Accuracy, Precision, and Method Detection Limits of Quantitative PCR for Airborne Bacteria and Fungi

DENINA HOSPODSKY (1), Naomichi Yamamoto (1,2), Jordan Peccia (1)

(1) Yale University (2) Japan Society for the Promotion of Science

     Abstract Number: 503
     Last modified: May 13, 2010

Abstract
Real Time quantitative PCR (qPCR) methods for rapid and specific enumeration of microbial agents are finding increased use in aerosol science. The goal of this study was to determine qPCR accuracy, precision, and method detection limits (MDL) within the context of indoor and ambient aerosol samples. Escherichia coli and Bacillus atrophaeus vegetative bacterial cells and Aspergillus fumigatus fungal spores were considered as test organisms. Efficiencies associated with recovery of DNA from aerosol samples were low and excluding these efficiencies in quantitative analysis may lead to under estimating the true aerosol concentration by 10 to 25 times. Precision near detection limits ranged from 28% to 79% coefficient of variation (COV) for the three test organisms and the majority of this variation was due to instrumental repeatability. Precision results suggest that qPCR is useful for determining dissimilarity between two samples only if the true differences are greater than 2.4 to 3.2 times ( 95% confidence, n=7). For MDLs, qPCR was able to produce a positive response with 99% confidence from the DNA of 5 B. atrophaeus cells and less than one A. fumigatus spore. Overall MDL values that included sample processing efficiencies ranged from 2,000 to 3,000 B. atrophaeus cells and 10 to 25 A. fumigatus spores. Applying the concepts of accuracy, precision, and MDL to qPCR aerosol measurements demonstrates that sample processing efficiencies must be accounted for in order to accurately estimate bioaerosol exposure, sets the statistical rigor necessary for appropriate application of qPCR to understand significant differences among separate aerosol samples, and allows for avoiding nondetect values at true aerosol concentrations that may be significant.