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Aerobiologia

, Volume 23, Issue 1, pp 35–45 | Cite as

Inhibition of quantitative PCR analysis of fungal conidia associated with indoor air particulate matter

  • James J. McDevitt
  • Peter S. J. Lees
  • William G. Merz
  • Kellogg J. SchwabEmail author
Original Paper

Abstract

Environmental samples analyzed by quantitative PCR (qPCR) are subject to interference by inhibitors present in the environment being sampled. A controlled determination of the effect of inhibitors associated with sampling indoor air and the ability of internal standard controls to detect inhibition was carried out by filter collection of air samples followed by spiking of the filters with green fluorescent protein-expressing Aspergillus fumigatus conidia. Microscopic conidial counts were compared with qPCR results and correlated with levels of particulate matter and viable airborne microorganisms. Our data showed that PCR can be inhibited by masses of particulate matter as low as 50 μg and that the amount of inhibition was positively correlated with the mass of particulate (r = 0.75) and the number of non-filamentous organisms (r = 0.73). The use of internal standard DNA identified the presence of inhibitors and indicated the need for additional sample processing or qualification of sample results.

Keywords

Airborne fungi Aspergillus fumigatus Conidia Quantitative PCR Particulate-matter Sample inhibition 

Notes

Acknowledgements

We thank Brian Schofield, The Johns Hopkins University Bloomberg School of Public Health for assistance with the microscopy. This research was supported in part by a Johns Hopkins Center in Urban Environmental Health pilot grant (ES03818) and a Johns Hopkins NIOSH Education and Research Center Pilot Project Research Training Award (T42/CCT31049-09).

References

  1. Alvarez, A. J., Buttner, M. P., Toranzos, G. A., Dvorsky, E. A., Toro, A., Heikes, T. B., Mertikas-Pifer, L. E., & Stetzenbach, L. D. (1994). Use of solid-phase PCR for enhanced detection of airborne microorganisms. Applied and Environmental Microbiology, 60, 374–376.Google Scholar
  2. Alvarez, A. J., Buttner, M. P., & Stetzenbach, L. D. (1995). PCR for bioaerosol monitoring: Sensitivity and environmental interference. Applied and Environmental Microbiology, 61, 3639–3644.Google Scholar
  3. Breysse, P. N., Buckley, T. J., Williams, D., Beck, C. M., Jo, S. J., Merriman, B., Kanchanaraksa, S., Swartz, L. J., Callahan, K. A., Butz, A. M., Rand, C. S., Diette, G. B., Krishnan, J. A., Moseley, A. M., Curtin-Brosnan, J., Durkin, N. B., & Eggleston, P. A. (2005). Indoor exposures to air pollutants and allergens in the homes of asthmatic children in inner-city Baltimore. Environmental Research, 98, 167–176.CrossRefGoogle Scholar
  4. Burge, H. A., Pierson, D. L., Groves T. O., Strawn K. F., & Mishra S. K. (2000). Dynamics of airborne fungal populations in a large office building. Current Microbiology, 40, 10–16.CrossRefGoogle Scholar
  5. 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. Journal of Microbiological Methods, 44, 263–269.CrossRefGoogle Scholar
  6. Courtney, B. C., Smith, M. M., & Henchal, E. A. (1999). Development of internal controls for probe-based nucleic acid diagnostic assays. Analytical Biochemistry, 270, 249–256.CrossRefGoogle Scholar
  7. Cruz-Perez, P., Buttner, M. P., & Stetzenbach, L. D. (2001). Detection and quantitation of Aspergillus fumigatus in pure culture using polymerase chain reaction. Molecular and Cellular Probes, 15, 81–88.CrossRefGoogle Scholar
  8. Griffiths, W. D., & DeCosemo, G. A. L. (1994). The assessment of bioaerosols – a critical review. Journal of Aerosol Science, 25, 1425–1458.CrossRefGoogle Scholar
  9. Haugland, R. A., Vesper, S. J., & Wymer, L. J. (1999). Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with the TaqMan fluorogenic probe system. Molecular and Cellular Probes, 13, 329–340.CrossRefGoogle Scholar
  10. Keswani, J., Kashon, M. L., & Chen, B. T. (2005). Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust. Journal of Environmental Monitoring, 7, 311–318.CrossRefGoogle Scholar
  11. Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W., & Deetz, K. (1995). Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications, 4, 357–362.Google Scholar
  12. Maher, N., Dillon, H. K., Vermund, S. H., & Unnasch, T. R. (2001). Magnetic bead capture eliminates PCR inhibitors in samples collected from the airborne environment, permitting detection of Pneumocystis carinii DNA. Applied and Environmental Microbiology, 67, 449–452.CrossRefGoogle Scholar
  13. McDevitt, J. J., Lees, P. S., Merz, W. G., & Schwab, K. J. (2004). Development of a method to detect and quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples. Mycopathologia, 158, 325–335.CrossRefGoogle Scholar
  14. McDevitt, J. J., Lees, P. S., Merz, W. G., & Schwab, K. J. (2005). Use of green fluorescent protein-expressing Aspergillus fumigatus conidia to validate quantitative PCR analysis of air samples collected on filters. Journal of Occupational and Environmental Hygiene, 2, 633–640.CrossRefGoogle Scholar
  15. Olive, D. M. (1989). Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. Journal of Clinical Microbiology, 27, 261–265.Google Scholar
  16. Roe, J. D., Haugland, R. A., Vesper, S. J., & Wymer, L. J. (2001). Quantification of Stachybotrys chartarum conidia in indoor dust using real time, fluorescent probe-based detection of PCR products. Journal of Exposure Analysis and Environmental Epidemiology, 11, 12–20.CrossRefGoogle Scholar
  17. Sachadyn, P., & Kur, J. (1998). The construction and use of a PCR internal control. Molecular and Cellular Probes, 12, 259–262.CrossRefGoogle Scholar
  18. Schwab, K. J., Neill, F. H., Le Guyader, F., Estes, M. K., & Atmar, R. L. (2001). Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of “Norwalk-like” viruses and hepatitis A virus in stool and shellfish. Applied and Environmental Microbiology, 67, 742–749.CrossRefGoogle Scholar
  19. Spiess, B., Buchheidt, D., Baust, C., Skladny, H., Seifarth, W., Zeilfelder, U., Leib-Mosch, C., Morz, H., & Hehlmann, R. (2003). Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients. Journal of Clinical Microbiology, 41, 1811–1818.CrossRefGoogle Scholar
  20. Wasylnka, J. A., & Moore, M. M. (2002). Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein. Infection and Immunity, 70, 3156–3163.CrossRefGoogle Scholar
  21. Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63, 3741–3751.Google Scholar
  22. Zhou, G., Whong, W. Z., Ong, T., & Chen, B. (2000). Development of a fungus-specific PCR assay for detecting low-level fungi in an indoor environment. Molecular and Cellular Probes, 14, 339–348.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • James J. McDevitt
    • 1
    • 2
  • Peter S. J. Lees
    • 1
  • William G. Merz
    • 3
  • Kellogg J. Schwab
    • 1
    Email author
  1. 1.Department of Environmental Health SciencesThe Johns Hopkins University, Bloomberg School of Public HealthBaltimoreUSA
  2. 2.Department of Environmental HealthThe Harvard School of Public HealthBostonUSA
  3. 3.Department of PathologyThe Johns Hopkins University School of MedicineBaltimoreUSA

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