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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 pair of specific primers and a TaqMan probe were designed and synthesized according to the conserved sequence of matrix protein (M) gene of swine influenza virus type A (SIV). A fluorescent RT-PCR method for detecting type A SIV was established. The results showed that the method for the H1N1, H3N2 and H9N2 subtypes SIV were amplified specifically for classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus and swine transmissible gastroenteritis virus pigs The most common linear detection range of MIV RNA was (SIV-M-RNA) was 3.8 × 10-1 ~ 3.8 × 10-8 copies / reaction. The standard curve equation Y = -3.4365X + 40.091, correlation coefficient (R ~ 2) was 0.999 8, and the lowest detection limit was 38 copies of SIV-M-RNA. Inter-group and in-group replicates of SIV-M-RNA in three different concentrations (3.8 × 10-3 ~ 3.8 × 10 ~ 5 copies / μL) showed that the CVs of each concentration were less than 1.5% Good reproducibility. Using this method, 860 imported swine nasal swab samples and 78 domestic swine swine nasal swab samples SIV test results of imported swine nasal swab samples SIV test were negative, 17 domestic swine swine nasal swab samples SIV Test positive. This study provides a rapid, sensitive and specific detection of type A swine influenza virus.