Comparison of RT-ddPCR and RT-qPCR platforms for SARS-CoV-2 detection: Implications for future outbreaks of infectious diseases

[Display omitted] •RT-ddPCR demonstrated superior quantification accuracy for SARS-CoV-2 detection compared to RT-qPCR and was particularly advantageous at low viral levels with better precision and sensitivity.•RT-ddPCR outperformed RT-qPCR for viral RNA detection in the complex wastewater matrix and yielded a significantly higher detection rate in large-volume SARS-CoV-2 positive wastewater samples.•RT-ddPCR exhibited more robust SARS-CoV-2 detection with the presence of a mismatch in the probe binding region when consensus assay was used.•RT-ddPCR showed superiority over RT-qPCR for virus genotyping with higher accuracy and detection rate, especially at low levels of allele frequencies and concentrations.•By taking SARS-CoV-2 as a model system, this study suggested RT-ddPCR as a promising tool in wastewater surveillance scenarios and other relevant applications for future infectious disease outbreaks. The increased frequency of human infectious disease outbreaks caused by RNA viruses worldwide in recent years calls for enhanced public health surveillance for better future preparedness. Wastewater-based epidemiology (WBE) is emerging as a valuable epidemiological tool for providing timely population-wide surveillance for disease prevention and response complementary to the current clinical surveillance system. Here, we compared the analytical performance and practical applications between predominant molecular detection methods of RT-qPCR and RT-ddPCR on SARS-CoV-2 detection in wastewater surveillance. When pure viral RNA was tested, RT-ddPCR exhibited superior quantification accuracy at higher concentration levels and achieved more sensitive detection with reduced variation at low concentration levels. Furthermore, RT-ddPCR consistently demonstrated more robust and accurate measurement either in the background of the wastewater matrix or with the presence of mismatches in the target regions of the consensus assay. Additionally, by detecting mock variant RNA samples, we found that RT-ddPCR outperformed RT-qPCR in virus genotyping by targeting specific loci with signature mutations in allele-specific (AS) assays, especially at low levels of allele frequencies and concentrations, which increased the possibility for sensitive low-prevalence variant detection in the population. Our study provides insights for detection method selection in the WBE applications for future infectious disease outbreaks.