一种基于CP204L基因的非洲猪瘟病毒Taq Man-MGB实时荧光PCR检测方法的建立

Development of a TaqMan-MGB probe real-time PCR assay for detection of african swine fever virus based on CP204L gene

基于非洲猪瘟病毒(ASFV)CP204L基因序列设计1对特异性引物和TaqMan-MGB探针,通过优化反应条件建立了一种检测ASFV的TaqMan-MGB实时荧光PCR方法。结果显示,该方法仅对ASFVDNA呈现特异性扩增,不与猪的其他常见病毒发生交叉反应,具有良好的特异性。该方法对标准对照质粒(PCR2.1-CP204L)的线性检测范围为4.2×10~1~4.2×10~9copies/μL,标准曲线方程为Y=-3.452 3X+39.014,相关系数(R^2)为0.998 8,检测下线为4.2个拷贝。对5个不同浓度(4.2×10~3~4.2×10~7copies/μL)的质粒标准品进行4次双份样品的重复检测,Ct值变异系数均小于1.5%,表明该方法具有良好的重现性。用该方法对总计164份的进口猪临床样品和生猪肌肉样品进行ASFV检测,结果均为阴性。该方法的建立为活猪临床样品和生猪肌肉样品中ASFV检测提供了一种快速、敏感和特异的技术手段。

By optimization of reacting conditions,a TaqMan-MGB probe real-time PCR was developed with a pair of specific primers and probe designed according to the conserved regions of the CP204 L gene sequences of African swine fever virus（ASFV）.The method had high specificity for the ASFV DNA and no cross-reactivity was observed among DNAs and cDNAs of other common swine viruses.The assay showed good linearity between Ct values and copies of the standarde plasmid from 4.2 × 10~1 to 4.2× 10~9copies/reaction（standard equation Y=-3.4523X＋39.014,correlation coefficient R^2=0.9988）,and the detection limit of the assay was as low as 42 copies of the standarde plasmid.Coefficiency of variation（CV） of Ct values of the standard plasmid tested in duplicate four times ranging from 4.2× 10~3 to 4.2 × 10~7 copies/μL by the assay were less than 1.5%.The 164 samples from the imported pigs and porks were detected to be negative by the assay.In conclusion,the method provides a useful tool for the detection of ASFV in the samples from the pigs and porks.