WO2002079512A2

WO2002079512A2 - Method for detection and quantification of the fungus aspergillus fumigatus using quantitative pcr

Info

Publication number: WO2002079512A2
Application number: PCT/US2002/006324
Authority: WO
Inventors: Patricia Cruz-Perez; Mark P. Buttner
Original assignee: NEVADA - LAS VEGAS, University of; University of Nevada Las Vegas
Current assignee: NEVADA - LAS VEGAS, University of; University of Nevada Las Vegas
Priority date: 2001-03-29
Filing date: 2002-02-28
Publication date: 2002-10-10
Anticipated expiration: 2003-09-29
Also published as: WO2002079512A3; AU2002306628A1

Abstract

A method for detecting the fungus Aspergillus fumigatus includes isolating DNA from a sample suspected of containing the fungus Aspergillus fumigatus. The method further includes subjecting the DNA to polymerase chain reaction amplification utilizing at least one primer. The at least one primer includes one of a 5'CGCGTCCGGTCCTCG3' and 5'TTAGAAAAATAAAGTTGGGTGTCGG3' base sequence. The method also includes detecting the fungus Aspergillus fumigatus by visualizing the product of the polymerase chain reaction.

Description

METHOD FOR DETECTION OF

Aspergillus fumigatus

IN PURE CULTURE AND FIELD SAMPLES USING

QUANTITATJNE POLYMERASE CHAIN REACTION RELATED APPLICATION

[0001] This application claims priority under 35 U.S.C. §119 based on U.S. Provisional Application No. 60/280,713 (Attorney Docket No. UNLV 00-02.PA) filed March 29, 2001, the disclosure of which is incorporated herein by reference. GOVERNMENT CONTRACT

[0002] This invention was made with Government support under DE-FG03-98ER62574 awarded by the U.S. Department of Energy. The Government has certain rights in this invention. FIELD OF THE INVENTION

[0003] The present invention relates generally to methods for detecting fungi and, more particularly, to methods for the detection and quantitation of the fungus Aspergillus fumigatus by means of genetic amplification of a specimen. BACKGROUND OF THE INVENTION

[0004] Aspergillus fumigatus is a ubiquitous, thermophilic fungus that grows in damp environments. It is an opportunistic pathogen that affects cystic fϊbrosis patients and the immunocompromised. In addition, it is allergenic and has been implicated in several respiratory diseases. Inhalation oi Aspergillus spores by susceptible individuals has been linked to the development of pulmonary disease. Researchers refer to A. fumigatus as the most common airborne fungal pathogen, and state that A. fumigatus is the predominant Aspergillus species involved in invasive infections. However, it is often underestimated in traditional culture analysis due to its specific growth temperature requirement and the expertise required in speciating members of the genus Aspergillus. Because numerous fungal spores exist in the air and on surfaces both indoors and outdoors, it is advantageous to accurately and rapidly detect organisms such as A. fumigatus in environmental samples. Due to the detection limitations associated with traditional culture analyses, molecular biology techniques can be utilized for the rapid and sensitive detection of target organisms in indoor environments. [0005] Polymerase chain reaction (PCR) is one such molecular biology technique and has been utilized to rapidly and accurately detect airborne virus, bacteria and fungi in indoor environments. PCR detects specific microorganisms by amplifying DNA sequences unique to the organism of interest. To use the PCR technique, sequence information must be identified for a specific target DNA segment. Once a unique DNA sequence has been identified, oligonucleotide primers are designed, synthesized, and then tested for sensitivity, specificity and selectivity. A fluorogenic nuclease assay in conjunction with a sequence detector (ABI PRISM 7700 Sequence Detection System, Applied Biosystems, Foster City, CA, USA) has been , developed as a means to amplify and quantitate PCR products, eliminating the need for post- PCR manipulations for visualization of results. This assay uses a fluorescently labeled oligonucleotide probe that anneals between the primers of choice as the amplification reaction proceeds, allowing for the determination of starting copy number of target DNA. The TaqMan™ assay that is integral to this quantitative technology has been previously validated by other researchers with DNA extracted from Listeria monocytogenes, Mycobacterium tuberculosis, and Salmonella.

[0006] Due to its potential as a health hazard and the importance of a fast diagnosis, several investigators have published on the rapid detection oi A. fumigatus in clinical samples using antigen assays and antibody production in response to these antigens. However, commercially available kits for the detection oi Aspergillus sp. that utilize the latex agglutination principle have the disadvantage of low sensitivity, and enzyme-linked immunosorbent assays can produce false positive results. More recently, researchers have utilized PCR as the detection mechanism for clinical or environmental samples but have targeted the Aspergillus genus rather than the species A. fumigatus.

[0007] Researchers have used post-PCR techniques such as denaturing gradient gel electrophoresis to differentiate A. fumigatus from niger (Reiss, E., K. Tanaka, G. Bruker, V. Chazalet, D. Coleman, P. Debeaupuis, R. Hanazawa, J.-P. Latge, J. Lortholary, K. Makimura, C. Morrison, S.Y. Murayama, S. Naoe, S. Paris, J. Sarfati, K. Shibuya, D. Sullivan, K. Uchida, and H. Yamaguchi. 1998. Molecular diagnosis and epidemiology of fungal infections. Med. Mycol. 36:249-257), or single-strand conformational polymorphism for the differentiation of A. fumigatus iromA.flavus and A. niger (Walsh, T.J., A. Francesconi, M. Kasai, and S.J. Chanock. 1995. PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi. J. Clin. Microbiol. 33:3216-3220). Girardin et al. (Girardin, H., J.-P. Latge, T. Srikantha, B. Morrow, and D.R. Soil. 1993. Development of DNA probes for fingerprinting Aspergillus fumigatus. J. Clin. Microbiol. 31:1547- 1554) used DNA hybridization to detect A. fumigatus, and Bart-Delabesse et al. (Bart-Delabesse, E., J. -F. Humbert, E. Delabesse, and S. Bretagne. 1998. Microsatellite markers for typing Aspergillus fumigatus isolates. J. Clin. Microbiol. 36:2413-2418) utilized microsatellites as PCR markers for differentiating A. fumigatus strains. Advances in the PCR detection of A. fumigatus have recently been reported; however, with identification of the target organism based on analysis of the PCR products by gel electrophoresis (Radford, S.A., E.M. Johnson, J.P. Leeming, M.R.

Millar, J.M. Cornish, A.B.M. Foot, and D.W. Warnock. 1998. Molecular epidemiological study oi Aspergillus fumigatus in a bone marrow transplantation unit by PCR amplification of ribosomal intergenic spacer sequences. J. Clin. Microbiol. 36:1294-1299). SUMMARY OF THE INVENTION [0008] Methods consistent with the invention employ quantitative PCR (QPCR) with novel primers for detecting and quantitating A. fumigatus. Quantitation of samples suspected of containing A. fumigatus, consistent with the invention, may be based on direct comparison to A. fumigatus standards (absolute quantitation). The primer and probe set used in QPCR consistent with the invention may include oligonucleotide primers and a fluorescent probe that were designed from the internal transcribed spacer region ITS2 of the 18S rRNA gene of the species A. fumigatus.

[0009] In accordance with the purpose of the invention as embodied and broadly described herein, a method for detecting the fungus Aspergillus fumigatus includes isolating DNA from a sample suspected of containing the fungus Aspergillus fumigatus; subjecting the DNA to polymerase chain reaction amplification utilizing at least one primer, wherein the at least one primer comprises one of a 5'CGCGTCCGGTCCTCG3' and 5'TTAGAAAAATAAAGTTGGGTGTCGG3' base sequence; and detecting the fungus Aspergillus fumigatus by visualizing the product of the polymerase chain reaction.

[0010] In another exemplary embodiment consistent with the present invention, a primer set for detecting Aspergillus fumigatus using polymerase chain reaction includes a first primer comprising a base sequence 5' CGCGTCCGGTCCTCG3'; and a second primer comprising a base sequence 5'TTAGAAAAATAAAGTTGGGTGTCGG3'.

[0011] In a further exemplary embodiment consistent with the present invention, a primer and probe set for detecting the fungus Aspergillus fumigatus using polymerase chain reaction includes a forward primer comprising a base sequence 5'CGCGTCCGGTCCTCG3'; a reverse primer comprising a base sequence 5' TTAGAAAAATAAAGTTGGGTGTCGG3'; and a probe comprising a base sequence 6-FAM-5' TGTCACCTGCTCTGTAGGCCCG 3'-TAMRA. [0012] In a further exemplary embodiment consistent with the present invention, a primer for use in polymerase chain reaction includes a base sequence comprising at least one of a first and second base sequence, wherein the first base sequence comprises 5' CGCGTCCGGTCCTCG3', and wherein the second base sequence comprises 5' TTAGAAAAATAAAGTTGGGTGTCGG3'.

[0013] In another embodiment consistent with the present invention, a method for detecting the presence of the fungus Aspergillus fumigatus includes obtaining a sample from the environment; extracting DNA from the sample; and amplifying the extracted DNA by polymerase chain reaction utilizing one or more primers to obtain an indication of the presence oi Aspergillus fumigatus in the sample, wherein the one or more primers comprise at least one of a 5' CGCGTCCGGTCCTCG3' and 5' TTAGAAAAATAAAGTTGGGTGTCGG3' base sequence.

[0014] In yet another embodiment consistent with the present invention, a method for identifying and quantifying the presence of the fungus Aspergillus fumigatus in a collected sample includes obtaining a primer set and probe that is specific for the fungal species

Aspergillus fumigatus; collecting the sample from the environment; extracting the collected sample's DNA; obtaining DNA standards from a culture oi Aspergillus fumigatus; determining the concentration oi Aspergillus fumigatus spores in the DNA standards; amplifying by polymerase chain reaction each of the DNA standards and the collected sample's DNA using the obtained primer set and probe; and comparing amplification plots obtained by polymerase chain reaction of each of the DNA standards and the collected sample's DNA to obtain an indication of the presence of the fungus Aspergillus fumigatus in the collected sample and a concentration ■ of the fungus Aspergillus fumigatus in the collected sample. BRIEF DESCRIPTION OF THE DRAWINGS [0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and, together with the description, explain the invention. In the drawings,

[0016] FIG. 1 is a flowchart that illustrates an exemplary process, consistent with the present invention, for preparing A. fumigatus PCR quantitation standards; [0017] FIG 2. is a flowchart that illustrates an exemplary process, consistent with the present invention, for enumerating A. fumigatus quantitation standards for use in quantitative PCR; [0018] FIGS. 3-5 are flowcharts that illustrate an exemplary process, consistent with the present invention, for DNA amplification of a collected sample suspected of containing A. fumigatus using QPCR; and

[0019] FIG. 6 is a plot of PCR cycle value (Ct) versus concentration for several^. fumigatus quantitation standards.

DETAILED DESCRIPTION

[0020] The following detailed description of the invention refers to the accompanying drawings. The same reference numbers in different drawings identify the same or similar elements. Also, the following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims.

[0021] Systems and methods consistent with the present invention permit the absolute quantitation of A. fumigatus in samples by performing QPCR with novel oligonucleotide primers. Through parallel amplifications of reference standards containing A. fumigatus with unknown samples suspected of containing A. fumigatus, an amplification plot of the reference standards can be produced from which the concentration of the unknown samples may be interpolated.

EXEMPLARY QUANTITATION STANDARD PREPARATION PROCESS

[0022] FIG. 1 is a flowchart that illustrates an exemplary process, consistent with the present invention, for preparing A. fumigatus PCR quantitation standards. The quantitation standards prepared in accordance with the exemplary process of FIG. 1 may be enumerated (see

FIG. 2 below) and then employed in QPCR for the absolute quantitation of A. fumigatus in a sample (see FIGS. 3-5 below).

[0023] To begin the illustrated exemplary process, at least four agar plates containing pure cultures oi A. fumigatus may first be obtained (step 105)(FIG. 1). A suitable growth medium such as malt extract agar (MEA, pH 4.7, Difco Laboratories, Detroit, MI, USA) or potato dextrose agar (PDA, pH 5.6, Difco) can be used. The pure cultures oi A. fumigatus can be incubated at 45°C for 2-3 days. The obtained agar plates may then be inverted onto a sterile glass funnel (step 110) and gently tapped to dislodge the spores into a sterile glass bottle (step

115). The spore harvest may be stored dry at 4° C until ready for use (step 120). EXEMPLARY QUANTITATION STANDARD ENUMERATION PROCESS

[0024] FIG. 2 is a flowchart that illustrates an exemplary process, consistent with the present invention, for enumerating quantitation standards for use in quantitative PCR. Enumeration of the quantitation standards prepared, for example, according to the exemplary process described with respect to FIG. 1 above, enables the construction of an amplification plot subsequent to QPCR DNA amplification (see FIGS. 3-5 below). Concentrations oi A. fumigatus in an amplified unknown sample may then be interpolated from the constructed amplification plot.

[0025] To begin the illustrated exemplary enumeration process, several loopfuls of dry spores, prepared according to the exemplary process of FIG. 1, maybe suspended in 3 ml of 0.01 M potassium phosphate buffer with 0.05% Tween 20 (Sigma Chemical Co.)(PBT, pH 7.0) (step 205)(FIG. 2). The resulting spore suspension may be vortexed at maximum speed for, for example, 1 minute (step 210). The spore suspension may then be diluted in filtered Isoton IT solution (Beckman Coulter, Inc., Miami, FL, USA) (step 215). The diluted spore suspension may be enumerated by counting particles in the spore-size range from several 50μL aliquots of the suspension (step 220). The particle count can be corrected using conventional coincidence correction techniques. The spore suspension may be enumerated using, for example a Coulter Multisizer II electronic particle counter that automatically performs coincidence correction of the particle count. The particle count data obtained using the electronic particle counter may be averaged and the concentration of total spores per ml in the spore suspension may be determined (step 225).

[0026] Several aliquots of the original spore suspension of known concentration (10° to 10⁵ spores/PCR reaction) may be serially diluted in PBT (step 230). Each of the several aliquots may be diluted such that they are exponentially separated. For example, each of the dilutions may be diluted using sequential dilution exponents (e.g., 1, 2, 3, 4, etc.). The serially diluted aliquots of the spore suspension may then be stored at -70° C for subsequent DNA extraction

(step 235). EXEMPLARY QUANTITATIVE POLYMERASE CHAIN REACTION PROCESS [0027] FIGS. 3-5 are flowcharts that illustrate an exemplary process for DNA amplification of A. fumigatus in a sample using QPCR, consistent with the present invention.

By employing novel oligonucleotide primers and a fluorescent probe, the exemplary process of

FIGS. 3-5 enables the detection and absolute quantitation oi A. fumigatus in a sample through parallel amplifications of the reference quantitation standards, prepared in accordance with the exemplary process of FIGS. 1-2, with an unknown sample suspected of containing A. fumigatus. [0028] The exemplary process may begin by obtaining a sample of fungus suspected of containing A. fumigatus (step 305)(FIG. 3). The sample may include, for example, a pure culture of a fungus or a field sample. A suitable growth medium such as MEA or PDA may be used. The agar-filled plates are inoculated with the fungal sample and incubated at 23° C for 3-7 days. [0029] The obtained pure culture may be sampled by gently swabbing the surface of the fungal colony with a sterile cotton swab (step 310). The swab may then be suspended in a buffer solution (step 315). The buffer solution may include, for example, 3ml of PBT. The buffer solution, with suspended swab, may be vortexed on maximum speed for one minute, for example, using a conventional vortex mixer (step 320). The suspended swab may be aseptically removed from the buffer solution (step 325). 500μl of the buffer solution containing the spore suspension may then be aliquotted and placed in, for example, a 2 ml microcentrifuge tube for DNA extraction (step 330).

[0030] The 500μl contained in the microcentrifuge tube may be treated with sodium dodecysulfate (0.5% final concentration) and proteinase K (20 μg/ml final concentration) (step 405)(FIG. 4). The treated spore suspension may then be incubated at, for example, 50° C for 10 minutes (step 410). Subsequent to incubation, the spore suspension may be boiled, for example, for 15 minutes to produce a DNA sample (step 415). The DNA sample may then be chilled on ice for, for example, 2 minutes (step 420). Bovine serum albumin (.05% final concentration) may further be added to the chilled DNA sample (step 425). The DNA sample, with bovine serum albumin added, may be incubated for 5 minutes at 37° C in a rotary shaker at, for example, a speed of 225 rpm (step 430). The DNA sample may be maintained at 4° C for immediate purification, or at -70° C for long-term storage (step 435).

[0031] Subsequent to extraction of the DNA from the spore suspension, the DNA sample may be concentrated and purified (step 505)(FIG. 5) using any conventional DNA purification kit in accordance with the manufacturer's instructions. Such kits may include the Pellet Paint™ Co-precipitant kit, (Novagen, Madison, WI, USA), the QIAamp Blood and Tissue kit (QIAGEN, Inc., Valencia, CA, USA), the DNeasy Plant kit (Qiagen, Inc., the Master Pure Plant Leaf DNA Purification kit (Epicentre Technologies, Madison, WI, USA), the EluQuik DNA Purification kit (Schleicher & Schuell, Keene, NH, USA), the Dynabeads DNA DIRECT System I (Dynal, Inc., Lake Success, NY, USA), and the QIAGEN Genomic tip 20/G in conjunction with the Genomic DNA Buffer set (QIAGEN, Inc.).

[0032] After concentration and purification of the spore suspension DNA, a minimum of four serially diluted concentrations of standards, in duplicate with replicate unknown samples, may be amplified by polymerase chain reaction (PCR) (step 510). The ABI Prism 7700 Sequence Detection System (7700 SDS; Applied Biosystems, Foster City, CA, USA) may be used, for example. Amplification conditions using, for example, Applied Biosystems reagents may include the following: fungal DNA template (5μl)

I X TaqMan™ buffer A

5 mM MgCl₂

O.l mM dATP O.l mM dCTP

O.l mM dGTP

0.2 mM dUTP

2.5 U Ampli Taq Gold

0.5 U AmpErase Uracyl N-Glycosylase 0.2 μM of each of the following forward and reverse A. fumigatus primers: forward primer: 5 'CGCGTCCGGTCCTCG3 ' reverse primer: 5 'TTAGAAAAATAAAGTTGGGTGTCGG3 '

0.2 μM of the following A. fumigatus probe: 6-FAM-5 'TGTCACCTGCTCTGTAGGCCCG3 '-TAMRA

IX Exogenous Internal Positive Control mix (IPC with VTC™-labeled probe, Applied Biosystems)

IX IPC DNA for a total reaction volume of 50μl. TaqMan™ cycling conditions may include the following: 2 minutes at 50° C; 10 minutes at 95° C; 40 cycles of 15 seconds at 95° C followed by 1 minute at 60° C

[0033] After amplification, the concentration of the serially diluted A. fumigatus standards maybe designated and a curve of Ct value versus concentration maybe constructed (step 515). The concentration of the serially diluted standards can be designated according to the concentration values determined at steps 225 and 230 above. Ct refers to the PCR cycle number where detectable amplification product is measured. The Ct value is inversely proportional to initial DNA template concentration. An exemplary curve 605 of Ct vs. concentration is illustrated in FIG. 6. Curve 605 depicts plots of the concentrations of each of the A. fumigatus standards (DR1, DR2, DR3, DR4 and DR5) (x-axis) and the Ct value (y-axis), corresponding to each A. fumigatus standard, at which fluorescence is first detected. Concentration values 610 of unknown samples may be interpolated from curve 605 by plotting the PCR cycle (Ct) 615 of the unknown sample where fluorescence is first detected (step 520). The internal positive control may further be analyzed, for example, according to the manufacturer's instructions (step 525). For example, the TaqMan® Exogenous Internal Positive Control Kit (IPC-Vic, Applied Biosystems) contains pre-optimized reagents that, when added to DNA samples for PCR amplification, can distinguish true negatives from false negative results due to inhibition. This kit contains control DNA, primers to amplify this control DNA, and a probe (labeled with the fluorescent dye Vic) that is specific for the control DNA. By adding all IPC kit reagents into the target sample ready for amplification, both the IPC and target DNA should amplify if no inhibitors are present. However, when inhibitors are present the JP C DNA is affected in the same way as the target DNA is affected, showing decreased or negative amplification results and therefore demonstrating the presence of inhibitors in the reaction. EXAMPLES:

TEST ORGANISMS AND CULTURE MEDIA [0034] The fungus A. fumigatus served as the test organism for this study. Forty-three fungal isolates of interest, representing 15 genera, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or from laboratory stocks, and cultured in the laboratory (See Table 1). Malt Extract agar (MEA, pH 4.7, Difco Laboratories, Detroit, MI, USA) and potato dextrose agar (PDA, pH 5.6, Difco) were used for the culture of all fungal species. Aspergillus fumigatus isolates were incubated at 45° C for 2-3 days. All other fungal isolates were incubated at 23° C for 3-7 days.

Table 1. Fungal isolates tested for PCR amplification using Aspergillus fumigatus primers and probe

ATCC, American Type Culture Collection; HRC/UNLV, Harry Reid Center for Environmental Studies, University of Nevada-Las Vegas; Harvard, Harvard School of Public Health, Boston, MA, courtesy of Harriet Burge.

SPORE HARVEST

[0035] Spores were harvested from pure cultures oi A. fumigatus (ATCC 36607) to prepare standards of known concentration. Dry spore harvests were performed by inoculating at least four Petri dishes with A. fumigatus and incubating as described above. The dishes were inverted onto a sterile glass funnel and gently tapped to dislodge the spores into a sterile glass bottle. The spore harvest was stored dry at 4°C until ready for use. PPJMER DESIGN AND PCR AMPLIFICATION

[0036] Aspergillus fumigatus sequences for the 18 S rRNA gene were obtained from GenBank and compared against all other sequences available on-line with the Basic Local Alignment Search Tool algorithm (BLAST, National Center for Biotechnology Information, National institutes of Health). Primers and probes were designed using the Primer Express software (Applied Biosystems) and obtained from commercial sources (Operon Technologies, Alameda, CA, USA; Synthetic Genetics, San Diego, CA, USA).

[0037] The ABI Prism 7700 Sequence Detection System (7700 SDS; Applied Biosystems, Foster City, CA, USA) was used for PCR analysis. Amplification conditions using the Applied Biosystems reagents were as follows: fungal DNA template (5 μl); 1 X TaqMan™ buffer A; 5mM MgCl₂; 0.1 mM dATP; 0.1 mM dCTP; O.lmM dGTP; 0.2 mM dUTP; 2.5 U Ampli Taq Gold; 0.5 U AmpErase Uracyl N-Glycosylase; 0.2 μM each primer (Operon Technologies); 0.2 μM probe (Synthetic Genetics), for a total reaction volume of 50 μl. An internal positive control (TPC-Vic Probe, Applied Biosystems) was incorporated into the PCR reaction to determine whether samples contained PCR inhibitors. The IP C kit contained control DNA, oligonucleotide primers and a fluorescent probe containing a dye different from that of the target DNA probe to allow for the differentiation of fluorescent signals generated during amplification. TaqMan™ cycling-conditions were as follows: 2 min. at 50° C; 10 min. at 95° C; 40 cycles of 15 s at 95° C followed by 1 min. at 60° C. Aspergillus fumigatus (ATCC 36607) DNA was used for testing the A. fumigatus primers. DNA EXTRACTION AND PURIFICATION

[0038] A boiling protocol developed for DNA extraction of bacterial endospores was used for the DNA extraction from A. fumigatus spores. This protocol consisted of treating the spore suspension with sodium dodecylsulfate (0.5% final concentration) and proteinase K (20 μg/ml final concentration), followed by incubation at 50°C for 10 min. and boiling for 15 min. The samples were chilled on ice for 2 min. and bovine serum albumin was added to a final concentration of 0.05%. The samples were incubated for 5 min. at 37°C in a rotary shaker at a speed of 225 rev./min. followed by concentration of A. fumigatus DNA with the Pellet Paint Co- precipitant (Novagen, Madison, WI, USA). Selected samples were further purified with the DNeasy Plant kit (QIAGEN, Inc., Valencia, CA, USA). The concentration and purification protocols were performed following manufacturer's specifications. SPECIFICITY TESTING [0039] For PCR primer specificity testing, pure cultures (see Table 1) were sampled by gently swabbing the surface of the fungal colony with a cotton swab and resuspending the swab in 3ml of 0.01 M phosphate buffer with 0.05% (v/v) Tween 20 (Sigma Chemical Company, St. Louis, MO, USA) (PBT, pH 7.0). After vortexing on maximum speed for 1 min., the swab was removed. Aliquots of 500μl were placed in 2 ml microcentrifuge tubes for subsequent DNA extraction and purification. Samples and aliquots were stored at -70°C Extraction was performed as described above. The extracted DNA samples were subjected to PCR amplification using the designed primers and probe for A. fumigatus. [0040] An ethidium bromide dot quantitation method was utilized for the determination of the presence of DNA in samples prepared for specificity testing. DNA controls were prepared by serial dilutions of a lOObp DNA ladder (Promega, Madison, WI, USA) in TrisEDTA buffer (TE, pH 8.0) to obtain concentrations of 1.3, 13.0 and 130.0 μg/ml. Four microliters of control or sample DNA was combined with an equal volume of ethidium bromide (1 μg/ml, final concentration) and mixed by vortexing. Negative controls were prepared by substituting TE buffer for DNA. Mixed samples were applied as droplets onto a piece of plastic wrap stretched over the surface of an UV transilluminator. A permanent record of the results was obtained by photography with a Polariod MP 4+ Instant Camera System (Fotodyne Inc., Hartland, WI, USA). PCR QUANTITATION STANDARDS AND ANALYSIS

[0041] Quantitation using the 7700 SDS was accomplished by the use of standards of known concentration. Total concentrations oi A. fumigatus (ATCC 36607) spore suspensions were determined using a Coulter Multisizer II electronic particle counter (Beckman Coulter, Inc., Miami, FL, USA). Several loopfuls of the dry A. fumigatus spores were resuspended in 3 ml of PBT and vortexed at maximum speed for 1 min. An aliquot of this spore suspension was diluted in filtered Isoton II solution (Beckman Coulter, Inc.) and enumerated using the Coulter Multisizer II. The data were automatically adjusted for coincidence correction by the instrument and the particles corresponding to the spore peak (1.9 to 3.5μm) were counted. The data from five 50 μl aliquots of the sample were averaged and the concentration of total spores per ml in the spore suspension was determined. Aliquots of the A. fumigatus spore suspension of known concentration (10° to 10⁵ spores/PCR reaction) were serially diluted in PBT and stored at -70°C for DNA extraction. Standards were treated using the same extraction and purification methods used to process samples to more accurately quantify fungal DNA in test samples.

[0042] Standards (10° to 10⁵ templates/reaction) were amplified in duplicate with replicate unknown samples. After amplification, the data were analyzed using the software provided with the 7700 SDS. The concentration of the standards was designated and the software constructed a standard curve of Ct value vs. concentration. Ct refers to the PCR cycle number where detectable amplification product is measured; the Ct value is inversely proportional to initial DNA template concentration. Concentration values for the unknown samples were interpolated from the standard curve by the software and reported as the mean of two replicates. ATCC and laboratory A. fumigatus isolates were enumerated with the Coulter Multisizer II and quantified with the 7700 SDS to test the efficiency of the quantitation standards prepared. RESULTS:

PRIMER SELECTION AND SPECIFICITY [0043] BLAST search results on the internal transcribed spacer regions (ITS1 and ITS2) of the 18 S rRNA gene oi A. fumigatus (GenBank accession # AF138288) revealed a high degree of sequence homology with several species of the genus Penicillium. Two regions unique to A. fumigatus were found at the beginning and the end of the ITS2 region. Primers and probes were designed for the ITS2 region using the Primer Express software (Applied Biosystems). One primer and probe set was selected. The forward primer (5'CGCGTCCGGTCCTCG3') was completely homologous with A. fumigatus and several Penicillium sp., and had one base difference with A. niger. The reverse primer (5'TTAGAAAAATAAAGTTGGGTGTCGG3') was unique for A. fumigatus. The fluorescent probe selected (6'FAM- 5'TGTCACCTGCTCTGTAGGCCCG3'-TAMRA) had one base difference with Penicillium chrysogenum and five-bases difference with the A. niger sequence. This primer set produced an 87-bp amplicon.

[0044] The A. fumigatus primers amplified one ATCC and 14 A. fumigatus laboratory isolates (Table 2). The primers did not amplify fungal DNA extracted from 28 other fungal species (comprising 15 other fungal genera), including eight non-fumigatus Aspergillus species and four Penicillium species. One of 14 A. fumigatus isolates from the Las Vegas area initially showed negative amplification with the A. fumigatus primer set. A 10-fold dilution of this DNA sample produced positive PCR results which indicated the presence of PCR inhibitors. Further purification of this sample through the DNeasy Plant Kit did not remove the PCR inhibitors present.

Table 2. PCR results obtained for the specificity testing oi Aspergillus fumigatus primers.

+, amplification; -, no amplification; *, results following sample dilution; ATCC, American Type Culture Collection; HRC/UNLV, Harry Reid Center for Environmental Studies, University of Nevada-Las Vegas.

[0045] All fungal extracts tested for presence of DNA with the dot quantitation method produced positive results with one exception (data not shown). The A. niger DNA sample, which contained a black pigment, did not emit fluorescence under UN. transillumination; however, a 10-fold dilution of this sample produced positive results. PCR QUANTITATION STANDARDS

[0046] Aspergillus fumigatus (ATCC 36607) PCR quantitation standards (10° to 10⁵ spores/PCR reaction) were prepared from spore suspensions enumerated with the Coulter Multisizer H. The DNA from spore suspensions of known concentration was extracted by the boiling/Pellet Paint protocol developed. The A. fumigatus quantitation standards produced a linear standard curve with a correlation coefficient (r²) of 0.982. QPCR sensitivity using these standards was <20 templates per PCR reaction. ATCC and laboratory A. fumigatus isolates enumerated with the Coulter Multisizer II and quantitated with the 7700 SDS showed comparable enumeration by the two methods (Table 3).

Table 3. QPCR results obtained for Aspergillus fumigatus samples enumerated with the Coulter Multisizer II and quantitated with the 7700 Sequence Detection System.

ATCC and laboratory A. fumigatus isolates were sampled and the DNA was extracted and purified with the developed protocol to test the comparability of the quantitation methods. HRC, Harry Reid Center for Environmental Studies; ATCC strain, American Type Culture Collection strain 36607. PCR ΓNHΓBITION

[0047] An internal positive control (IPC) produced a Ct of 26-27 (Table 4) when amplified alone but a higher Ct when PCR inhibitors were present. A previously amplified A. fumigatus sample (UNLV/HRC 3) produced negative PCR results when undiluted and positive results when diluted. Amplification of this undiluted DNA using the TPC produced negative results of both the sample and the internal positive control, indicating total inhibition. Amplification of 11 of the A. fumigatus isolates using the TPC produced positive results of the undiluted samples but Ct's greater than 26 for the internal positive control, indicating partial inhibition. Inhibitors were removed in one of these samples (UNLV/HRC 4) that was further purified with the DNeasy Plant Kit (Table 4).

Table 4. PCR results oiAspergil lus fumigatus samples amplified with an internal positive

prevent amp ficat on o IPC DNA (Ct of 40 = negat ve resu t). CONCLUSION

[0048] Methods consistent with the present invention, therefore, enable the detection and absolute quantitation of A. fumigatus using QPCR. By employing oligonucleotide primer sequences to amplify A. fumigatus quantitation standards in parallel with unknown samples, an amplification curve can be constructed from which the concentrations oi A. fumigatus in the unknown samples may be interpolated.

[0049] The foregoing description of exemplary embodiments of the present invention provides illustration and description, but is not intended to be exhaustive or to limit the invention to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. Also, while series of steps have been described with regard to FIGS. 1-5, the order of the steps may not be critical.

[0050] No element, act, or instruction used in the description of the present application should be construed as critical or essential to the invention unless explicitly described as such. Also, as used herein, the article "a" is intended to include one or more items. Where only one item is intended, the term "one" or similar language is used. The scope of the invention is defined by the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting the fungus Aspergillus fumigatus, comprising: isolating DNA from a sample suspected of containing the fungus Aspergillus fumigatus; subjecting the DNA to polymerase chain reaction amplification utilizing at least one primer, wherein the at least one primer comprises one of a 5'CGCGTCCGGTCCTCG3' and

5'TTAGAAAAATAAAGTTGGGTGTCGG3' base sequence; and detecting the fungus Aspergillus fumigatus by visualizing the product of the polymerase chain reaction.

2. The method of claim 1 , wherein the subjecting the DNA to polymerase chain reaction further utilizes a probe comprising a base sequence 6-FAM- 5 'TGTCACCTGCTCTGTAGGCCCG3 '-TAMRA.

3. A primer set for detecting Aspergillus fumigatus using polymerase chain reaction, comprising: a first primer comprising a base sequence 5' CGCGTCCGGTCCTCG3'; and a second primer comprising a base sequence 5 'TTAGAAAAATAAAGTTGGGTGTCGG3 ' .

4. The primer set of claim 3, wherein the first primer comprises a forward primer.

5. The primer set of claim 3, wherein the second primer comprises a reverse primer.

6. A primer and probe set for detecting the fungus Aspergillus fumigatus using polymerase chain reaction, comprising: a forward primer comprising a base sequence 5'CGCGTCCGGTCCTCG3'; a reverse primer comprising a base sequence 5' TTAGAAAAATAAAGTTGGGTGTCGG3'; and ' a probe comprising a base sequence 6-FAM-5' TGTCACCTGCTCTGTAGGCCCG 3'- TAMRA.

7. A primer for use in polymerase chain reaction, comprising: a base sequence comprising at least one of a first and second base sequence, wherein the first base sequence comprises 5' CGCGTCCGGTCCTCG3', and wherein the second base sequence comprises 5' TTAGAAAAATAAAGTTGGGTGTCGG3'.

8. A method for detecting the presence of the fungus Aspergillus fumigatus, comprising: obtaining a sample from the environment; extracting DNA from the sample; and amplifying the extracted DNA by polymerase chain reaction utilizing one or more primers to obtain an indication of the presence oi Aspergillus fumigatus in the sample, wherein the one or more primers comprise at least one of a 5' CGCGTCCGGTCCTCG3' and 5' TTAGAAAAATAAAGTTGGGTGTCGG3' base sequence.

9. The method of claim 8, wherein amplifying the sample by polymerase chain reaction further utilizes a probe comprising a base sequence 6-FAM-5 '

TGTCACCTGCTCTGTAGGCCCG3 '-TAMRA.

10. A method for identifying and quantifying the presence of the fungus Aspergillus fumigatus in a collected sample, comprising: obtaining a primer set and probe that is specific for the fungal species Aspergillus fumigatus; collecting the sample from the environment; extracting the collected sample's DNA; obtaining DNA standards from a culture oi Aspergillus fumigatus; determining the concentration oi Aspergillus fumigatus spores in the DNA standards; amplifying by polymerase chain reaction each of the DNA standards and the collected sample's DNA using the obtained primer set and probe; and comparing amplification plots obtained by polymerase chain reaction of each of the DNA standards and the collected sample's DNA to obtain an indication of the presence of the fungus Aspergillus fumigatus in the collected sample and a concentration of the fungus Aspergillus fumigatus in the collected sample.