环介导等温扩增技术检测痰标本中结核分枝杆菌的研究

Detecting My cobacterium Tuberculosis in Sputum Specimens by Technique of Loop-mediated Isothermal Amplification

〔目的〕寻找1种快速的,且敏感度高,特异性强的结核分枝杆菌的检测方法。〔方法〕环介导等温扩增基因检测(Loop-mediated isothermal amplification LAMP)是一种便捷、灵敏度高而又特异性强的核酸基因扩增检测技术。本课题组根据结核分枝杆菌gyrB特征性序列设计3对特征性引物对痰液中和培养基中的结核分枝杆菌进行检测。其中1对环状引物结合于特征性序列中环状结构部分,可极大地加快LAMP反应速度,且不干扰内引物在LAMP反应中作用。〔结果〕实验结果表明:使用环状引物的LAMP反应可在0.5h内完成,总共分析时间不超过1h。LAMP检测技术的灵敏度比经典PCR技术高100倍左右,LAMP检测技术具有与实时PCR(Real-timePCR)技术相同的特异度。另外,由于LAMP检测技术是在恒温的情况下进行,不需要特别昂贵的仪器设备,检测方法简单,结果判定直接。〔结论〕该方法的特点和优势可以使之在基层实验室及现场监测方面广泛使用,在快速检测方面具有一定的开发潜力。

Objective To find out a method of high specificity, efficiency and rapidity to detect Myeobaeterium tuberculosis in speutam specimens. Method Loop-mediated isothermal amplification （LAMP） is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. Myeobacterium tuberculosis complex was detected by lAMP directly from sputum specimens and culture isolates grown in a liquid medium, meanwhile, the species-specific primers were designed by targeting the gyrB gene. A method to accelerate the I,AMP reaction was developed by using additional primers and termed loop primers. Of which, one loop primer hybridized to the stem-loops, except for the loops that were hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers reacted via the loops, the reactions were of different mechanisms. Results The reaction time by using LAMP with loop primers was less than 0.5 h eamparing with that of the original lAMP method. Since the total time of analysis including detection was less than 1 h, the sensitivity of I,AMP with Myeobaeterium tuberculosis was more 100 times than PCR, similar to real-time PCR. In addition, as lAMP method was performed under isothermal conditions without special apparatus needed, which makes ith more economical and practical than PCR or real-time PCR.. Conclusion This new method might facilitate genetic analysis applied in clinical laboratory and field surveillance. Meanwhieh, it also has some potentials for rapid test.