College of Animal Science and Veterinary Medicine, Jilin University, No. 5333, Xi'an Road, Changchun 130062, China.
J Virol Methods. 2010 Nov;169(2):391-6. doi: 10.1016/j.jviromet.2010.08.011. Epub 2010 Aug 17.
The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10(8) copies of HRV RNA. Regression analysis demonstrated a significant correlation with an R(2) value of 0.9963 and a slope of -3.18 between the mean C(t) values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37-1.72% and 1.37-3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.
本研究旨在开发一种使用 TaqMan 探针检测和定量虹鳟鱼呼肠孤病毒（HRV）的实时定量逆转录聚合酶链反应（qRT-PCR）检测方法。结果表明，该检测方法的检测限为每个反应 100 个 RNA 拷贝，HRV RNA 的对数线性范围高达 10(8)个拷贝。回归分析表明，平均 C(t)值与 HRV cRNA 之间存在显著相关性，R(2)值为 0.9963，斜率为-3.18。该检测方法比传统的一步法 RT-PCR 检测方法灵敏 100 倍。qRT-PCR 检测方法具有高度可重复性，内和间试验的变异系数分别为 0.37-1.72%和 1.37-3.79%。该引物和 TaqMan 探针特异性针对 HRV，与鲤鱼春病毒血症病毒（SVCV）、传染性胰腺坏死病毒（IPNV）、传染性造血器官坏死病毒（IHNV）、水产动物双RNA 病毒（MABV）、病毒性出血性败血症病毒（VHSV）或病毒性神经坏死病毒（VNNV）均无反应。该方法用 40 个鱼类样本进行了评估，表明与目前用于 HRV 监测的经典病毒分离方法相比，该方法具有很大的优势。总之，开发的 qRT-PCR 检测方法是一种可靠、特异和敏感的鱼类样本中 HRV 定量诊断工具。
