Xu Shuxia, Wang Dapeng, Yang David, Liu Huashan, Tian Peng
College of Agronomy, Henan Agricultural University, No.63, Nongye Road, Zhengzhou, 450002, China; Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA.
Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA; Dept. of Food Science and Technology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
Food Microbiol. 2015 Jun;48:22-7. doi: 10.1016/j.fm.2014.12.004. Epub 2014 Dec 19.
Culturable animal caliciviruses are widely-used as surrogates for human norovirus (HuNoV). The infectivity of a culturable virus was traditionally determined by plaque assay and/or 50% tissue culture infectious dose (TCID50) assay, both of which are time-consuming and labor-intensive. Molecular approaches, such as quantitative real time RT-PCR (qRT-PCR) and RT-PCR, could be used for detection of the viral genome but yet fail to determine the infectivity of a virus. In this study, we evaluated different assays for determination of infectivity of Tulane virus (TV), a surrogate for HuNoV. The infectivity of TV was measured by RNase exposure assay, RT-PCR assays, cellular-receptor-mediated capture qRT-PCR assay, receptor-mediated in situ capture qRT-PCR assay, cell-culture-mediated amplification qRT-PCR, and confirmed by TCID50 assay. RNase exposure assay was only useful for measuring TV inactivation caused by heat. Short template RT-PCR assay did not reflect inactivation status of TV. Partial reduction in viral RNA signal could be measured by long-template RT-PCR only when TV was inactivated by thermal or chlorine treatments at full-inactivation levels. Cellular-receptor-mediated capture qRT-PCR exhibited low sensitivity and specificity for the evaluation of virus infectivity. The in situ capture qRT-PCR assay could be used to evaluate virus inactivation deriving from damage to viral capsid caused by heat and chlorine. The cell-culture-mediated amplification qRT-PCR could be used as an alternative method to rapidly determine the infectivity of TV.
可培养的动物杯状病毒被广泛用作人类诺如病毒（HuNoV）的替代物。传统上，可培养病毒的感染性通过噬斑测定和/或50%组织培养感染剂量（TCID50）测定来确定，这两种方法都耗时且费力。分子方法，如定量实时RT-PCR（qRT-PCR）和RT-PCR，可用于检测病毒基因组，但无法确定病毒的感染性。在本研究中，我们评估了不同的测定方法来确定图兰病毒（TV）的感染性，TV是HuNoV的替代物。通过核糖核酸酶暴露测定、RT-PCR测定、细胞受体介导的捕获qRT-PCR测定、受体介导的原位捕获qRT-PCR测定、细胞培养介导的扩增qRT-PCR来测量TV的感染性，并通过TCID50测定进行确认。核糖核酸酶暴露测定仅用于测量热引起的TV失活。短模板RT-PCR测定不能反映TV的失活状态。只有当TV通过热或氯处理完全失活时，长模板RT-PCR才能测量病毒RNA信号的部分降低。细胞受体介导的捕获qRT-PCR在评估病毒感染性方面表现出低灵敏度和特异性。原位捕获qRT-PCR测定可用于评估由热和氯对病毒衣壳造成的损伤导致的病毒失活。细胞培养介导的扩增qRT-PCR可作为一种替代方法来快速确定TV的感染性。
