猕猴结核分枝杆菌PCR检测方法的建立与初步应用

Establishment of Touchdown PCR Method for Detection of Mycobacterium from Rhesus monkey and Its Preliminary Application

目的建立猕猴结核分枝杆菌PCR快速检测方法。方法根据GenBank中报道的人型结核分枝杆菌标准株H37RV全基因序列,针对其中致病性结核分枝杆菌所特有的保守区域ESAT-6设计一对引物进行Touchdown PCR反应,利用此方法对100份野生来源猕猴全血标本进行检测,并与传统的PPD实验和咽拭子抗酸染色实验结果作比对。结果抽取33份野生来源猕猴的外周血,提取DNA进行扩增,扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的序列基本相同,检测标本中有4份(4/33)为阳性,其中3份(3/33)标本与结核菌素试验和咽拭子抗酸染色试验结果相符合。结论建立了从猴全血中直接检测猴结核分枝杆菌DNA的Touchdown PCR方法,具有敏感性和特异性高,且简便的优点。

Objective To establish a rapid PCR detective method of the laboratory monkeys. Methods Two primers were designed according to the conserved sequence （ESAT-6） from the standard strain H37RV in the GenBank, and the positive plasimid control we constructed, and 33 samples of rhesus monkey blood were detected with touchdown PCR we have built. Also, we compared this new method with traditional PPD （Purified protein derivative ） test and acid-fast stain of throat swab from monkeys. Result The results indicated both the positive plasimid control we constructed and 4 samples were postive, while the traditional PPD test and acid-fast stain method can only test 3 samples positive which PCR also showed positive result. The amplified fragment of samples was highly homology comparing with H37RV gene sequence in the GenBank by using BLAST software. Conclusion A sensitive, specifical and rapid method of touchdown PCR was established for detection of Mycobaeterium from rhesus monkey.