TaqMan~T-PCR对水疱性口炎病毒的鉴定检测

TaqMan~ RT-PCR for the Detection and Differentiation of Indiana and New Jersey Strains of Vesicular Stomatitis Virus

水疱性口炎分为印第安那型 ( Indiana)和新泽西型 ( New Jersey)。这两种血清型病毒的快速和可靠的鉴别对该病的诊断、检疫、分子流行病学调查和监测至关重要。文章按照 VSV核蛋白基因序列 ,设计了一对两型通用引物和两型各自特异性探针。研究建立了 VSV实时荧光定量 PCR检测方法 ,对 VSV细胞培养物、人工感染实验动物组织、血清样品 ,以及系列稀释的不同 TCID50 样品、其它相关或相似病毒进行鉴定 ,同时与常规 PCR、病毒分离试验作了比较。Taq Man○R RT-PCR的特异性和敏感性相当于或优于对照方法。重复性和稳定性试验证实 ,该方法可靠。每个试验中设立阳性、阴性对照和标准稀释度对照 ,使试验结果可对病毒 RNA作准确定量 ,并可在 4h内获得结果。研究结果表明 ,Taq Man○R RT-PCR方法是一种特异性强、敏感性高、快速安全的定量检测方法。因此 ,可以作为 VSV的快速检测和定型。

Object Vesicular stomatitis virus (VSV) is responsible for enzootic and occasionally epizootic disease in cattle, swine, and horses. Human beings are also readily susceptible to VSV infection with the production of influenza-like illness. In cattle, swine, and horses, VSV infections cause severe oral and pedal erosive and ulcerative lesions, resulting in considerable economic damage. These lesions are easily mistaken for those produced during etiologically unrelated viral diseases, including foot-and-mouth disease, swine vesicular disease, and other exanthemous viral livestock diseases. Their rapid and reliable detection and differentiation is essential for disease surveillance. Method A quantitative TaqMan^((r)) reverse transcription-polymerase chain reaction (RT-PCR) is described for VSV detection and strain differentiation. The VSV-New Jersey and VSV-Indiana serotypes could be differentiated through the selective use of the appropriate primers and probes. Result Sensitivity and specificity were compared with a conventional VSV RT-PCR and virus isolation, and to the detection of both VSV types in cell cultures and both were found to be equal or superior to the reference methods. Reproducibility was tested and proved that the assay was very reliable. Standard dilutions included in each test allowed absolute quantitation of the amount of viral RNA. Conclusion The TaqMan^((r)) assay described is time-saving, easy to handle, exhibits a decreased risk of cross-contamination and is highly sensitive and specific. It is, therefore, considered to be a powerful tool for the rapid detection and differentiation of VSV. This assay is ideal for the study of VSV pathogenesis and persistence.