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<p>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 10<sup>3</sup> copies/μL, 10<sup>3</sup> copies/μL, 10<sup>2</sup> copies/μL, and 10<sup>3</sup> 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&#xa0;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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年8月21日

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  • Yan Wang,
  • Jinfeng Wang,
  • Yujing Li,
  • Maolin Wei,
  • Wei Wang,
  • Libing Liu,
  • Bolong Liang,
  • Xiangdong Xu,
  • Jianchang Wang

摘要

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.