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猪瘟病毒野毒株TaqMan-MGB荧光定量PCR、鉴别方法的建立与应用
引用本文:刘俊,王琴,范学政,徐璐,赵启祖,黄伟,汤波,沙莎,周远成,陈蕾,邹兴启.猪瘟病毒野毒株TaqMan-MGB荧光定量PCR、鉴别方法的建立与应用[J].中国农业科学,2009,42(12):4366-4371.
作者姓名:刘俊  王琴  范学政  徐璐  赵启祖  黄伟  汤波  沙莎  周远成  陈蕾  邹兴启
作者单位:1. 中国兽医药品监察所,国家猪瘟参考实验室,北京,100081;西南大学荣昌校区,重庆,402460
2. 中国兽医药品监察所,国家猪瘟参考实验室,北京,100081
3. 西南大学荣昌校区,重庆,402460
基金项目:国家支撑计划课题,北京市自然科学基金 
摘    要: 【目的】建立一种快速、敏感、特异地检测猪瘟病毒野毒的一步TaqMan-MGB 荧光定量PCR方法,为临床鉴别猪瘟野毒和HCLV疫苗毒提供了准确可靠的工具。【方法】在猪瘟病毒基因组5′端非编码保守区设计一对猪瘟病毒通用引物和一条特异性MGB探针,通过优化,得到最佳反应体系和反应条件,进行特异性、灵敏度和临床样本符合检验。【结果】该方法在100~10-7范围内线性相关系数为0.998,检测极限达5.3×10-2pg 病毒核酸;对24个质控样本检测结果显示,该方法能检测除猪瘟兔化弱毒疫苗(HCLV)以外的猪瘟流野毒株,其它猪相关致病病原检测阴性;对122份疑似猪瘟样本检测的结果与本实验室建立的RT-nPCR方法的符合率为94.3%(115/122),阳性和阴性符合率分别为86.4%(38/44)和98.7%(77/78),Kappa值为0.87>0.75。【结论】建立的一步TaqMan-MGB 荧光定量PCR方法能鉴别猪瘟野毒和HCLV疫苗毒,且特异性好、灵敏度高、与笔者先前建立的RT-nPCR方法对临床样本的检测结果具有高度的一致性。

关 键 词:猪瘟  荧光定量PCR  TaqMan-MGB  兔化弱毒疫苗
收稿时间:2009-01-05;

Differentiation of Wild-Type Viruses and HCLV Vaccine of Classical Swine Fever Virus by One-Step Fluorescent Quantitative PCR Using TaqMan-MGB Probe Technology
LIU Jun,WANG Qin,FAN Xue-zheng,XU Lu,ZHAO Qi-zu,HUANG Wei,TANG Bo,SHA Sha,ZHOU Yuan-cheng,CHEN Lei,ZOU Xing-qi.Differentiation of Wild-Type Viruses and HCLV Vaccine of Classical Swine Fever Virus by One-Step Fluorescent Quantitative PCR Using TaqMan-MGB Probe Technology[J].Scientia Agricultura Sinica,2009,42(12):4366-4371.
Authors:LIU Jun  WANG Qin  FAN Xue-zheng  XU Lu  ZHAO Qi-zu  HUANG Wei  TANG Bo  SHA Sha  ZHOU Yuan-cheng  CHEN Lei  ZOU Xing-qi
Institution:(China Institute of Veterinary Drug Control, National Classical Swine Fever Reference Lab )
Abstract:Objective] A rapid one step fluorescent quantitative PCR assay using TaqMan minor-groove-binding (MGB)probe was developed and evaluated to discriminate wild-type stains from wild-type stains and hog cholera lapinized virus (HCLV)strain of classical swine fever virus (CSFV).Method]A pair of universal primer and a wild-type specific MGB probe were designed from the conservative region of CSFV genome.Then the method was optimized.Specificity,sensitivity,and conformity were tested. Results] The detection results of 24 quality-control samples showed good specificity.The sensitivity of the assay was 5.3×10~(-2)pg viral RNA per reaction,and showed a good linear relationship at a range of 10~0-10~(-7).The coincidence rate was 94.3% in detecting 122 clinical samples compared with RT-nPCR (115/122),and the positive coincidence rate and negative coincidence rate were 86.4% (38144) and 98.7% (77/78),respectively,Kappa value was 0.87>0.75. Conclusion] The one-step TaqMan-MGB PCR is able to differentiate wild-type and HCLV of CSFV strain,which shows good specificity,sensitivity and also shows high consistency of TaqMan-MGB Probe Technology and RT-nPCR method.This assay may serve as a useful diagnostic tool for CSF.
Keywords:TaqMan-MGB  classical swine fever  fluorescent quantitative PCR  TaqMan-MGB  CSFV
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