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.pdfVIP

中国人兽共患病学报 2012 , 28 (6) Chinese Journal of Zuono:;es 579 001: 10. 3969/cjz. j. issn. 1002 - 2694. 2012. 06. 013 基于 TaqMan 探针的猪繁殖与呼吸障碍综合症病毒 实时荧光客量 PCR 方法的建立祷 祝秀梅，马全美，社乎，王凡，吕志慧，牟克斌 摘 要: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p(双方法。方法 根 据 PRRSV 的 ORF7 基因保守区的核苦酸序列设计引物和 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. 关键词:猪繁殖与呼吸综合征病毒，ORF7 基因;TaqMan 荧光定量 PCR 中图分类号:R373 文献标识码:Ä 文章编号: 1002- 2694(2012)06- 0579-04 Development of real-time f1 uorescent quantitative PCR assay for detection of PRRSV based on TaqMan probe ZHU Xiu-mei.MA Quan-yin.DU Pin , WANG Fan , LÜ Zhi-hui , MU Ke-bin (China Agricultural Veterinary Biological Science and Technology Co. • Ltd. Lanzhou 730046. China) ABSTRACT: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 estahlished quantitative RT-PCR as- say. 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 O. 4 ,._mol. and detection limit was as low as 3.51 copies/μ1. The results hy the TaqMan real-time PCR method were 100% consistent with the viral isolation te