Shan Lei, Yang David, Wang Dapeng, Tian Peng
Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA.
MOST-USDA Joint Research Center for Food Safety & Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China.
J Virol Methods. 2016 May;231:1-7. doi: 10.1016/j.jviromet.2016.01.012. Epub 2016 Feb 10.
In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.
在本研究中，我们使用杜兰病毒（TV）作为人诺如病毒（HuNoV）的替代物，以评估两种基于细胞的检测方法与三种基于聚合酶链反应（PCR）的检测方法之间的相关性。具体而言，基于细胞的噬斑和半数组织培养感染剂量（TCID50）检测方法用于测量感染性病毒颗粒，而基于PCR的核糖核酸酶暴露检测、猪胃粘蛋白原位捕获定量逆转录聚合酶链反应（PGM-ISC-qRT-PCR）检测以及抗体原位捕获定量逆转录聚合酶链反应（Ab-ISC-qRT-PCR）检测则用于测量衣壳化病毒基因组内的扩增子。使用十批病毒储备液，其噬斑形成单位（PFU）范围为3.41×10⁵至6.67×10⁶，以PFU作为参考进行并行比较。结果表明，1个PFU相当于6.69±2.34个TCID50单位、9.75±10.87个未用核糖核酸酶处理的基因组拷贝（GC）、2.87±3.05个用核糖核酸酶处理的GC、0.07±0.07个PGM-ISC-qRT-PCR GC以及0.52±0.39个Ab-ISC-qRT-PCR GC。我们观察到，虽然基于细胞的检测方法之间相互一致，但TCID50检测比噬斑检测更敏感。相比之下，基于PCR的检测方法与基于细胞的检测方法并不总是一致。两种原位捕获定量逆转录聚合酶链反应（ISC-RT-qPCR）检测方法所测得的GC变化非常大，这使得它们难以与基于细胞的检测方法所测得的PFU或TCID50单位中相对较小的变化（<20倍）相关联。
