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

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。

Development of rea1-time fluorescent quantitative PCR assay based on TaqMan probe for detection of PRRSV

In order to 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,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 was compared with that of routine RT-PCR and viral isolation tests. TaqMan fluorescent quantitative PCR for detection of PRRSV was established successfully with the optimal probe concentration 0.4μmol and detection limit as low as 3.51copies/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 routine PCR(25/30). The results indicated this method showed high specificity, sensitivity and reproducibility and could be used for the diagnosis of PRRSV infection.