the test and calibrator samples is given by 2 −ΔΔCT . (If the efficiency is less than one, then the new amplification efficiency value is used instead of 2.) For example, a ratio of 0.1, calculated in this manner, would indicate that the target sequence level in the test sample is one-tenth the level found in the calibrator sample. A direct comparison (ΔC T ) approach should allow the discrimination of 1-fold differences in the quantities of target sequences in different samples with 95% confidence. 
     SPECIFIC EXAMPLES 
     Example 1 
     Quantitative Measurement of  Stachybotrys chartarum  conidia using Real Time Detection of PCR Products with the TaqMan™ Fluorogenic Probe System 
     Conidial stocks of the target fungus, e. g.  Stachybotrys chartarum,  and the reference target, e.g.  Geotrichum candidum,  were prepared to act as calibrator and internal standard, respectively. 
     Genomic DNAs were extracted from 20 μl conidial suspensions using a glass bead milling and glass milk adsorption method. Briefly, this method involved mixing test and reference conidia suspensions (10 μl ea.) with 0.3 g of acid-washed glass beads (G-1277; Sigma, St. Louis, Mo.) and 10 μl, 100 μl and 300 μl, respectively, of glass milk suspension, lysis buffer and binding buffer from an Elu-Quik DNA purification kit (Schleicher and Schuell, Keene, N.H.) in sterile 2 ml conical bottom, screw cap tubes (506-636; PGC Scientifics, Gaithersburg, Md.). The tubes were shaken in a mini beadbeater (Biospec Products, Bartlesville, Okla.) for one minute at maximum rate and DNAs were recovered in final volumes of 200 μl distilled water after performing a slight modification of the small-scale protocol provided with the Elu-Quik purification kit. 
     The TaqMan probes and primers were obtained from the custom oligonucleotide synthesis facility at PE-Applied Biosystems (Foster City, Calif.). TaqMan probes contained a TAMRA group conjugated to their 3′-terminal nucleotide and a FAM group linked to their 5′-terminal nucleotides as the quencher and reporter fluorochromes, respectively. For  Geotrichum candidum,  the forward primer is NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:86), the reverse primer is GcandR1: 5′-AGAAAAGTTGCCCTCTCCAGTT (SEQ ID NO:87), and the probe is GeoP2: 5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:88). For  Stachybotrys chartarum,  the forward primer is StacF4 5′-TCCCAAACCCTTATGTGAACC (SEQ ID NO:186), the reverse primer is StacR5 5′-GTTTGCCACTCAGAGAATACTGAAA (SEQ ID NO:187), and the probe is StacP2 5′-CTGCGCCCGGATCCAGGC (SEQ ID NO:188). 
     PCR reactions were prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Applied Biosystems, Foster City Calif.) by addition of the following components: 12.5 μl of TaqMan Universal Master Mix, a 2×concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs with UTP, passive reference dye and optimized buffer components (PE Applied Biosystems, Foster City Calif.); 2.5 μl of a mixture of forward and reverse primers (10 nM each); 2.5 μl of 400 nM TaqMan probe; 2.5 μl of 2 mg/ml bovine serum albumin (fraction V, Sigma Chemical, St. Louis, Mo.) and 5 μl of DNA template. Standard procedures for the operation of the model 7700, as described in the instrument&#39;s manual, were followed. This included the use of all default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisting of two min at 50° C., 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Cycle threshold (C T ) determinations, i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were automatically performed by the instrument for each reaction using default parameters. Assays for  S. chartarum  (target) sequences and  G. candidum  (reference) sequences in the same DNA samples were performed in separate reaction tubes. 
     Quantitation of  S. chartarum  conidia using the comparative C T  method. was performed by first subtracting mean reference sequence C T  values from mean target sequence C T  values for both test samples and a pre-specified calibrator sample to obtain ΔC T  values. Calibrator sample ΔC T  values are then subtracted from ΔC T  values of the test samples to obtain ΔΔC T  values. 
     Calibrator samples were DNA extracts from mixtures of approximately 2×10 4    S. chartarum  (strain UMAH 6417) and 2×10 5    G. candidum  conidia. Test samples were mixed with the same quantity of  G. candidum  conidia prior to DNA extraction. Ratios of target sequences determined in the test and calibrator samples were then multiplied by the known quantities of  S. chartarum  conidia in the calibrator samples to obtain estimates of the absolute quantities of these conidia in the test samples. 
     Each series of DNA extracts was also analyzed using only  S. chartarum  target sequence assay results. In these calculations, calibrator sample C T  values were subtracted directly from corresponding test sample C T  values to obtain ΔC T,STAC  values. These values were used in place of ΔΔC T  values to determine the ratio of target sequences in the test and calibrator samples and to quantify  S. chartarum  conidia in the test samples as described above. 
     Air sampling was performed in rooms that had previously been occupied by infants diagnosed with PH from three homes in the Cleveland, Ohio area. Airborne particles were recovered in sterile BioSampler® vials (SKC Inc., Eighty Four, Pa.) connected to an AirCon-2 High Flow Sampler pump (Gilian Instrument Co., Clearwater, Fla.). Air samples were taken over an eight hour time period under passive conditions (i.e. with no activity occurring in the rooms) at a flow rate of 10 liter per min., for a total collection volume of 4.8 m 3 . Two additional air samples were taken in the same manner over a twelve hour period from the basement of a home in the Cincinnati, Ohio area that was also determined to contain extensive  S. chartarum  growth. One of these samples was collected under passive conditions, as described above, while the other was collected under aggressive sampling conditions (i.e. during and after a cleaning effort in the contaminated area). 
     Each of the BioSampler vessels was rinsed three times with 5 ml of sterile water. The pooled rinses from each vial were transferred to sterile 50 ml capped test tubes (25330-50; Corning Inc., Corning, N.Y.) and centrifuged for 15 min at 1,000×g in a Sorval RC2 centrifuge using the SS-34 rotor (DuPont Instruments, Newton, Conn.). After carefully pipeting off the top 13 to 14 ml of the supernatants, the pelleted materials in each tube were resuspended in the remaining liquid and transferred to 2 ml microfuge tubes (16-8100-27; PGC Scientific, Frederick, Md.). These suspensions were centrifuged at 14,000 rpm for 3 min in an Eppendorf micro-centrifuge (5415C; Brinkman Instruments, Westbury, N.Y.) and the majority of the supernatants were again removed by pipetting. The pellets and small amounts of liquid remaining in each tube were adjusted to either to 100 or 200 μl with sterile water. 
     Direct counts of putative  S. chartarum  conidia in 10 μl aliquots of the recovered samples were made in a hemocytometer chamber. Separate counts of up to six separate aliquots of each sample were taken over the entire grid portion of the chamber and the mean counts were converted to cell concentrations based on the instrument manufacturer&#39;s specified total volume of this portion of the chamber. Presumptive identification and scoring of particles as  S. chartarum  conidia were based on recognition of the characteristic size, shape and pigmentation of these conidia. Three additional 10 μl aliquots of each recovered sample were mixed with  G. candidum  reference conidia and subjected to total genomic DNA extraction for subsequent analysis in the model 7700 as specified above. 
     Yields of target sequences extracted from these conidia samples and from calibrator samples were determined from their respective C T  results in the model 7700 and compared using both the ΔΔC T  (including Geotrichum reference sequence data) and ΔC T,STAC  (not including reference sequence data) versions of the comparative C T  method. Quantities of conidia estimated from these analyses were then compared with those determined from direct microscopic counts of the samples taken in a hemocytometer. 
     Results obtained by the ΔC T,STAC  analysis method and from direct counting showed good agreement for most of the samples (Example 1, FIG.  1 ). In thirteen of these fourteen instances, the estimate of the ΔC T,STAC  method was within a 1-fold range of the direct counting result. The results further indicated that this level of presumed accuracy and precision (i.e. within a 50-200% range of direct counts) may be expected to occur in 95% of all analyses performed by the ΔC T,STAC  method. Based on comparisons of results obtained by the ΔΔC T  analysis method for the same samples (data not shown), it was estimated that this method would provide the same level of accuracy in only about 70% of all analyses. Conidia from each of the different strains examined appeared to be quantified with similar degrees of precision and accuracy using the ΔC T,STAC  analysis method. 
     The sensitivity of the TaqMan assay and the functional dynamic range of the ΔC T,STAC  quantitation method were further examined using ten-fold serial dilutions of  S. chartarum  strain UMAH 6417 conidia stock suspensions as test samples. These samples contained expected quantities of cells that ranged from 2 to 2000, based on direct counting analyses of the starting stock suspensions. The results of these analyses were again in good agreement with the expected results (Example 1, FIG.  2 ). Five of the eight measurements gave estimates that coincided with the expected quantities of conidia in the samples within the relative errors of the analyses. The mean results of these analyses were within a 1-fold range of the expected values in all instances. In one of these two experiments, a low level of signal (equivalent to an estimated mean quantity of 0.27 conidia) was observed in the negative control samples. Parallel samples taken from one of the two dilution series of conidia (cf. experiment 2 in Example 1, FIG. 2) were also subjected to DNA extractions in the absence of Geotrichum cells. Although these extracts yielded slightly lower quantitative results than those obtained for the corresponding samples extracted with the normal amendment of Geotrichum cells, the difference in results was not statistically significant (p=0.35&gt;0.05, data not shown). 
     A final evaluation of the TaqMan assay and ΔC T,STAC  method was made by analyzing particulate samples collected from the inside air of four homes with known colonization by  S. chartarum.  TaqMan-based results were again compared with those obtained by direct microscopic observations of the samples in a hemocytometer. The two methods again gave similar mean determinations of the quantities of  S. chartarum  conidia in these samples with four of the five results agreeing within the relative errors of the TaqMan analyses (Example 1, Table 1). No  S. chartarum  conidia were found in the fifth sample by direct microscopic observation, however, this sample also appeared to approach the detection limits of the TaqMan assay with only two of the three replicate DNA extracts producing signals above background. 
     Example 1, Table 1. Quantification of  S. chartarum  conidia recovered from indoor air samples by direct microscopic counting and the ΔC T,STAC  method as determined from TaqMan analysis. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Quantification of  S. chartarum  conidia recovered from 
               
               
                 indoor air samples by direct microscopic counting and the 
               
               
                 ΔC T,STAC  method as determined from TaqMan analysis. 
               
             
          
           
               
                   
                 Direct Count Estimate 
                 ΔC T,STAC  TaqMan Estimate 
               
             
          
           
               
                 Sample 
                 Sampling 
                   
                   
                 Relative 
               
               
                 Source 
                 Conditions a   
                 Conidia/m 3  air 
                 Conidia/m 3  air 
                 error 
               
               
                   
               
               
                 Home 1 b   
                 Passive 
                  46 c   
                  23 b   
                 7.5-69 
               
               
                 Home 2 b   
                 Passive 
                  15 c   
                  14 c   
                 5.2-37 
               
               
                 Home 3 b   
                 Passive 
                  31 c   
                  26 c   
                 9.4-68 
               
               
                 Home 4 d   
                 Passive 
                   0 e   
                    2.2 e   
                 0.3-19 
               
               
                 Home 4 d   
                 Aggressive 
                 5600 e   
                 4300 e   
                 2600-7300 
               
               
                   
               
               
                   a Defined in materials and methods  
               
               
                   b Located in Cleveland, Ohio  
               
               
                   c Value based on a total air sample volume of 4.8 m 3   
               
               
                   d Located in Cincinnati, Ohio  
               
               
                   e Value based on a total air sample volume of 7.2 m 3   
               
             
          
         
       
     
     Example 2 
     Quantification of Fungus From Dust Using Real Time, Fluorescent Probe-Based Detection of PCR Products 
     Dust samples from the home of an infant with pulmonary hemosiderosis in Cleveland, Ohio (Home 1) were collected using 37-mm filter cassettes, pore size 0.8 μm, as the collection device. Samples were obtained from two rooms in the basement, the living room, and the dining room. Additional dust samples were obtained in a similar manner from the basement of a home in Cincinnati, Ohio (Home 2) containing a significant, but localized, growth of  S. chartarum  as determined by surface sample analysis. One sample was taken from the floor directly beneath the area of growth, a second from another location in the same room and a third from an adjacent room in the basement. All of these dust samples were sieved through a 75 μm mesh and stored in a −20° C. freezer. 
     Total DNAs were extracted from dust samples using glass bead milling and glass milk adsorption method. Weighed dust samples were added directly to sterile 2 ml conical bottom, screw cap tubes (506-636; PGC Scientifics, Gaithersburg, Md.), containing 0.3 g of glass beads (G-1277; Sigma, St. Louis, Mo.) and 100 and 300 μl of lysis and binding buffer, respectively from an Elu-Quik DNA Purification Kit (Schleicher and Schuell, Keene, N.H.). Ten μl aliquots of a 2×10 7  conidia/ml suspension of  G. candidum  in 0.5% Tween 20 were also routinely added to the tubes as a potential source of reference DNA sequences. Ten μl aliquots of  S. chartarum  conidia suspensions in water were also added as needed. The tubes were shaken in a mini beadbeater (Biospec Products, Bartlesville, Ohio) for one minute at a maximum speed. To bind the DNA, 25 μl of Elu-Quik glass milk suspension (Schleicher and Schuell, Keene, HN) was added to the samples and the tubes were placed on a mini-rotating mixer (Glas-Col, Terre Haute, Ind.) for 20 minutes. The samples were transferred to SPIN™ filter and catch tube assemblies (BIO 101, Vista Calif.) and centrifugation at 7500×g for 1.5 min to remove binding and lysis buffers. The retained particulates, including glass milk with adsorbed nucleic acids, were washed twice in the filter cartridges with 0.5 ml Elu-Quik wash buffer and once with 0.5 ml Elu-Quik salt reduction buffer and centrifuged as above after each wash. Nucleic acids were desorbed from the glass milk particles by two successive washes with 100 μl distilled water and collected by centrifuging the washes into clean catch tubes. Calibrator samples, used in the analytical method as standards for the quantification of  S. chartarum  conidia in the test samples, contained 2×10 4    S. chartarum  and 2×10 5    G. candidum  conidia with no dust and DNA extractions from these samples was performed in the same manner. 
     PCR reactions were prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Biosystems, Foster City Calif.). Each reaction contained 12.5 μl of “Universal Master Mix”- a 2×concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs, passive reference dye and optimized buffer components. (PE Biosystems, Foster City Calif.), 0.5 μl of a mixture of forward and reverse primers at 50 mM each, 2.5 μl of 400 nM TaqMan probe (PE Biosystems, Foster City, Calif.), 2.5 μl of 2 mg/ml fraction V bovine serum albumin (Sigma Chemical, St. Louis, Mo.) and 2 μl of autoclaved water. Five μl of purified DNA extract was added to complete the 25 μl reaction mix. 
     The TaqMan probes and primers were obtained from the custom oligonucleotide synthesis facility at PE-Applied Biosystems (Foster City, Calif.). TaqMan probes contained a TAMRA group conjugated to their 3′-terminal nucleotide and a FAM group linked to their 5′-terminal nucleotides as the quencher and reporter fluorochromes, respectively. For  Geotrichum candidum,  the forward primer is NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:86), the reverse primer is GcandRl: 5′-AGAAAAMAAGTTGCCCTCTCCAGTT (SEQ ID NO:87), and the probe is GeoP2: 5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:88). For  Stachybotrys chartarum,  the forward primer is StacF4 5′-TCCCAAACCCTTATGTGAACC (SEQ ID NO:186), the reverse primer is StacR5 5′-GTTTGCCACTCAGAGAATACTGAAA (SEQ ID NO:187), and the probe is StacP2 5′-CTGCGCCCGGATCCAGGC (SEQ ID NO;188). 
     Standard procedures for the operation of the model 7700, as described in the instrument&#39;s manual, were followed using all of the default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisted of 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Cycle threshold (C T ) determinations were automatically performed by the instrument for each assay using default parameters. Assays for  S. chartarum  sequences and  G. candidum  sequences in the same DNA samples were performed in separate reaction tubes. 
     To quantify conidia the mean  S. chartarum  calibrator C T  value was subtracted from the mean  S. chartarum  sequence C T  values in the sample extracts to obtain ΔC T,STAC  values. Ratios of target sequences in the test and calibrator samples were multiplied by the known quantities of  S. chartarum  conidia in the calibrator samples to obtain measurements of the quantities of these conidia in the test samples. similar calculations were performed in parallel using  G. candidum  sequence C T  values from the same calibrator and test samples to determine ΔC T,GEO  values and quantities of these conidia in the test samples. 
     Then  G. candidum  sequence C T  values were subtracted from mean  S. chartarum  sequence C T  values for both test and calibrator sample extracts to obtain ΔC T  values. Calibrator sample ΔC T  values were then subtracted from the test sample ΔC T  values to obtain ΔΔC T  values. These values were used in place of ΔC T,STAC  values to determine the ratios of  S. chartarum  target sequences in the test and calibrator samples and to quantify  S. chartarum  conidia in the test samples as indicated above. 
     Variances of ΔC T  were estimated from the results of the replicate extracts of each sample by: S ΔC     T     2 =S Target   2 +S Ref   2 −2rS Target S ref[ 1], where S Target  and S Ref  are the standard deviations (SD) of the  S. chartarum  and  G. candidum  assay results, respectively, and r is the correlation coefficient between these results. Variances of ΔΔC T  were estimated by S ΔΔC     T     2 =S ΔC     T     (C)   2 +S ΔC     T     (S)   2 [2], where S ΔCT(C)  is given by Equation [1] applied to the calibrator results, and S ΔCT(S) , by Equation [1] applied to the test sample results. Since calibrator and test sample C T  values were independent of one another; variances of ΔC T,STAC  results were estimated by: S ΔC     T,STAC     2 =S Calib   2 +S Target   2 [2], where S Calib  was the SD of the C T  for the calibrator. Variances of ΔC T,GEO  values were calculated in the same manner. Standard errors of difference were determined from the appropriate standard deviation divided by the square root of the number of replicate observations (extractions), and confidence intervals for the differences were constructed using these standard errors. 
     With N 0  representing the number of cells in the calibrator sample, the corresponding cell numbers in test samples were estimated by N 0 2 −ΔY [4], where ΔY was the estimator ΔΔC T , ΔC T,STAC , or ΔC T,GEO . In this paper the term “relative error” refers the range implied by one standard deviation about ΔY, i.e. N 0 2 −ΔY±S     ΔY   , in which S ΔY  is given by equation [2] or [3]. Confidence intervals were constructed around the estimated cell numbers by N 0 2 −ΔY±t·S     ΔY       /{square root over (3)}   , where t is the appropriate Student t-value and three replicate extractions were used. 
     In method evaluation experiments, conidia quantities determined by the ΔΔC T , ΔC T,STAC , or ΔC T,GEO  methods (N T ) were compared to “known” quantities of conidia added to the dust samples. The “known” quantities were determined from hemocytometer cell counts of three replicate aliquots (at least 400 total counts) of the conidia stock suspensions used for dilution and sample amendment. The “known” value for ΔC T  (N H ) was calculated from equation [4] based on the hemocytometer counts and dilution factors, and the differences: d=ΔC T −known value were evaluated via analysis of variance to test the null hypothesis: d=0. The 95% confidence level range for individual observations of d was constructed, assuming d to be normally distributed, and used to characterize the precision of a single estimate utilizing TaqMan quantification. Note that when antilogs are taken, the confidence interval describes lower and upper limits to the ratio N T /N H . 
     The direct enumeration of Stachybotrys conidia in dust samples was performed by weighing dust samples, suspending them in 0.5% Tween 20 to a concentration of 1 mg/ml and, with constant mixing of the suspensions, aliquots were applied to a hemocytometer chamber. Nine replicate aliquots, or fewer if this was sufficient to enumerate at least 400 conidia, were counted in this manner for each suspension. The volumes of the examined grids were used to calculate conidia numbers per ml of suspension and these values converted to numbers per mg of dust. For comparability with relative error of the TaqMan estimates, one standard deviation ranges for direct count estimates were calculated. Conidia were assumed to be randomly distributed within each grid. Under this assumption the corresponding relative error is a range such that the observed count represents an observation one standard deviation above or one standard deviation below a Poisson variable with mean given by the lower or upper limit, respectively. 
     Quantitative measurements of  S. chartarum  conidia in dust samples taken from two contaminated homes were obtained by ΔΔC T  analyses of TaqMan assay results and compared with the results of presumptive direct microscopic enumeration of these conidia. Mean estimates obtained from the TaqMan assays fell within, or very close to the 0.24 to 1.04 range of direct counts that was predicted by the method evaluation experiments (Example 2, Table 1). 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Quantities of  S. chartarum  conidia in home dust samples determined by 
               
               
                 ΔΔC T  TaqMan analysis and direct microscopic enumeration. 
               
             
          
           
               
                 Location in 
                 Proximity to 
                 ΔΔCT TaqMan Estimate 
                 Direct count estimate 
               
             
          
           
               
                 Home 
                 Fungal Growth 
                 Conidia/5 mg dust 
                 Relative Error g   
                 Conidia/5 mg dust 
                 Relative Error g   
               
               
                   
               
             
          
           
               
                 Living Rm 
                 Remote Room d   
                   6 
                  4-10 
                 667 
                  444-1000 
               
               
                 Basement 
                 Same Room 
                  1100 f   
                 — 
                 2333 
                 1877-2901 
               
               
                 Basement 
                 Same Room 
                  9200 
                  7100-12000 
                 26889 
                 25215-28674 
               
               
                 Dining Rm 
                 Remote Room 
                  560 
                 420-740 
                 1444 
                 1096-1904 
               
               
                 Basement 
                 Same Room 
                 23800 
                 18300-31000 
                 30444 
                 28660-32340 
               
               
                 Basement 
                 Adjacent Room 
                  300 
                 260-340 
                 778 
                  534-1133 
               
               
                 Basement 
                 Same Room e   
                 77200 
                  56700-105000 
                 68286 
                 50737-55597 
               
               
                 HVAC 
                   
                   1.7 
                  0.3-11.2 
                 556 
                 357-866 
               
               
                   
               
               
                   a Home 1 located in Cleveland, Ohio  
               
               
                   b Home 2 located in Cincinnati, Ohio  
               
               
                   c Composite HVAC system dust as described in Methods section of text  
               
               
                   d Small amount of fungal growth, no confirmed  Stachybotrys    
               
               
                   e Sample collected directly beneath area of fungal growth  
               
             
          
         
       
     
     Analyses of known numbers of Stachybotrys conidia over a range from 2×10 1  to 2×10 4  in the presence of 10 mg of composite HVAC system dust were found to provide 95% occurrence results within a range from 25% to 104% of expected values using this approach. 
     A second type of matrix effect that can affect PCR-based analyses of dust samples is the influence of PCR inhibitory compounds. Retention of such compounds through the DNA extraction and purification procedures occurred in only one sample in this study. A simple procedure, involving the dilution and re-analysis of a DNA extract from this sample was used to identify this matrix effect and to obtain a corrected estimate of conidia quantities. This procedure should be generally applicable so long as the concentrations of target sequences in the samples are sufficiently high to still be detectable after the inhibitor&#39;s effects are negated by dilution. In practice, however, such follow-up analyses are only likely to be necessary when significant differences are observed in the reference sequence assay results of test and calibrator samples in the initial analyses of the samples. 
     Example 3 
     Evaluation of  Stachybotrys chartarum  in the House of an Infant with Pulmonary Hemorrhage: Quantitative Assessment Before, During and After Remediation 
     Air samples (Example 3, Table 1) were taken in a home under remediation for mold damage in two ways; either using a cassette filter (37 mm with 0.8 mm filter) or with a BioSampler (SKC, Eighty Four, Pa.) connecting to an AirCon-2 High Flow Sampler pump (Gilian Instrument Co., Clearwater, Fla.) calibrated at a flow rate of 10 liter per min. These samples were taken for a period between 6 and 90 hrs at 10 liter per min (L/min). During the remediation process itself, one of the workers wore a personal monitoring pump (PMP) for about 6 h a day which also used a cassette filter (37 mm with 0.8 mm filter). 
     Conidial stocks of the target fungus, i.e.  Stachybotrys chartarum,  and the reference target, i. e.  Geotrichum candidum,  were prepared to act as calibrator and internal standard, respectively. 
     Genomic DNAs were extracted from 20 μl conidial suspensions using a glass bead milling and glass milk adsorption method. Briefly, this method involved mixing test and reference conidia suspensions (10 μl ea.) with 0.3 g of acid-washed glass beads (G-1277; Sigma, St. Louis, Mo.) and 10 μl, 100 μl and 300 μl, respectively, of glass milk suspension, lysis buffer and binding buffer from an Elu-Quik DNA purification kit (Schleicher and Schuell, Keene, N.H.) in sterile 2 ml conical bottom, screw cap tubes (506-636; PGC Scientifics, Gaithersburg, Md.). The tubes were shaken in a mini beadbeater (Biospec Products, Bartlesville, Okla.) for one minute at maximum rate and DNAs were recovered in final volumes of 200 μl distilled water after performing a slight modification of the small-scale protocol provided with the Elu-Quik purification kit. 
     The TaqMan probes and primers were obtained from the custom oligonucleotide synthesis facility at PE-Applied Biosystems (Foster City, Calif.). TaqMan probes contained a TAMRA group conjugated to their 3′-terminal nucleotide and a FAM group linked to their 5′-terminal nucleotides as the quencher and reporter fluorochromes, respectively. For  Geotrichum candidum,  the forward primer is NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:86), the reverse primer is GcandR1: 5′-AGAAAAGTTGCCCTCCAGTT (SEQ ID NO:87), and the probe is GeoP2: 5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:88). For  Stachybotrys chartarum,  the forward primer is StacF4 5′-TCCCAAACCCTTATGTGAACC (SEQ ID NO:186), the reverse primer is StacR5 5′-GTTTGCCACTCAGAGAATACTGAAA (SEQ ID NO:187), and the probe is StacP2 5′-CTGCGCCCGGATCCAGGC (SEQ ID NO:188). 
     PCR reactions were prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Applied Biosystems, Foster City Calif.) by addition of the following components: 12.5 μl of TaqMan Universal Master Mix, a 2×concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs with UTP, passive reference dye and optimized buffer components (PE Applied Biosystems, Foster City Calif.); 2.5 μl of a mixture of forward and reverse primers (10 nM each); 2.5 μl of 400 nM TaqMan probe; 2.5 μl of 2 mg/ml bovine serum albumin (fraction V, Sigma Chemical, St. Louis, Mo.) and 5 μl of DNA template. Standard procedures for the operation of the model 7700, as described in the instrument&#39;s manual, were followed. This included the use of all default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisting of two min at 50° C., 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Cycle threshold (C T ) determinations, i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were automatically performed by the instrument for each reaction using default parameters. Assays for  S. chartarum  (target) sequences and  G. candidum  (reference) sequences in the same DNA samples were performed in separate reaction tubes. 
     Results of air sampling with either filters or BioSamplers indicated that the number of airborne  S. chartarum  spores in this PH house was low before the remediation began (Example 3, Table 1). The number of  S. chartarum  spores in the air, when the furnace blower was activated (typical condition for the winter months), increased by a factor of 17-47 in the living room. During demolition, the number of  S. chartarum  spores in the air increased by four orders of magnitude in the basement, about three orders of magnitude in the dining room and about two orders of magnitude in the upstairs bedroom (Example 3, Table 1). Thus this technology, under actual conditions, can detect the target fungus over four orders of magnitude. 
     
       
         
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Results of air sampling for  S. chartarum  (S.c.) 
               
               
                 spores in the mold contaminated home. 
               
             
          
           
               
                   
                   
                   
                 Sampling 
                   
               
               
                   
                   
                 Location 
                 Time 
                 S.c. Spores 
               
               
                 Date 
                 Sample Method 
                 (Room = RM) 
                 (H) 
                 (#/m 3  air) 
               
               
                   
               
             
          
           
               
                 Pre-remediation 
               
             
          
           
               
                 12/29-30 
                 Filter (passive) 1   
                 Living Rm 
                 25.5 
                 0.2 
               
               
                   
                 BioSampler 
                 Living Rm 
                 25.5 
                 0.3 
               
               
                   
                 (passive) 
               
               
                 12/30-31 
                 Filter 
                 Living Rm 
                 24 
                 9.3 
               
               
                   
                 (active) 2   
               
               
                   
                 BioSampler 
                 Living Rm 
                 24 
                 5.0 
               
               
                   
                 (active) 
               
               
                 12/31 
                 Filter 
                 Dining Rm 
                 90 
                 0.1 
               
               
                   
                 (active) 
               
               
                   
                 Biosampler 
                 Dining rm 
                 90 
                 1.7 
               
               
                   
                 (active) 
               
               
                 12/31-1/4 
                 Filter (active) 
                 Basement 
                 90 
                 0.6 
               
             
          
           
               
                 During Remediation 3   
               
             
          
           
               
                 1-19 
                 Filter 
                 Basement 
                 6.6 
                 1.1 × 10 3   
               
               
                   
                 Biosampler 
                 Basement 
                 6.6 
                 1.6 × 10 3   
               
               
                   
                 Filter PMP 4   
                 Basement 
                 6.5 
                 2.0 × 10 3   
               
               
                 1-20 
                 Filter 
                 Dining Rm 
                 6.25 
                 1.8 × 10 3   
               
               
                   
                 BioSampler 
                 Dining Rm 
                 6.25 
                 2.7 × 10 3   
               
               
                   
                 Filter PMP 
                 Dining Rm 
                 5.75 
                 4.0 × 10 3   
               
               
                 1-21 
                 Filter 
                 N. Bedrm 
                 7.75 
                 0.1 × 10 3   
               
               
                   
                 BioSampler 
                 N. Bedrm 
                 7.75 
                 0.1 × 10 3   
               
               
                   
               
               
                   1 “passive” means furnace blower off, furnace sealed and inoperable.  
               
               
                   2 “active” means furnace blower on, furnace operable.  
               
             
          
         
       
     
     Example 4 
     Identification and Quantification of  Helicobacter pylori    
     Culturing of  Helicobacter pylori  from environmental sources continues to be an obstacle in detecting and enumerating this organism. Selection of primer and probe sequences for the ureA gene was performed based on comparative sequence analyses of 16 strains of  H. pylori  and other Helicobacter species. For  Helicobacter pylorii,  the forward primer is HpylF1: 5′-GGGTATTGAAGCGATGTTTCCT (SEQ ID NO:223), the reverse primer is HpylR1: 5′-GCTTTTTTGCCTTCGTTGATAGT (SEQ ID NO:224), and the probe is HpylR1: 5′-AAACTCGTAACCGTGCATACCCCTATTGAG (SEQ ID NO:225). 
     DNA was extracted from aliquots of ten-fold serial dilutions of  H. pylori  by EluQuick kits from Schleeicher and Schuell, Inc. The cells were lysed, DNA bound to glass beads and washed with alcohol and salt reduction solutions followed by elution from filters with water. One set of extraction tubes contained only  H. pylori.  A second set also received 10 7 /ml  E. coli.  Portions of some DNA extracts were subjected to agarose gel electrophoresis and GelStar staining. Yields of high molecular weight total DNA (appearing as bands on the 1.5% gels) were estimated by comparisons of their fluorescence signals with those of a series of known mass standards (Gibco/BRL) using a model Sl fluorimager (Molecular Dynamics). 
     The more commonly identified non-pylori Helicobacter species were tested with  H. pylori  primers and probe (Example 4, Table 1). Results show that when compared to the negative extraction control all of these species were also negative. All obtained C T  values in the range of 37 to 39. A 40 C T  is the lowest negative value obtainable. Counts of the bacteria were high. They ranged from 10 7  to 10 8  per assay. The  H. pylori  strain also was initially in this range with a C T  value was 15. 
     
       
         
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                   
                   
                 C T   
               
               
                   
                 Bacteria 
                 Dilution 
                 Cells/TaqMan 
                 Values 
               
               
                   
                   
               
             
             
               
                   
                 
                   Campylobacter jejuni 
                 
                 10 0   
                 8.75 × 10 6   
                 36.55 
               
               
                   
                   
                 10 −1   
                 8.75 × 10 5   
                 36.86 
               
               
                   
                   
                 10 −2   
                 8.75 × 10 4   
                 38.78 
               
               
                   
                 
                   Helicobacter felis 
                 
                 10 0   
                  6.8 × 10 5   
                 37.55 
               
               
                   
                   
                 10 −1   
                  6.8 × 10 4   
                 36.17 
               
               
                   
                   
                 10 −2   
                  6.8 × 10 3   
                 38.21 
               
               
                   
                 
                   Helicobacter hepaticus 
                 
                 10 0   
                  2.3 × 10 7   
                 36.68 
               
               
                   
                   
                 10 −1   
                  2.3 × 10 6   
                 37.14 
               
               
                   
                   
                 10 −2   
                  2.3 × 10 5   
                 39.74 
               
               
                   
                 
                   Helicobacter mustelae 
                 
                 10 0   
                  1.9 × 10 8   
                 34.66 
               
               
                   
                   
                 10 −1   
                  1.9 × 10 7   
                 36.37 
               
               
                   
                   
                 10 −2   
                  1.9 × 10 6   
                 37.65 
               
               
                   
                 
                   Helicobacter pylori 
                 
                 10 0   
                  2.1 × 10 7   
                 14.93 
               
               
                   
                   
                 10 −1   
                  2.1 × 10 6   
                 18.23 
               
               
                   
                   
                 10 −2   
                  2.1 × 10 5   
                 21.45 
               
               
                   
                   
                 10 −3   
                  2.1 × 10 4   
                 25.24 
               
               
                   
                   
                 10 −4   
                  2.1 × 10 3   
                 32.73 
               
               
                   
                   
                 10 −5   
                  2.1 × 10 2   
                 34.63 
               
               
                   
                   
                 10 −6   
                  2.1 × 10 1   
                 35.24 
               
               
                   
                   
                 10 −7   
                  2.1 × 10 0   
                 39.58 
               
               
                   
                 Negative Extraction 
                 — 
                 — 
                 37.65 
               
               
                   
                 Control 
               
               
                   
                 Positive Calibrator 
                 10 −1   
                  9.8 × 10 5   
                 17.3  
               
               
                   
                 Control 
               
               
                   
                   
               
             
          
         
       
     
     Samples of serially diluted  H. pylori  cells spanning a 6-log concentration range were subjected to DNA extraction and TaqMan analysis. 
     Estimated cell quantities in the extracted samples ranged from 20 to 2×10 6  based on direct microscopic counts following staining with DAPI. Results from 5 replicate experiments showed a good correlation (r2=0.99) between TaqMan assay results (expressed as cycle threshold values) and the logarithms of expected cell numbers based on direct counts over the entire cell quantity range tested. Similar results were seen for two Helicobacter pylori strains. It was concluded that the TaqMan quantitative PCR method has the potential to provide accurate quantification of  H. pylori  cells in environmental samples. 
     Ten-fold dilutions of a single DNA extract are shown in FIG. 4 along with the corresponding regression analysis. This curve is linear all the way to a negative C T  of 40. The R-squared is 0.999. Counts that correspond to the initial dilution can be extrapolated for the other dilutions and can be included on the X-axis. FIG. 4 shows a linear range from 1.5×10 5  to 1.5 genome equivalents. 
     Example 4, FIG.  4 . Log (base 10) H. pylori  counts per assay are plotted against the cycle threshold values. 
     Conclusions 
     Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing form the spirit and scope of the invention and without undue experimentation. 
     While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the intention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims. 
     REFERENCES 
     Haugland, R. A. and Heckman J. L. 1998. Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species  Stachybotrys chartarum.  Molec. Cellular Probes. 12:387-396. 
     Haugland, R. A, J. L. Heckman, and L. J. Wymer. 1999. Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR. J. Microbiol. Methods. 37:165-176. 
     Haugland, Vesper, Wymer. 1999. Quantitative Measurement of  Stachybotrys chartarum  conidia Using Real Time Detection of PCR Products with the TaqMan™ Fluorogenic Probe System. Molecular and Cellular Probes. Molecular and Cellular Probes. 13:332-340. 
     Vesper, Dearborn, Yike, Allan, Sobolewski, Hinkley, Jarvis, Haugland. 2000. Evaluation of  Stachybotrys chartarum  in the House of an Infant with Pulmonary Hemorrhage: Quantitative Assessment Before, During and After Remediation. Journal of Urban Health. 77:68-85.