猪瘟病毒野毒株TaqMan-MGB荧光定量PCR鉴别方法的建立与应用  

Differentiation of Wild-Type Viruses and HCLV Vaccine of Classical Swine Fever Virus by One-Step Fluorescent Quantitative PCR Using TaqMan-MGB Probe Technology

作　　者：刘俊[1,2] 王琴[1] 范学政[1] 徐璐[1] 赵启祖[1] 黄伟[2] 汤波[1,2] 沙莎[2] 周远成[1] 陈蕾[1] 邹兴启[1] 

机构地区：[1]中国兽医药品监察所/国家猪瘟参考实验室,北京100081 [2]西南大学荣昌校区,重庆402460

出　　处：《中国农业科学》2009年第12期4366-4371,共6页Scientia Agricultura Sinica

基　　金："十一五"国家支撑计划课题(2006BAD06A12;2006BAD06A18);北京市自然科学基金(5072041)

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。【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^-2pg 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%（38/44） 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.

关 键 词：猪瘟 荧光定量PCR TaqMan-MGB 兔化弱毒疫苗 

分 类 号：S852.65[农业科学—基础兽医学]