Inhibition of quantitative PCR analysis of fungal conidia associated with indoor air particulate matter
- 180 Downloads
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.
KeywordsAirborne fungi Aspergillus fumigatus Conidia Quantitative PCR Particulate-matter Sample inhibition
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).
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63, 3741–3751.Google Scholar