AUTHOR=Yang Liu , Long Yunzhi , Li Qianqian , Song Wenbo , Huang Ying , Liang Gong , Yu Daobing , Zhou Mingguang , Xu Gaoyuan , Chen Yao , Huang Chao , Tang Xibiao TITLE=Detection of suid herpesvirus 1 infectivity in pigs by propidium monoazide-qPCR JOURNAL=Frontiers in Veterinary Science VOLUME=9 YEAR=2022 URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2022.975726 DOI=10.3389/fvets.2022.975726 ISSN=2297-1769 ABSTRACT=

At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50–100 μM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.