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

Real-time quantitative PCR (qPCR) for rapid and specific enumeration of microbial agents is finding increased use in aerosol science. The goal of this study was to determine qPCR accuracy, precision, and method detection limits (MDLs) within the context of indoor and ambient aerosol samples. Escherichia coli and Bacillus atrophaeus vegetative bacterial cells and Aspergillus fumigatus fungal spores loaded onto aerosol filters were considered. Efficiencies associated with recovery of DNA from aerosol filters were low, and excluding these efficiencies in quantitative analysis led to underestimating the true aerosol concentration by 10 to 24 times. Precision near detection limits ranged from a 28% to 79% coefficient of variation (COV) for the three test organisms, and the majority of this variation was due to instrument repeatability. Depending on the organism and sampling filter material, precision results suggest that qPCR is useful for determining dissimilarity between two samples only if the true differences are greater than 1.3 to 3.2 times (95% confidence level at n = 7 replicates). For MDLs, qPCR was able to produce a positive response with 99% confidence from the DNA of five 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 per filter and 10 to 25 A. fumigatus spores per filter. 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, provides guidance on the necessary statistical rigor required to understand significant differences among separate aerosol samples, and prevents undetected (i.e., nonquantifiable) values for true aerosol concentrations that may be significant.Real-time quantitative PCR (qPCR) is an analytical method for the rapid and potentially sensitive enumeration of broad and specific microbial populations in environmental samples (17). For bioaerosol analysis, this method allows for detection and enumeration independent of culturing, thereby circumventing the significant concerns surrounding the unculturability of environmental microorganisms and loss of culturability due to aerosol sampling (1, 2, 18, 34, 46, 55). Over the last decade, the application of qPCR has advanced research in the human health, environmental, and the national security arenas by enabling the specific measurement of airborne allergenic mold, pathogenic bacteria, and human viruses (6, 7, 9, 13, 37, 45).The quantitative nature of this technique as well as the documented advantages over culturing provides the potential for integrating microbial measurements with physical and chemical aerosol processes to understand exposure and to describe the fate and sources of biological aerosols in indoor environments and the atmosphere. However, the logarithmic amplification that is the basis of qPCR results in significant standard deviations among repeated qPCRs (25, 50). This variability rarely constrains the use of qPCR in aquatic and terrestrial systems, where biological growth typically dictates concentrations above detection limits and multiple order-of-magnitude differences in microorganism concentrations between treatments. However, the volume concentrations of biological agents in air (10³ to 10⁶ per m³ of air) are dramatically more dilute than those measured in environmental waters (10¹² to 10¹⁴ per m³ of water) (4, 5, 10, 12, 20, 52), and processes that result in indoor and atmospheric bioaerosol concentrations are growth independent. These processes include aerosol infiltration and exfiltration, resuspension, and deposition and typically result in less than an order of magnitude of variability in aerosol or biological particulate matter (PM) concentrations (22, 29, 41). As qPCR becomes more commonly used in indoor and outdoor air quality research, it is necessary to know the analytical variability and method detection limits (MDLs) to determine whether the method is suitable for estimating exposure and delineating the experimental differences observed in aerosol processes.The goal of this study was to estimate the accuracy, precision, and MDLs associated with qPCR of aerosol samples. These concepts were applied to air sampling filters loaded with three test organisms, including spores of Aspergillus fumigatus and vegetative bacterial cells from the Gram-negative Escherichia coli and Gram-positive Bacillus atrophaeus. The efficiencies associated with DNA extraction and with extraction of whole cells from aerosol filters were measured to describe the statistical accuracy of common qPCR bioaerosol protocols. Overall precision (reproducibility) as well as instrument repeatability were determined, and a binary method for describing MDLs was developed and applied to fungal spores and bacterial cells. Such experimental and statistical treatment of qPCR-based aerosol measurements is expected to guide improved estimates of human exposure, incorporate limits of qPCR precision into experimental design, and provide a context for undetected (i.e., nonquantifiable) values.