中国畜牧兽医2019，Vol.46Issue(1)：239-246,8.DOI:10.16431/j.cnki.1671-7236.2019.01.028
猪繁殖与呼吸综合征病毒TaqMan-MGB实时荧光定量RT-PCR方法的建立及应用
Establishment and Application of TaqMan-MGB Real-time RT-PCR Assay for Detection of Porcine Reproductive and Respiratory Syndrome Virus
摘要
Abstract
In order to quickly and accurately detect porcine reproductive and respiratory syndrome (PRRSV) , one TaqMan-MGB Real-time RT-PCR assay for detection of PRRSV was established using specific primers and probe based on PRRSV gene.The developed Real-time RT-PCR method could detect PRRSV classic strain, highly pathogenic mutant strain, as well as the new NADC30-like strain at the same time.The specificity, sensitivity and repeatability of the established method were detected.120 clinical samples were detected using the developed Real-time RT-PCR method and the conventional PCR method.The results showed that TaqMan-MGB Realtime RT-PCR method was specific for PRRSV classic strain, highly pathogenic mutant strain, aswell as the NADC30-like strain, and the test results were negative with CSFV, PEDV, TGEV, RV, PRV, PPV and PCV2.The method showed a good linear relationship within the template ranges from 101 to 108 copies/μL, and its sensitivity was 100 times that of routine PCR assay.The correlation of the standard curve was 0.9999, and the efficiency was 93％.The limit of detection concentration was 101 copies/μL.The coefficient of variation in the intra-and inter-assays were0.17％to 0.90％ and 0.65％ to 2.34％, respectively.The positive detection rate of PRRSV in clinical samples using the developed Real-time RT-PCR method (59.2％) was higher than that using the conventional PCR method (44.2％) .The establishment of Real-time RT-PCR method provided a rapid and accurate detection means for early diagnosis and epidemiological investigation of the disease.关键词
猪繁殖与呼吸综合征病毒 (PRRSV)/TaqMan-MGB实时荧光定量方法/临床样品Key words
porcine reproductive and respiratory syndrome virus (PRRSV) /TaqMan-MGB Real-time RT-PCR assay/clinical samples分类
农业科技引用本文复制引用
李晓菲,陈婷,魏笑笑,孙爱荣,黄艳,葛晓杰,徐婷,王彩宏,秦立廷..猪繁殖与呼吸综合征病毒TaqMan-MGB实时荧光定量RT-PCR方法的建立及应用[J].中国畜牧兽医,2019,46(1):239-246,8.基金项目
国家重点研发计划项目(2016YFD0501506、2017YFD0500600) （2016YFD0501506、2017YFD0500600）
