柯萨奇病毒B组1型TaqMan一步法荧光定量 RT-PCR检测方法的建立

Establishment of a one-step TaqMan quantitative real-time RT-PCR assay to detect coxsackievirus B1

目的 建立柯萨奇病毒B组1型(coxsackievirus B1,CV-B1)特异的TaqMan一步法荧光定量RT-PCR检测方法。方法 根据CV-B1的VP1序列,设计特异性引物和TaqMan探针。将目的基因插入pMD19;载体,在DH5α感受态细胞中扩增,体外转录后获得RNA标准品,建立标准曲线。并对检测方法的灵敏度、特异性、重复性进行评价。结果 该方法在10^(2)-10^(11)拷贝/μl的模板范围内具有良好的线性关系(R^(2)=1.000),扩增效率E=107.5%。灵敏度达1×10^(2)拷贝/μl,只对CV-B1有特异性扩增曲线,且重复性较好。结论 建立的实时荧光定量RT-PCR方法具有较高的灵敏度、特异性和重复性,可作为CV-B1的快速检测和绝对定量分析。

Objective To establish a TaqMan one-step fluorescence quantitative RT-PCR method for coxsackievirus B1(CV-B1) specific detection. Methods Specific primers and the TaqMan probe were designedaccording to the VP1 sequence of CV-B1. The target gene was inserted into the pMD19;vector and amplified in DH5α competent cells.After in vitro transcription, an RNA standard was obtained and the standard curve was established. The sensitivity, specificity, and reproducibility of the assay were evaluated, and the viral load of the CV-B1 strain was detected at different time points after inoculation in Vero cells. Results In10^(2)-10^(11) copy/μl, the method showed a good linear correlation within the limits of the template(R^(2)=1.000). The amplification efficiency E=107.5%. The sensitivity was 1×10^(2) copies/μl, the specific amplification curve was only for CV-B1, and the reproducibility was good. The RNA load of CV-B1 could be detected at different days after infection. Conclusions The established real-time fluorescent quantitative RT-PCR shows high sensitivity, specificity, and reproducibility, and can be used for rapid detection and absolute quantitative analysis of CV-B1.