一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.02.009

›› 2016, Vol. 43 ›› Issue (2): 348-355.doi: 10.16431/j.cnki.1671-7236.2016.02.009

一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立

王建华, 董志珍, 王玉玲, 肖妍, 赵祥平

天津出入境检验检疫局动植物与食品检测中心, 天津 300456

收稿日期:2015-08-04 出版日期:2016-02-20 发布日期:2016-03-02
通讯作者: 赵祥平 E-mail:zhaoxp@tjciq.gov.cn
作者简介:王建华(1965-),男,黑龙江林口人,博士,高级兽医师,研究方向:动物疫病检测与诊断试剂研制,E-mail:wjhwqy1965@163.com
基金资助:
天津市科技支撑项目(13ZCZDNCO1300);天津市滨海新区科技计划项目(2013-BK15H013)

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

WANG Jian-hua, DONG Zhi-zhen, WANG Yu-ling, XIAO Yan, ZHAO Xiang-ping

Animal, Plant and Foodstuff (APF) Inspection Center, Tianjin Entry-exit Inspection and Quarantine Bureau, Tianjin 300456, China

Received:2015-08-04 Online:2016-02-20 Published:2016-03-02

摘要/Abstract

摘要： 为建立一种快速、敏感和特异地鉴别尼帕病毒(NiV)和高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的检测方法,本试验以NiV M基因和HP-PRRSV nsp2基因为靶序列,通过优化反应条件建立了一种二重荧光RT-PCR检测方法,并对该方法的特异性、定量线性范围、敏感性和重复性进行了评价及初步应用.结果显示,用该方法检测NiV M基因和HP-PRRSV nsp2基因的RNA标准对照(NiV-M-RNA和HP-PRRSV-nsp2-RNA),线性范围分别为4.6×10¹~4.6×10⁷和4.1×10¹~4.1×10⁸拷贝/μL;最低检出限分别为46和4.1拷贝;该方法组内试验和组间试验的变异系数均小于2.0%,显示出良好的可重复性;该方法仅对NiV和HP-PRRSV呈现特异性扩增曲线,不与猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪流感病毒(SIV)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV2)发生交叉反应.用该方法对236份猪实际样品进行NiV和HP-PRRSV核酸检测,所有样本的NiV检测结果均为阴性,8份样本的HP-PRRSV检测结果为阳性.本研究建立的方法为猪实际样本中NiV和HP-PRRSV的鉴别检测提供了一种快速、敏感和特异的技术手段.

关键词: 尼帕病毒; 高致病性猪繁殖与呼吸综合征病毒; TaqMan-MGB探针; 二重荧光RT-PCR方法

Abstract: To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.

Key words: Nipha virus; highly pathogenic porcine reproductive and respiratory syndrome virus; TaqMan-MGB probe; duplex Real-time RT-PCR

中图分类号:

王建华, 董志珍, 王玉玲, 肖妍, 赵祥平. 一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立[J]. , 2016, 43(2): 348-355.

WANG Jian-hua, DONG Zhi-zhen, WANG Yu-ling, XIAO Yan, ZHAO Xiang-ping. Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus[J]. , 2016, 43(2): 348-355.

参考文献

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一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

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