《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (1): 239-246.doi: 10.16431/j.cnki.1671-7236.2019.01.028

• 预防兽医 • 上一篇    下一篇

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

李晓菲, 陈婷, 魏笑笑, 孙爱荣, 黄艳, 葛晓杰, 徐婷, 王彩宏, 秦立廷   

  1. 山东新希望六和集团有限公司, 青岛 266000
  • 收稿日期:2018-05-21 出版日期:2019-01-20 发布日期:2019-01-19
  • 通讯作者: 秦立廷 E-mail:qinlt2013@163.com
  • 作者简介:李晓菲(1988-),女,山东青岛人,硕士,研究方向:诊断试剂研发,E-mail:965232411@qq.com
  • 基金资助:

    国家重点研发计划项目(2016YFD0501506、2017YFD0500600)

Establishment and Application of TaqMan-MGB Real-time RT-PCR Assay for Detection of Porcine Reproductive and Respiratory Syndrome Virus

LI Xiaofei, CHEN Ting, WEI Xiaoxiao, SUN Airong, HUANG Yan, GE Xiaojie, XU Ting, WANG Caihong, QIN Liting   

  1. Shandong New Hope Liuhe Group Co., Ltd., Qingdao 266000, China
  • Received:2018-05-21 Online:2019-01-20 Published:2019-01-19

摘要:

为了快速、准确地检测猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome,PRRSV),本研究根据PRRSV基因序列设计特异性引物和探针,建立了一种可同时检测PRRSV经典毒株、高致病性变异毒株以及近几年中国新出现的NADC30-like毒株的TaqMan-MGB实时荧光定量方法,并对该方法的特异性、敏感性和重复性进行验证,同时用建立的实时荧光定量方法与常规PCR方法对临床收集的120份疑似PRRSV样品进行检测。结果表明,该方法特异性良好,对PRRSV的经典毒株、高致病性变异毒株及NADC30-like毒株均有良好的扩增,但对猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(RV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)的检测结果均为阴性,无交叉反应;模板浓度在101~108拷贝/μL范围内具有良好的线性关系,标准曲线结果显示其扩增的相关系数为0.9999,扩增效率为93%;敏感性高,约是常规PCR方法的100倍,最低可以检测到101拷贝/μL的模板;重复性好,批内和批间重复性试验变异系数分别为0.17%~0.90%和0.65%~2.34%;用本研究所建立的实时荧光定量检测方法对120份临床样品进行检测,PRRSV阳性检出率为59.2%(71/120),而常规PCR方法的PRRSV阳性检出率为44.2%(53/120)。该方法的建立为PRRSV的实验室诊断、流行病学调查,以及预防和控制中国PRRSV的流行提供了快速、准确的检测手段。

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

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 Real-time RT-PCR method was specific for PRRSV classic strain,highly pathogenic mutant strain,as well 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 were 0.17% 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.

Key words: porcine reproductive and respiratory syndrome virus (PRRSV); TaqMan-MGB Real-time RT-PCR assay; clinical samples

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