Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust.
Advances in polymerase chain reaction (PCR) have permitted accurate, rapid and quantitative identification of microorganisms in pure cultures regardless of viability or culturability. In this study, a simple sample processing method was investigated for rapid identification and quantification of fungal spores from dust samples using both conventional and real-time PCR. The proposed method was evaluated for susceptibility to interference from environmental dust samples. Stachybotrys chartarum and Aspergillus fumigatus were used as test organisms. The sensitivity of detection in pure culture was 0.1 spore DNA equivalents per PCR reaction corresponding to 20 spores ml(-1) in the sample. However, 1 spore DNA equivalent per PCR reaction corresponding to 200 spores ml(-1) in the sample was the lowest amount of spores tested without interference in dust samples spiked with spores of either fungal species. The extent of inhibition was calculated using conventional and real-time PCR reactions containing fungal spores, specific primers, specific probes (for real-time PCR) and various amounts of dust. The results indicate that the extent of inhibition by dust on PCR varies with the type and amount of dust, and number of spores. No interference in the analysis of spiked samples was detected from 0.2 mg ml(-1) of four real-life dust samples at p-value >0.05 using 2 x 10(4) spores for conventional PCR and 2 x 10(5) spores for real-time PCR. However, samples containing >0.2 mg ml(-1) real-life dust compromised the PCR assay. These results suggest the potential usefulness of a simple sample processing method in conjunction with PCR for monitoring the fungal content of aerosols collected from indoor environments.
Keswani
Jyoti
J
Health Effects Laboratory Division, Exposure Assessment Branch, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, 1095 Willowdale Road, M/S L-3030, Morgantown, WV 26505, USA. JKeswani@cdc.gov
Kashon
Michael L
ML
Chen
Bean T
BT
D016902:Air Pollution, Indoor / Q000032:analysis*; D001232:Aspergillus fumigatus / Q000302:isolation & purification; D015169:Colony Count, Microbial; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis; D004271:DNA, Fungal / Q000032:analysis*; D004391:Dust / Q000032:analysis*; D004784:Environmental Monitoring / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013171:Spores, Bacterial / Q000302:isolation & purification; D013191:Stachybotrys / Q000302:isolation & purification; D013997:Time Factors
D015342:DNA Probes; D004269:DNA, Bacterial; D004271:DNA, Fungal; D004391:Dust