A型猪流感病毒TaqMan探针荧光RT-PCR检测方法的建立

发布时间：2018-06-13 14:16

本文选题：A型猪流感病毒 + TaqMan探针　；参考：《中国动物检疫》2016年12期

【摘要】：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。
[Abstract]:A pair of specific primers and a Taq Man probe were designed and synthesized according to the conserved sequence of the matrix protein (MN) encoding gene of Swine Influenza virus type A (SIV), and a fluorescent RT-PCR method for detecting SIV was established. The results showed that the method was specific to H1H3N2 and H9N2 subtype SIV, but not cross-amplified to other common viruses such as swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus and porcine infectious gastroenteritis virus. The optimal linear detection range for M gene RNA control SIV-M-RNAs is 3.8 脳 10 ~ (-1) ~ 3.8 脳 10 ~ (-8) copy number / response. The standard curve equation is Y-3.4365X 40.091, the correlation coefficient is 0.999 ~ (2), and the lowest detection limit is 38 copies of SIV-M-RNA. Three SIV-M-RNAs with different concentrations of 3. 8 脳 10 ~ (3) 3. 8 脳 10 ~ (5) copies/ 渭 L) were tested by repeated experiments. The coefficients of variation of Ct values of each concentration were less than 1. 5%, which showed that SIV-M-RNA had good reproducibility. This method was used to detect the SIV of 860 imported pig nasal swab samples and 78 domestic pig farm nasal swabs samples. The results showed that the SIV test of imported pig nasal swab samples were all negative and 17 domestic pig farm nasal swabs samples were positive for SIV detection. This study provides a rapid, sensitive and specific detection method for swine influenza A virus.
【作者单位】：天津出入境检验检疫局动植物与食品检测中心;
【基金】：天津市科技支撑项目(13ZCZDNCO1300) 天津市滨海新区惠民项目(2013-BK15H013)
【分类号】：S852.65

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