实时荧光定量PCR方法检测青藏高原喜马拉雅旱獭血样中布鲁氏菌DNA

Detection of Brucella DNA in blood samples of Marmota himalayana in Qinghai-Tibet Plateau by real-time fluorescence quantitative PCR

目的应用实时荧光定量PCR(real-time PCR)方法检测青藏高原喜马拉雅旱獭血样中布鲁氏菌DNA。方法2019—2020年收集青海省青藏高原喜马拉雅旱獭全血,进行布鲁氏菌病血清学试验,包括虎红平板凝集试验(RBT)、试管凝集试验(SAT)微量法、胶体金免疫试验(GICA),阳性标本进行real-time PCR检测。分析real-time PCR方法的灵敏度、特异度等指标,评价其结果的准确性。结果共收集全血1466份,RBT检出阳性64份,GICA检出阳性28份,SAT检出阳性18份。64份RBT阳性标本进行real-time PCR检测,其中56份标本及阳性对照菌株有特异的荧光扩增曲线,8份标本及阴性对照无特异的扩增曲线。real-time PCR与SAT、GICA比较灵敏度为100%,与RBT比较特异度为99.93%,Kappa值为0.81,高度一致。结论与传统方法相比,real-time PCR方法具有快速、特异等优点,可以用于青藏高原喜马拉雅旱獭样本检测。

Objective To detect Brucella DNA in blood samples of Marmota himalayana in Qinghai-Tibet Plateau by realtime fluorescence quantitative PCR(real-time PCR).Methods The whole blood samples of Marmota himalayana in Qinghai-Tibet Plateau were collected for serological tests of Brucella,including tiger red plate agglutination test(RBT),tube agglutination test(SAT)and colloidal gold immune test(GICA).The positive samples of serological tests were detected by real-time PCR.The SAT results was chosen as a diagnostic criteria to analyze the sensitivity and specificity of the improved degree of tube real-time PCR and evaluate the accuracy of the micro real-time PCR results.Results We collected 1466 whole blood samples of Marmota himalayana,in which 64 were positive in RBT,28 were positive in GICA,and 18 were positive in SAT.Sixty four RBT positive samples were detected by real-time PCR,in which 56 samples and positive control strains had specific amplification fluorescence curve,8 samples and negative control strains had no specific amplification curve.Real time PCR had a sensitivity of 100%compared with SAT and GICA and a specificity of 99.93%compared with RBT,the Kappa value was 0.81,which were highly consistent.Conclusion Compared with traditional methods,real-time PCR is rapid and highly specific,which is suitable for the detection of Marmota himalayana samples.