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

IF 2.7 2区农林科学 Q2 ENVIRONMENTAL SCIENCES

Food and Environmental Virology Pub Date : 2025-08-21 DOI:10.1007/s12560-025-09661-3

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

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引用次数: 0

Abstract

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.

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一种新型实时RT-qPCR方法同时检测双壳贝类中HEV、HAstV和SaV

戊型肝炎病毒（HEV）、人类星状病毒（HAstV）和萨波病毒（SaV）是与食源性疾病暴发相关的病原体。我们以MS2噬菌体作为过程控制病毒，建立了一种快速灵敏的四倍实时荧光定量PCR （RT-qPCR）方法，用于同时检测HEV、HAstV和SaV。优化实验参数，对HEV、HAstV、SaV和MS2 RNA的检出限分别为103拷贝/μL、103拷贝/μL、102拷贝/μL和103拷贝/μL，方法内变异系数和方法间变异系数均小于3.0%，重复性好，总检测时间小于90 min。在河北省不同地区采集双壳贝类标本354份。优化蛋白酶K-PEG 8000沉淀-氯仿萃取法提取病毒核酸后，采用四联实时RT-qPCR同时检测。HEV阳性率为9.60% (34/354)，HAstV阳性率为3.67% (13/354)，SaV阳性率为6.78% (24/354)，HEV和HAstV阳性率为0.28%，HEV和SaV阳性率为2.54%，HAstV和SaV阳性率为0.56%。随机选取200份样本进行单次实时RT-qPCR，结果与四重法总体吻合98.67%，阳性吻合100%，阴性吻合98.54%，Kappa值为0.922。总之，这种四重实时RT-qPCR方法可以快速筛选双壳贝类中HEV、HAstV和SaV。

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来源期刊

Food and Environmental Virology ENVIRONMENTAL SCIENCES-MICROBIOLOGY

CiteScore

6.50

自引率

2.90%

发文量

审稿时长

1 months

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.