Application Research on the Triplex RT-ddPCR Assay for the Simultaneous Detection of Human Astrovirus and Sapovirus in Bivalve Shellfish

Human astrovirus (HAstV) and sapovirus (SaV) are significant pathogens associated with acute gastroenteritis in humans. This study established a triple reverse transcription-droplet digital polymerase chain reaction (PCR) (RT-ddPCR) assay incorporating MS2 phage as a process control virus for the simultaneous quantification of HAstV and SaV. The assay was validated using 240 bivalve samples, comprising five shellfish species: <i>Ostreidae</i> (<i>n</i> = 43), <i>Ruditapes philippinarum</i> (<i>n</i> = 84), <i>Sinonovacula constricta</i> (<i>n</i> = 27), <i>Scapharca subcrenata</i> (<i>n</i> = 23), and <i>Pectinidae</i> (<i>n</i> = 63). The results indicated that the developed RT-ddPCR assay had a good exclusivity, with detection limits of 5.15 copies/reaction for HAstV, 7.71 copies/reaction for SaV, and 6.13 copies/reaction for MS2 RNA. Viral screening revealed HAstV in 1.25% (3/240) of samples, with a maximum load of 22,140 copies/2 g, while SaV exhibited a higher prevalence of 13.33% (32/240) and a peak concentration of 68,700 copies/2 g. Different species of bivalve shellfish exhibited varying detection rates; the highest SaV detection rate was found in <i>Ostreidaes</i> at 20.93% (9/43), followed by <i>Ruditapes philippinarums</i> at 14.29% (12/84), <i>Scapharca subcrenatas</i> at 13.04% (3/23), <i>Pectinidaes</i> at 11.11% (7/63), and <i>Sinonovacula constrictas</i> at 3.70% (1/27). HAstV was only detected in <i>R. philippinarums</i> and <i>Pectinidaes</i>, with detection rates of 1.19% (1/84) and 3.17% (2/63), respectively. Additionally, both HAstV and SaV were detected in a single <i>Pectinidae</i> sample (0.42%, 1/240). The triple RT-ddPCR assay developed in this study is reliable, accurate, and highly sensitive, providing effective technical support for the quantitative detection of HAstV and SaV in bivalve shellfish.