猪瘟病毒野毒株TaqMan-MGB荧光定量PCR鉴别方法的建立与应用

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BCLV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10~0～10~(-7)范围内线性相关系数为0.998,检测极限达5.3×10~(-2)pg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~(-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.