小鼠仙台病毒TaqMan-MGB探针荧光定量RT-PCR检测方法的建立

Development of A RT-qPCR Method Using A TaqManTM-MGB Fluorescent Probe for Detection of Sendai Virus in Mice

仙台病毒(Sendai virus, SeV)能够引起啮齿类动物呼吸道疾病,引起免疫应答,严重影响SPF级动物模型实验结果,实验动物质量国家标准T/CALAS49-2017也将其作为SPF级大小鼠质量检测必检项目之一。为了开发用于快速检测及日常监测SeV的分子生物学诊断方法,本研究基于TaqMan-MGB探针荧光技术,根据GenBank中SeV的L基因的保守区段(8 556-15 242)序列,设计了1对特异性引物与1条5‵端携带羧基荧光素(FAM)与3‵端为淬灭基团(Eclipse)标记的TaqMan探针,标准品进行稀释建立标准曲线,并经反应条件优化,建立了TaqMan-MGB探针荧光定量RT-PCR检测方法,并对其特异性、敏感性与重复性进行评估。结果显示,最佳优化反应条件为:反应体系,20μmol/L;引物和探针浓度,10μmol/L和15μmol/L;退火温度,54℃;在特异性试验中,除SeV检测出现特异性曲线外,其他鼠类常见病毒均未出现特异性曲线;在敏感性试验中,Sev最低检测限为5.29×10^(1)拷贝/μL。在重复性试验中,其批内变异系数为0.44%~0.77%,批间变异系数为0.44%~1.03%。对临床样本进行检测,检出率高于常规的RT-PCR方法。因此,以SeV L基因保守序列为靶基因建立的TaqMan探针荧光定量RT-PCR方法具有敏感性高、特异性强、良好的重复性等优点,适用于对SPF级实验动物的SeV定期监测以及对野生鼠类的SeV日常监测和流行病学调查,为相关研究提供了良好的技术支持。

The Sendai virus(SeV) can cause respiratory diseases, induce an immune response, and seriously affect the results of experiments in specific pathogen-free(SPF) animals. The national standard in China for laboratory-animal quality(T/CALAS49-2017) is used for SPF rats and mice. We wished to develop a diagnostic method based on molecular biology for rapid detection and daily monitoring of the SeV. This study was based on a TaqMan-MGB fluorescent probe as well as the sequence of the conserved segment(8,556-15,242) of the L gene of the SeV in GenBank. First, we diluted specific primers. Then, a TaqMan ? probe labeled with carboxyfluorescein at the 5’ end and a quenching group(Eclipse) at the 3’ end was created. We diluted standards to establish a standard curve. The reaction conditions were optimized to establish a real-time reverse transcription-quantitative polymerase chain reaction(RT-qPCR) detection method. The specificity, sensitivity, and repeatability of this RT-qPCR method were evaluated. We ascertained the optimal reaction conditions: reaction system = 20 μmol/L;primer concentration = 10 μmol/L;probe concentration = 15μmol/L;annealing temperature = 54°C. In the sensitivity test, the lower limit of detection of the SeV standard was 5.29×10^(1) copies/μL, which was about the same order of sensitivity as that of the SYBR Green RT-qPCR protocol. In the repeatability test, the intra-assay coefficient of variation was 0.44%–0.77% and the inter-assay coefficient of variation was 0.44% – 1.03%. For clinical samples, the detection rate of this method was 15%.Therefore, we established a RT-qPCR method using a TaqMan fluorescent probe employing the conserved sequence of the L gene of the SeV as the target gene. This method had the advantages of high sensitivity, strong specificity, and good reproducibility. Our method is suitable for regular detection of the SeV and disease in SPF experimental animals.