Maolin Wei, Jinfeng Wang, Yan Wang, Libing Liu, Xiangdong Xu, Jianchang Wang
{"title":"三联RT-ddPCR法在双壳贝类中同时检测人类星状病毒和萨波病毒的应用研究。","authors":"Maolin Wei, Jinfeng Wang, Yan Wang, Libing Liu, Xiangdong Xu, Jianchang Wang","doi":"10.1089/fpd.2025.0011","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":12333,"journal":{"name":"Foodborne pathogens and disease","volume":" ","pages":""},"PeriodicalIF":1.9000,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Application Research on the Triplex RT-ddPCR Assay for the Simultaneous Detection of Human Astrovirus and Sapovirus in Bivalve Shellfish.\",\"authors\":\"Maolin Wei, Jinfeng Wang, Yan Wang, Libing Liu, Xiangdong Xu, Jianchang Wang\",\"doi\":\"10.1089/fpd.2025.0011\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":12333,\"journal\":{\"name\":\"Foodborne pathogens and disease\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":1.9000,\"publicationDate\":\"2025-04-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Foodborne pathogens and disease\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.1089/fpd.2025.0011\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"FOOD SCIENCE & TECHNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Foodborne pathogens and disease","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1089/fpd.2025.0011","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"FOOD SCIENCE & TECHNOLOGY","Score":null,"Total":0}
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: Ostreidae (n = 43), Ruditapes philippinarum (n = 84), Sinonovacula constricta (n = 27), Scapharca subcrenata (n = 23), and Pectinidae (n = 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 Ostreidaes at 20.93% (9/43), followed by Ruditapes philippinarums at 14.29% (12/84), Scapharca subcrenatas at 13.04% (3/23), Pectinidaes at 11.11% (7/63), and Sinonovacula constrictas at 3.70% (1/27). HAstV was only detected in R. philippinarums and Pectinidaes, with detection rates of 1.19% (1/84) and 3.17% (2/63), respectively. Additionally, both HAstV and SaV were detected in a single Pectinidae 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.
期刊介绍:
Foodborne Pathogens and Disease is one of the most inclusive scientific publications on the many disciplines that contribute to food safety. Spanning an array of issues from "farm-to-fork," the Journal bridges the gap between science and policy to reduce the burden of foodborne illness worldwide.
Foodborne Pathogens and Disease coverage includes:
Agroterrorism
Safety of organically grown and genetically modified foods
Emerging pathogens
Emergence of drug resistance
Methods and technology for rapid and accurate detection
Strategies to destroy or control foodborne pathogens
Novel strategies for the prevention and control of plant and animal diseases that impact food safety
Biosecurity issues and the implications of new regulatory guidelines
Impact of changing lifestyles and consumer demands on food safety.