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Title: Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

item STOKDYK, JOEL - Us Geological Survey (USGS)
item FIRNSTAHL, AARON - Us Geological Survey (USGS)
item Spencer, Susan
item BURCH, TUCKER - Us Geological Survey (USGS)
item Borchardt, Mark

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2016
Publication Date: 3/11/2016
Publication URL:
Citation: Stokdyk, J.P., Firnstahl, A.D., Spencer, S.K., Burch, T.R., Borchardt, M.A. 2016. Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR. Applied and Environmental Microbiology. 96:105-113.

Interpretive Summary: For many laboratory analyses, an important measure of quality and performance is the limit of detection, or LOD. Ideally, 95% LOD is reported (the lowest concentration at which 95% of the time a positive sample will be detected and not incorrectly reported as negative). In studies of environmental microbiology it has become standard to detect microorganisms by a genetic method called quantitative polymerase chain reaction or qPCR. Unfortunately, in many of these studies the 95% LOD is not reported because it can be difficult and time-consuming. We developed a streamlined approach for determining the 95% LOD for qPCR analysis of waterborne pathogens. Using this approach we found the 95% LODs for detecting waterborne Salmonella, adenovirus, and enterovirus in a 1500 liter water sample are 1.3, 1.5, and 4.0 gene copies per liter, respectively. Having researchers determine the 95% LOD for their qPCR analyses of waterborne microorganisms is important for comparing methods, designing meaningful studies, and helping with data interpretation.

Technical Abstract: The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay rather than the entire sample process. Our objective was to develop a method to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) in the extraction step, secondary concentration step, and primary concentration step (i.e., working backwards), which established a concentration that was detectable following losses of the pathogen due to processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then a third, concentration. If two concentrations had the same fraction of positive replicates, we selected an additional concentration. We calculated the LOD using probit analysis. For the entire process from dead-end ultrafiltration to qPCR, we determined the 95% LOD for Salmonella typhimurium, adenovirus group 41, and poliovirus Sabin III as 11, 12, and 6 genomic copies (gc) per reaction, respectively (equivalent to 1.3, 1.5, and 4.0 gc L-1 based on the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple targets simultaneously. An LOD determined this way can facilitate study design, aid method evaluation, and inform data interpretation.