A Streamlined qPCR Method for Characterization ofEnterococcusspp. Levels in Ambient Surface Water Samples

Abstract Measurement of Enterococcus spp. levels with qPCR allows for same-day advisory notification of recreational water quality conditions, representing a major advance over traditional culture-based methods that require 18 or more hours to obtain results. In 2015, the United States Environmental Protection Agency released an Enterococcus qPCR protocol for recreational water quality testing. Over the past decade, there have been multiple advances in qPCR-based environmental testing, affording the opportunity to update the current methodology. A streamlined Enterococcus qPCR protocol is introduced that simplifies the mathematical model to estimate target sequence concentrations (TSC), reduces sample testing time by 20 min, incorporates a certified control material for standard curve generation, and introduces an inactivated E. faecalis whole cell DNA standard (WCDS) control material. A series of experiments were conducted 1) to compare results of the two Enterococcus qPCR protocols in analysis of marine, estuarine, and freshwater samples (n = 60), 2) to investigate alternative practices to adjust results due to potential water sample matrix interference, control material degradation, and/or analyst inconsistencies, and 3) to evaluate the performance, homogeneity, and stability of an inactivated E. faecalis cell preparation as a WCDS control material. Findings indicate a strong correlation between water sample mean log10 TSC per reaction results (R2 = 0.980) and 100% agreement in amplification and sample processing control tests. A Bayesian approach that accounts for uncertainty in qPCR measurements confirmed statistical equivalence for all water samples yielding paired measurements in the range of quantification, with 72.7% of samples exhibiting reduced error with the new streamlined protocol. Evaluation of three alternative practices to adjust for variation in Enterococcus qPCR measurements indicated no significant difference in water sample log10 TSC per reaction results with varying concentrations of treated sewage influent. Systematic testing of an inactivated WCDS control material yielded statistically equivalent performance compared to viable E. faecalis cell preparations. Homogeneity and stability experiments indicated that Enterococcus qPCR measurements of inactivated WCDS are reproducible across multiple preparations and that the material is stable at −20°C for at least 38 weeks. Together, experiments demonstrate that the streamlined protocol and alternative practices should make Enterococcus qPCR faster, easier to implement, safer, and more reproducible. Competing Interest Statement The authors have declared no competing interest. Funding Statement This study did not receive any funding Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors