Persson Sofia, Molin Ramia, Eriksson Ronnie, Lavander Moa, Widén Frederik, Ellström Patrik, Simonsson Magnus
European Union Reference Laboratory for Foodborne Viruses, Swedish Food Agency, Dag Hammarskjölds väg 56 A, 752 37, Uppsala, Sweden.
Department of Medical Sciences, Zoonosis Science Centre, Uppsala University, Husargatan 3, 752 37, Uppsala, Sweden.
Food Environ Virol. 2025 Jun 2;17(2):33. doi: 10.1007/s12560-025-09645-3.
Hepatitis E virus (HEV) infection is an emerging zoonosis that can be transmitted to humans through the consumption of raw or undercooked pork products. Several methods have been described to detect the virus in food, but there are few data on qualitative and quantitative performance characteristics. In this study, we have developed an optimised method for quantitative detection of HEV in pork sausage based on a combination of previously published protocols. The method utilises sample disruption and phase separation with tri-reagent and 1-bromo-3-chloropropane followed by RNA concentration with isopropanol precipitation. We validated the method for use on reverse transcription quantitative real-time PCR (RT-qPCR) and reverse transcription droplet digital (RT-ddPCR). The 95% limit of detection and limit of quantification was 200 copies/g for both RT-qPCR and RT-ddPCR. RT-ddPCR technology has previously shown promise as a more precise alternative to RT-qPCR. However, we found no evidence for improved performance using RT-ddPCR instead of RT-qPCR for this method. Additionally, we further verified the performance of the HEV RT-PCR assay using the WHO International Standard and Reference Panel for HEV RNA. Finally, we assessed different combinations of RNA concentration protocols and RT-PCR detection strategies. This showed that isopropanol precipitation of viral RNA was at least twice as efficient as magnetic silica bead-based extraction when an inhibitor-tolerant RT-qPCR detection strategy was used. In summary, we present an efficient and well-characterised method for quantitative detection of HEV in pork sausage. Such methods are valuable to provide high-quality data for risk assessments and food monitoring.
戊型肝炎病毒（HEV）感染是一种新出现的人畜共患病，可通过食用生的或未煮熟的猪肉制品传播给人类。已有多种方法用于检测食品中的该病毒，但关于定性和定量性能特征的数据较少。在本研究中，我们基于先前发表的方案组合，开发了一种优化的定量检测猪肉香肠中HEV的方法。该方法利用三试剂和1-溴-3-氯丙烷进行样品破碎和相分离，随后用异丙醇沉淀进行RNA浓缩。我们验证了该方法在逆转录定量实时PCR（RT-qPCR）和逆转录液滴数字PCR（RT-ddPCR）上的应用。RT-qPCR和RT-ddPCR的95%检测限和定量限均为200拷贝/克。RT-ddPCR技术此前已显示出作为RT-qPCR更精确替代方法的潜力。然而，我们发现对于该方法，使用RT-ddPCR而非RT-qPCR并没有性能改善的证据。此外，我们使用世界卫生组织戊型肝炎病毒RNA国际标准品和参考品进一步验证了戊型肝炎病毒RT-PCR检测方法的性能。最后，我们评估了RNA浓缩方案和RT-PCR检测策略的不同组合。结果表明，当使用耐抑制剂的RT-qPCR检测策略时，病毒RNA的异丙醇沉淀效率至少是基于磁珠提取的两倍。总之，我们提出了一种高效且特征明确的定量检测猪肉香肠中HEV的方法。此类方法对于为风险评估和食品监测提供高质量数据具有重要价值。
