Simultaneous Detection of HEV, HAstV and SaV in Bivalve Shellfish Using a Novel Real-Time RT-qPCR Method

Hepatitis E virus (HEV), human astrovirus (HAstV), and sapovirus (SaV) are pathogens associated with foodborne disease outbreaks. We developed a rapid and sensitive quadruple real-time fluorescence quantitative PCR (RT-qPCR) method for the simultaneous detection of HEV, HAstV, and SaV, using MS2 phage as a process control virus. We optimized the experimental parameters, detection limits for HEV, HAstV, SaV, and MS2 RNA were 10³ copies/μL, 10³ copies/μL, 10² copies/μL, and 10³ copies/μL, respectively, with intra-method and inter-method coefficients of variation below 3.0%, indicating good reproducibility and a total detection time of less than 90 min. We collected 354 bivalve shellfish samples from various regions in Hebei Province. After optimizing the proteinase K-PEG 8000 precipitation-chloroform extraction method for viral nucleic acid extraction, we applied the quadruple real-time RT-qPCR for simultaneous detection. The positive rates were 9.60% (34/354) for HEV, 3.67% (13/354) for HAstV, and 6.78% (24/354) for SaV, with mixed contaminations observed for HEV and HAstV (0.28%), HEV and SaV (2.54%), and HAstV and SaV (0.56%). In addition, a single real-time RT-qPCR was performed on 200 randomly selected samples and showed an overall agreement with the quadruple method of 98.67%, 100% positive agreement, 98.54% negative agreement and a Kappa value of 0.922. In conclusion, this quadruple real-time RT-qPCR method offers rapid screening for HEV, HAstV, and SaV in bivalve shellfish.