基于TaqMan探针的猪繁殖与呼吸障碍综合症病毒实时荧光定量PCR方法的建立

Development of real-time fluorescent quantitative PCR assay for detection of PRRSV based on TaqMan probe

目的建立一种能检测猪繁殖与呼吸障碍综合症病毒(PRRSV)的TaqMan探针荧光定量PCR方法。方法根据PRRSV的ORF7基因保守区的核苷酸序列设计引物和TaqMan探针,通过探针浓度的优化,建立检测PRRSV的TaqMan探针荧光定量PCR方法。用该方法对30份临床疑似病料进行检测,并与常规RT-PCR方法和病毒分离方法进行比较。结果 TaqMan荧光PCR检测PRRSV的最佳探针浓度为0.4μmol,检测灵敏度可达3.51拷贝/μL。检测的30份样品与病毒分离结果的符合率为100%,与普通PCR的检测结果(25/30)比较,本方法对临床样品的检出率(28/30)更高。结论建立的方法特异性强、敏感性高、重复性好,可用于临床样品的检测。

We establish a TaqMan real-time PCR assay for detection of PRRSV.The specific primers and probes were designed in the conserved region of the ORF7 gene for PRRSV,and the real-time fluorescent quantitative PCR was established by optimizing the probe concentration.Thirty clinical samples were detected by using the established quantitative RT-PCR assay,and the results were compared with that of conventional RT-PCR and viral isolation tests.TaqMan fluorescent quantitative PCR for detection of PRRSV was established successfully with the optimal probe concentration 0.4 μmol,and detection limit was as low as 3.51 copies/μl.The results by the TaqMan real-time PCR method were 100% consistent with the viral isolation tests.Sensitivity and positive rate（28/30） for clinical samples of TaqMan fluorescent quantitative PCR were relatively higher than conventional PCR（25/30）.The results indicated this method has high specificity,sensitivity and reproducibility,and could be used for the diagnosis of PRRSV infection.