Development of an in-house, one-step RT-qPCR mix and optimized MS2 detection primers for hepatitis A virus and...

One-step, reverse transcriptase-quantitative PCR (RT-qPCR) is the primary method for detecting foodborne viruses in food matrices. The ISO 15216-2:2019 serves as the international standard for detecting human norovirus GI, GII, and hepatitis A virus. Some food matrices, such as berries, tend to co-purify PCR inhibitors with viral RNA, which can lead to false-negative results. To prevent this, the protocol includes extensive control approaches. However, the high cost of commercial RT-qPCR kits makes large-scale virus testing expensive and inaccessible. To address this, we developed an in-house, one-step RT-qPCR mix using commercial, next-generation enzymes with improved resistance to PCR inhibitors and with enhanced performance. The in-house mix offers a more cost-effective alternative to expensive and outdated commercial mixes. In this paper, we describe: • the development of an in-house, one-step multiplexable RT-qPCR protocol and optimization process as a reference for laboratories seeking to develop their own in-house protocols. • altered and optimized, previously described primers for the MS2 virus, further improving the efficiency of its detection and its reliability as a process control virus.