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...
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MDPI AG
2025-03-01
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| Online Access: | https://www.mdpi.com/2076-2607/13/4/776 |
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| author | Lorena Casado-Martín Marta Hernández Nadine Yeramian Daniel Pérez José M. Eiros Antonio Valero David Rodríguez-Lázaro |
| author_facet | Lorena Casado-Martín Marta Hernández Nadine Yeramian Daniel Pérez José M. Eiros Antonio Valero David Rodríguez-Lázaro |
| author_sort | Lorena Casado-Martín |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-37a1594ec91b4cab81ffd5056b3c6db7 |
| institution | DOAJ |
| issn | 2076-2607 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
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| series | Microorganisms |
| spelling | doaj-art-37a1594ec91b4cab81ffd5056b3c6db72025-08-20T03:13:32ZengMDPI AGMicroorganisms2076-26072025-03-0113477610.3390/microorganisms13040776The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater SurveillanceLorena Casado-Martín0Marta Hernández1Nadine Yeramian2Daniel Pérez3José M. Eiros4Antonio Valero5David Rodríguez-Lázaro6Microbiology Area, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, SpainMicrobiology Area, Faculty of Medicine, University of Valladolid, 47002 Valladolid, SpainMicrobiology Area, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, SpainMicrobiology Area, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, SpainMicrobiology Area, Faculty of Medicine, University of Valladolid, 47002 Valladolid, SpainDepartment of Food Science and Technology, Faculty of Veterinary, Agrifood Campus of International Excellence (ceiA3), University of Cordoba, 14014 Córdoba, SpainMicrobiology Area, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, SpainQuantitative 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.https://www.mdpi.com/2076-2607/13/4/776wastewaterepidemiologyvirus surveillanceOne HealthRT-qPCRstandard curves |
| spellingShingle | Lorena Casado-Martín Marta Hernández Nadine Yeramian Daniel Pérez José M. Eiros Antonio Valero David Rodríguez-Lázaro The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater Surveillance Microorganisms wastewater epidemiology virus surveillance One Health RT-qPCR standard curves |
| title | The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater Surveillance |
| title_full | The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater Surveillance |
| title_fullStr | The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater Surveillance |
| title_full_unstemmed | The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater Surveillance |
| title_short | The Impact of the Variability of RT-qPCR Standard Curves on Reliable Viral Detection in Wastewater Surveillance |
| title_sort | impact of the variability of rt qpcr standard curves on reliable viral detection in wastewater surveillance |
| topic | wastewater epidemiology virus surveillance One Health RT-qPCR standard curves |
| url | https://www.mdpi.com/2076-2607/13/4/776 |
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