A型猪流感病毒TaqMan探针荧光RT-PCR检测方法的建立

Development of a Real-time RT-PCR Assay with TaqMan Probe for Detecting Swine Influenza A Virus

根据A型猪流感病毒(SIV)的基质蛋白(M)编码基因保守序列设计合成一对特异性引物和一条Taq Man探针,建立了一种检测A型SIV的荧光RT-PCR方法。结果显示,该方法对H1N1、H3N2和H9N2亚型SIV均呈特异性扩增,对猪瘟病毒、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒和猪传染性胃肠炎病毒等猪的其他常见病毒无交叉扩增反应;该方法对M基因RNA对照(SIV-M-RNA)的最适线性检测范围为3.8×10~1~3.8×10~8拷贝数/反应,标准曲线方程为Y=-3.4365X+40.091,相关系数(R^2)为0.999 8,最低检出限为38个拷贝数的SIV-M-RNA。对3个不同浓度(3.8×10~3~3.8×10~5 copies/μL)的SIV-M-RNA进行组间和组内重复试验,每个浓度的Ct值变异系数均小于1.5%,具有良好的重现性。用该方法对860份进口猪的鼻拭子样本和78份国内猪场猪鼻拭子样本进行SIV检测,结果进口猪鼻拭子样本的SIV检测均为阴性,17份国内猪场猪鼻拭子样本SIV检测为阳性。本研究提供了一种快速、敏感和特异的A型猪流感病毒检测方法。

A Taqman probe-based real-time RT-PCR was developed with a pair of primers and a probe designed according to the conserved region of M gene sequence of swine influenza A virus（SIV）. Results showed the assay was specific to detect subtype H1N1,H3N2 and H9N2 of swine influenza A virus and had no cross-reaction with classical swine fever virus（CSFV）,porcine reproductive and respiratory syndrome virus（PRRSV）,porcine epidemic diarrhea virus （PEDV）and transmissible gastroenteritis virus（TGEV）. The real-time RT-PCR assay has a broad linear detection range for RNA standard control of M gene of SIV（SIV-M-RNA）from 3.8×101 copies/μL to 3.8×108 copies/μL,the standard curve equation was Y=-3.4365X＋40.091,and the correlation coefficient of the assay was 0.999 8. This assay could detect 38 copies/μL of SIV-M-RNA at the lowest level. Three different concentrations of SIV-M-RNA were used to test the repeatability,and the coefficients of variation（CVs）of both inter-assay and intra-assay were less than 1.5%, showing good repeatability. 938 nasal swab samples were tested for SIV detection by the assay. No positivereaction was found for all 860 samples from imported pigs. 17 of 78 samples from domestic pigs were found to be positive for SIV detection. These results suggested that the newly established real-time RT-PCR would provide a rapid,sensitive and specific detection for swine influenza A virus.