Yan Wang, Jinfeng Wang, Yujing Li, Maolin Wei, Wei Wang, Libing Liu, Bolong Liang, Xiangdong Xu, Jianchang Wang
{"title":"一种新型实时RT-qPCR方法同时检测双壳贝类中HEV、HAstV和SaV","authors":"Yan Wang, Jinfeng Wang, Yujing Li, Maolin Wei, Wei Wang, Libing Liu, Bolong Liang, Xiangdong Xu, Jianchang Wang","doi":"10.1007/s12560-025-09661-3","DOIUrl":null,"url":null,"abstract":"<div><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 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></div>","PeriodicalId":563,"journal":{"name":"Food and Environmental Virology","volume":"17 3","pages":""},"PeriodicalIF":2.7000,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Simultaneous Detection of HEV, HAstV and SaV in Bivalve Shellfish Using a Novel Real-Time RT-qPCR Method\",\"authors\":\"Yan Wang, Jinfeng Wang, Yujing Li, Maolin Wei, Wei Wang, Libing Liu, Bolong Liang, Xiangdong Xu, Jianchang Wang\",\"doi\":\"10.1007/s12560-025-09661-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><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 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></div>\",\"PeriodicalId\":563,\"journal\":{\"name\":\"Food and Environmental Virology\",\"volume\":\"17 3\",\"pages\":\"\"},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2025-08-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Food and Environmental Virology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s12560-025-09661-3\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"ENVIRONMENTAL SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Food and Environmental Virology","FirstCategoryId":"97","ListUrlMain":"https://link.springer.com/article/10.1007/s12560-025-09661-3","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ENVIRONMENTAL SCIENCES","Score":null,"Total":0}
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.
期刊介绍：
Food and Environmental Virology publishes original articles, notes and review articles on any aspect relating to the transmission of pathogenic viruses via the environment (water, air, soil etc.) and foods. This includes epidemiological studies, identification of novel or emerging pathogens, methods of analysis or characterisation, studies on survival and elimination, and development of procedural controls for industrial processes, e.g. HACCP plans. The journal will cover all aspects of this important area, and encompass studies on any human, animal, and plant pathogenic virus which is capable of transmission via the environment or food.
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