TaqMan探针荧光PCR检测副猪嗜血杆菌方法的建立和应用

Development and application of real-time PCR with TaqMan probe for detection of Hemophilus parasuis

为了建立基于TaqMan探针荧光PCR技术检测副猪嗜血杆菌的方法,本试验参考GenBank已发表的副猪嗜血杆菌(Hemophilus parasuis,HPS)转录起始因子infB基因序列,利用生物学软件Primer Express 2.0设计特异性引物和探针;并对副猪嗜血杆菌标准菌株提取DNA后进行PCR扩增,将产物测序鉴定后克隆到pMD18-T载体,再转化到大肠杆菌感受态细胞,细菌培养后对菌液进行PCR扩增,把产物测序鉴定后获得的重组质粒作为标准阳性对照;最后将建立的TaqMan探针荧光PCR方法进行灵敏度、特异性、稳定性试验。结果显示,该检测方法可以达到1μl 1.0×101拷贝数量级的灵敏度;另外,该方法可以将HPS和大肠杆菌O157、沙门氏菌、金黄色葡萄球菌、猪链球菌分开,特异性良好;稳定性试验显示,2次批内变异系数分别为0.65%和0.92%,批间变异系数为1.31%,稳定性良好。对口岸送检的56份猪肉和12份血浆蛋白粉样品进行检测,结果表明TaqMan探针荧光PCR方法和细菌分离方法的检测效果一致。

A method of real-time PCR with TaqMan probe for detection of Hemophilus parasuis（HPS） was developed.Primers and probes were designed by biology software Primer Express 2.0 based on transcription initiation factor 2 sequences of HPS published in GenBank.DNA of HPS was extracted and amplified by PCR.The PCR product was cloned into pMD18-T and transferred into the competent cells of Escherichia coli.The recombinant plasmid was set as standard positive control.The sensitivity,specificity,and stability of the method were tested.This method could detect 10 copies per μl and could distinguish HPS from E.coli O157,Salmonella,Staphylococcus aureus and Streptococcus suis.The stability test showed that the intraassay coefficients of variation（CV） were 0.65% and 0.92%,and interassay CV was 1.31%.56 porks and 12 plasma protein powder samples were detected for HPS by real-time PCR,bacteria isolation and routine PCR,and the former two got the same result.