title: Comparison of Enterococcus quantitative polymerase chain reaction analysis results from fresh and marine waters on two real-time instruments.
abstract: The U.S. Environmental Protection Agency will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for implementation of this and other qPCR methods is whether the results are comparable on different PCR instruments. In this study, quantitative estimates of Enterococcus densities from marine and freshwater samples were determined by the qPCR method from cycle threshold (Ct) measurements obtained on Applied Biosystems StepOnePlus and Cepheid SmartCycler instruments. Three variations of a comparative Ct model, differing in their sources of calibration data, were used in the estimations. Both traditional and Bayesian statistical modeling approaches were examined in the instrument comparisons. The traditional analysis of variance (ANOVA) approach indicated no significant differences (p>0.05) between mean density estimates from the instruments in two of the three model variations. The Bayesian approach indicated that the 95% Bayesian credible intervals of density estimates from the instruments overlapped in all models; however, the uncertainty of the estimates varied depending on the model. These results support the interchangeable use of the two instruments in the method and also illustrate the importance of defining the source of calibration data used in the comparative Ct model.
authors.affiliation: National Risk Management Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH 45268, USA.
authors.forename: Mano
authors.initials: M
authors.lastname: Sivaganensan
authors.forename: Manju
authors.initials: M
authors.lastname: Varma
authors.forename: Richard A
authors.initials: RA
authors.lastname: Haugland
mesh_terms: D001499:Bayes Theorem; D002138:Calibration; D016983:Enterococcus / Q000235:genetics* / Q000302:isolation & purification*; D005618:Fresh Water / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D012623:Seawater / Q000382:microbiology*; D060753:Water Quality