Liang Gong, Long Yunzhi, Li Qianqian, Yang Liu, Huang Ying, Yu Daobing, Song Wenbo, Zhou Mingguang, Xu Gaoyuan, Huang Chao, Tang Xibiao
Diagnostic Center Department, Wuhan Keqian Biology Co., Ltd, Wuhan, China.
Front Vet Sci. 2022 Jul 15;9:931392. doi: 10.3389/fvets.2022.931392. eCollection 2022.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.
逆转录定量聚合酶链反应（RT-qPCR）可灵敏检测流行样本中的病毒颗粒和病毒，但无法区分非感染性病毒和感染性病毒。评估了单叠氮溴化丙锭（PMA）与定量聚合酶链反应（qPCR）联用检测感染性病毒的效果。目前，尚无成熟的检测猪流行性腹泻病毒（PEDV）感染的方法。在本研究中，PMA与qPCR联用可检测PEDV的感染性。我们从引物选择、PMA工作浓度以及热或紫外线（UV）灭活方法等方面对该方法进行了优化。在qPCR之前用PMA处理的病毒，通过热或UV进行灭活。然而，单独添加PMA并不影响活病毒的检测，这表明病毒衣壳破裂可能是PMA与基因组结合所必需的。对裸露的PEDV RNA重复该方法表明，它可用于检测潜在感染性的PEDV。结果表明，PMA的最佳方案对于评估感染性和非感染性病毒可能极为有用。
