副结核荧光PCR试剂盒研制与应用

采用TaqMan荧光标记探针技术原理, 建立副结核分枝杆菌特异的实时荧光PCR快速检测鉴定方法并组装形成临床诊断试剂盒。试剂盒提供荧光PCR与样品核酸提取试剂, 检测全程包括样品处理可在1 d内完成。特异性试验结果表明, 试剂盒对8株副结核分枝杆菌标准菌株的检测均呈典型阳性反应, 对牛分枝杆菌、结核分枝杆菌等其它12种分枝杆菌标准菌株以及大肠杆菌、肺炎链球菌等多种常见微生物均呈阴性反应。试剂盒检测灵敏度可达单个菌细胞、15个基因拷贝, 比常规PCR检测灵敏度提高100倍。对每份添加50~100个菌细胞的20份阴性牛奶样品进行检测, 均呈阳性反应。重复性试验结果显示, 试剂盒组内变异系数为1.41%, 组间变异系数为2.42%。采用所研制的副结核分枝杆菌荧光PCR试剂盒对来自广东地区7个奶牛场的250份牛奶样品和粪便样品、来自10个养猪场的143份猪血清样品, 以及3批次进口食蟹猴共100份血清样品进行检测, 检出牛奶样品中副结核分枝杆菌阳性率为7.7%, 牛粪样品中阳性率为3.7%, 猪血清样品中阳性率为8.2%, 进口猴血清样品中阳性率为3.0%。

Development and Application of a Real Time PCR Detection Kit Specific for Mycobactium paratuberculosis

The real time PCR method based on TaqMan fluorescent DNA probe that specific for Mycobacterium paratuberculosis detection was established and developed into testing kit for rapid clinical diagnosis. The kit provided reagents for real time PCR and DNA extraction. The whole detection procedure included sample treatment and real time amplification could complete within 1 d. The testing kit could specifically identify 8 reference strains of M. paratuberculosis among various environmental existing microorganisms and 12 strains of other mycobacteria including M. tuberculosis, M. bovis and M. avium. Tests on M. paratuberculosis culture samples and serial ten fold dilution of recombinant plasmid containing target template showed that, the assay could detect single bacterium and 15 copies of target gene, which was 100 fold increase of sensitivity than the gel-based PCR using primers of the same sequences. And the test on 20 mimic infected milk sample containing 50~100 bacteria of M. paratuberculosis all yielded positive results. The inter-assay CV% and intra-assay CV% of the kit was 1.41%, 2.42% respectively. We used the testing kit to investigate infection of M. paratuberculosis on domestic and imported animals. Nature samples including 250 fecal and milk samples from 7 cattle farms, 143 serum samples from 10 pig farms within Guangdong province, and 100 serum samples from three shipments of imported monkey, were collected and tested. The positive rate of M. paratuberculosis was 7.7% in cattle serum, 3.7% in cattle fecal, 8.2% in pig serum and 3.0% in monkey serum respectively.