Development of a method to detect and quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples Article Received: 21 October 2003 Accepted: 16 June 2004 DOI:
Cite this article as: Mcdevitt, J.J., Lees, P.S.J., Merz, W.G. et al. Mycopathologia (2004) 158: 325. doi:10.1007/s11046-004-2304-8 Abstract
Aspergillus fumigatus is linked with respiratory diseases such as asthma, invasive aspergillosis, hypersensitivity pneumonitis, and allergic bronchopulmonary aspergillosis. Molecular methods using quantitative PCR (qPCR) offer advantages over culture and optical methods for estimating human exposures to microbiological agents such as fungi. We describe an assay that uses lyticase to digest A. fumigatus conidia followed by TaqMan™ qPCR to quantify released DNA. This method will allow analysis of airborne A. fumigatus samples collected over extended time periods and provide a more representative assessment of chronic exposure. The method was optimized for environmental samples and incorporates: single tube sample preparation to reduce sample loss, maintain simplicity, and avoid contamination; hot start amplification to reduce non-specific primer/probe annealing; and uracil- N-glycosylase to prevent carryover contamination. An A. fumigatus internal standard was developed and used to detect PCR inhibitors potentially found in air samples. The assay detected fewer than 10 A. fumigatus conidia per qPCR reaction and quantified conidia over a 4−log 10 range with high linearity ( R 2 > 0.99) and low variability among replicate standards (CV=2.0%) in less than 4 h. The sensitivity and linearity of qPCR for conidia deposited on filters was equivalent to conidia calibration standards. A. fumigatus DNA from 8 isolates was consistently quantified using this method, while non-specific DNA from 14 common environmental fungi, including 6 other Aspergillus species, was not detected. This method provides a means of analyzing long term air samples collected on filters which may enable investigators to correlate airborne environmental A. fumigatus conidia concentrations with adverse health effects. Keywords airborne fungi Aspergillus fumigatus conidia filter air monitoring quantitative PCR sample inhibition References Burge, HA, Otten, JA 1999 Fungi Macher, JM eds. Bioaerosols: Assessment and Control. Cincinnati. ACGIH Ohio 19.1 19.13 Google Scholar Wijnands, LM, Deisz, WD, Leusden, FM 2000 Marker antigens to assess exposure to molds and their allergens. I. Aspergillus fumigatus Allergy 55 850 855 PubMed CrossRef Google Scholar
Greenberger PA. Allergic bronchopulmonary aspergillosis. In: Adkinson NF, Ellis EE, Yunginger JW, Reed CE, Busse WW, eds. Allergy: Principles and practice. Mosby-Year Book Inc., 2000: 981–992.
Vernon, DR, Allan, F 1980 Environmental factors in allergic bronchopulmonary aspergillosis. Clin Allergy 10 217 227 PubMed Google Scholar Cruz-Perez, P, Buttner, MP, Stetzenbach, LD 2001 Detection and quantitation of Aspergillus fumigatus in pure culture using polymerase chain reaction Mol Cell Probes 15 81 88 PubMed Google Scholar Denning, DW 1998 Invasive aspergillosis Clin Infect Dis 26 781 803 PubMed Google Scholar Denning, DW 1996 Therapeutic outcome in invasive aspergillosis Clin Infect Dis 23 608 615 PubMed Google Scholar Latge, JP 1999 Aspergillus fumigatus and aspergillosis Clin Microbiol Rev 12 310 350 PubMed Google Scholar Griffiths, WD, DeCosemo, GAL 1994 The assesment of bioaerosols-A critical review J Aerosol Sci 25 1425 1458 Google Scholar Mukoda, TJ, Todd, LA, Sobsey, MD 1994 PCR and gene probes for detecting bioaerosols. J Aerosol Sci 25 1523 1532 Google Scholar Chazalet, V, Debeaupuis, JP, Sarfati, J, Lortholary, J, Ribaud, P, Shah, P, Cornet, M, Vu, TH, Gluckman, E, Brucker, G, Latge, JP 1998 Molecular typing of environmental and patient isolates of Aspergillus fumigatus from various hospital settings J Clin Microbiol 36 1494 1500 PubMed Google Scholar Wu, PC, Su, HJ, Ho, HM 2000 A comparison of sampling media for environmental viable fungi collected in a hospital environment Environ Res 82 253 257 PubMed CrossRef Google Scholar Laitinen, S, Linnainmaa, M, Laitinen, J, Kiviranta, H, Reiman, M, Liesivuori, J 1999 Endotoxins and IgG antibodies as indicators of occupational exposure to the microbial contaminants of metal-working fluids Int Arch Occup Environ Health 72 443 450 PubMed CrossRef Google Scholar Rath, PM, Ansorg, R 1997 Value of environmental sampling and molecular typing of aspergilli to assess nosocomial sources of aspergillosis J Hosp Infect 37 47 53 PubMed CrossRef Google Scholar Leenders, A, Belkum, A, Janssen, S, Marie, S, Kluytmans, J, Wielenga, J, Lowenberg, B, Verbrugh, H 1996 Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward J Clin Microbiol 34 345 351 PubMed Google Scholar Hospenthal, DR, Kwon-Chung, KJ, Bennett, JE 1998 Concentrations of airborne Aspergillus compared to the incidence of invasive aspergillosis: lack of correlation Med Mycol 36 165 168 PubMed CrossRef Google Scholar Thio, CL, Smith, D, Merz, WG, Streifel, AJ, Bova, G, Gay, L, Miller, CB, Perl, TM 2000 Refinements of environmental assessment during an outbreak investigation of invasive aspergillosis in a leukemia and bone marrow transplant unit Infect Control Hosp Epidemiol 21 18 23 PubMed Google Scholar Schwab, KJ, Leon, R, Sobsey, MD 1996 Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR Appl Environ Microbiol 62 2086 2094 PubMed Google Scholar Haugland, RA, Vesper, SJ, Wymer, LJ 1999 Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with the TaqMan (TM)fluorogenic probe system Mol Cell Probes 13 329 340 PubMed CrossRef Google Scholar Kuske, CR, Banton, KL, Adorada, DL, Stark, PC, Hill, KK, Jackson, PJ 1998 Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil Appl Environ Microbiol 64 2463 2472 PubMed Google Scholar Schwab, KJ, Neill, FH, Fankhauser, RL, Daniels, NA, Monroe, SS, Bergmire-Sweat, DA, Estes, MK, Atmar, RL 2000 Development of methods to detect “Norwalk-like viruses” (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak Appl Environ Microbiol 66 213 218 PubMed CrossRef Google Scholar Cruz-Perez, P, Buttner, MP, Stetzenbach, LD 2001 Specific detection of Stachybotrys chartarum in pure culture using quantitative polymerase chain reaction Mol Cell Probes 15 129 138 PubMed Google Scholar Costa, C, Vidaud, D, Olivi, M, Bart-Delabesse, E, Vidaud, M, Bretagne, S 2001 Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum J Microbiol Methods 44 263 269 PubMed CrossRef Google Scholar Roe, JD, Haugland, RA, Vesper, SJ, Wymer, LJ 2001 Quantification of Stachybotrys chartarum conidia in indoor dust using real time, fluorescent probe-based detection of PCR products J Expo Anal Environ Epidemiol 11 12 20 PubMed CrossRef Google Scholar Haugland, RA, Heckman, JL 1998 Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum Mol Cell Probes 12 387 396 PubMed CrossRef Google Scholar Velegraki, A, Kambouris, M, Kostourou, A, Chalevelakis, G, Legakis, NJ 1999 Rapid extraction of fungal DNA from clinical samples for PCR amplification. Med Mycol 37 69 73 PubMed CrossRef Google Scholar Haugland, RA, Heckman, JL, Wymer, LJ 1999 Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis J Microbiol Methods 37 165 176 PubMed CrossRef Google Scholar Muller, FM, Werner, KE, Kasai, M, Francesconi, A, Chanock, SJ, Walsh, TJ 1998 Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption J Clin Microbiol 36 1625 1629 PubMed Google Scholar Burik, JA, Schreckhise, RW, White, TC, Bowden, RA, Myerson, D 1998 Comparison of six extraction techniques for isolation of DNA from filamentous fungi Med Mycol 36 299 303 PubMed Google Scholar Loeffler, J, Hebart, H, Schumacher, U, Reitze, H, Einsele, H 1997 Comparison of different methods for extraction of DNA of fungal pathogens from cultures and blood J Clin Microbiol 35 3311 3312 Google Scholar Loeffler, J, Schmidt, K, Hebart, H, Schumacher, U, Einsele, H 2002 Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system J Clin Microbiol 40 2240 2243 PubMed CrossRef Google Scholar Bir, N, Paliwal, A, Muralidhar, K, Reddy, P, Sarma, PU 1995 A rapid method for the isolation of genomic DNA from Aspergillus fumigatus Prep Biochem 25 171 181 PubMed Google Scholar Haugland, RA, Brinkman, N, Vesper, SJ 2002 Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis J Microbiol Meth 50 319 323 CrossRef Google Scholar Sambrook, J, Russell, DW 2001 Appendix 8. Commonly Used Techniques in Molecular Cloning Argentine, J eds. Molecular Cloning a Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor A8.1 A8.54. Google Scholar Turenne, CY, Sanche, SE, Hoban, DJ, Karlowsky, JA, Kabani, AM 1999 Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system J Clin Microbiol 37 1846 1851 PubMed Google Scholar Loeffler, J, Henke, N, Hebart, H, Schmidt, D, Hagmeyer, L, Schumacher, U, Einsele, H 2000 Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system J Clin Microbiol 38 586 590 PubMed Google Scholar Einsele, H, Hebart, H, Roller, G, Loffler, J, Rothenhofer, I, Muller, CA, Bowden, RA, Burik, J, Engelhard, D, Kanz, L, Schumacher, U 1997 Detection and identification of fungal pathogens in blood by using molecular probes J Clin Microbiol 35 1353 1360 PubMed Google Scholar Peberby, JF 1990 Fungal cell walls—a review Kuhn, PJ Trinci, APJ Jung, MJ Goosey, MW Cooping, LG eds. Biochemistry of Cell Walls and Membranes in Fungi Springer-Verlag Germany 5 30 Google Scholar Nobel, JG, Klis, FM, Munnik, T, Priem, J, Den, EH 1990 An assay of relative cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe Yeast 6 483 490 PubMed Google Scholar Copyright information
© Kluwer Academic Publishers 2004