The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater Surveillance

Quantitative Polymerase Chain Reaction (qPCR) is a molecular technique that has become a gold standard in various disciplines, including environmental microbiology, due to its high sensitivity and specificity. In recent years, it has been extensively used in wastewater-based epidemiology to monitor the prevalence of different viruses in the population. In this study, we evaluated whether the no inclusion of a standard curve in each single experiment to reduce time and costs could have an impact on the accuracy of the results. Thirty independent RT-qPCR standard curve experiments using quantitative synthetic RNA material were conducted for seven different viruses, which include two targets of the novel SARS-CoV-2, hepatitis A and E, noroviruses genogroups I and II, human astrovirus, and rotavirus. Results showed that although all the viruses presented adequate efficiency rates (>90%), variability was also observed between them, independently of the viral concentration tested. NoVGII was the virus that presented the higher inter-assay variability in terms of efficiency while showing better sensitivity. In terms of heterogeneity in results, the two targets of SARS-CoV-2 showed the highest rates, being N2 the gene that presented the largest variability (CV 4.38-4.99%) and the lowest efficiency (90.97%). These findings indicate that including a standard curve in every experiment is recommended to obtain reliable results.