实时荧光环介导等温扩增在快速检测结核分枝杆菌中的应用

Real-time fluorescence loop-mediated isothermal amplicication for the rapid detection of Mycobacterium tuberculosis

【摘要】目的建立实时荧光环介导等温扩增快速检测结核分枝杆菌的方法。方法针对结核分枝杆菌特异序列建立实时荧光环介导等温扩增技术；对其特异性和敏感性进行评估；通过敏感性和特异性等指标验证其稳定性。结果建立的实时荧光环介导等温扩增法对结核和非结核分枝杆菌标准菌株检测的敏感性和特异性均为100％，灵敏度可达102个菌／mL；痰标本结核分歧杆菌检测的敏感性为94．64％（53，56），荧光定量聚合酶链式反应方法检测的敏感性为92．86％（52／56）；检测的特异性为96．55％（28，29），荧光定量聚合酶链式反应方法检测的特异性为96．55％（28／29）。结论建立实时荧光环介导等温扩增检测结核分枝杆菌的方法，敏感性和特异性强，灵敏度高，稳定性好，可用于痰液中结核分枝杆菌的快速检测。

Objective To establish a rapid method of real-time fluorescence loop-mediated isothermal amplification （LAMP） for detection of Mycobacterium tuberculosis. Methods According to the specific sequence of IS6110 in Mycobacterium tuberculosis, 6 primers of loop-mediated isothermal amplification were designed and synthesized. With the ESE-Quant tube scanner platform, a real-time fluorescence loop-mediated isothermal amplification was established. 14 strains of non-tuberculous mycobacteria standard strains and 14 strains of Mycobacterium tuberculosis isolated from cases were used to evaluate specificity and sensitivity of the method. Sputum samples were collected from 56 positive cases for Mycobacterium tuberculosis and 29 negative cases for Mycobaeterium tuberculosis. The stability of the method was validated by the sensitivity and specificity compared to the real-time fluorescent PCR method. Results Real-time fluorescence loop-mediated isothermal amplification method was established, the specificity and＇sensitivity of Mycobacterium tuberculosis and non-tuberculous myeobacterial standard strains reflected 100%. In 56 cases of sputum with Mycobacterium tuberculosis, the sensitivity of real-time fluorescence LAMP method was 94.64% （53/56）, while the sensitivity of real-time fluorescence polymerase chain reaction （PCR） method was 92.86% （52/56）. In 56 eases of sputum without Myeobacterium tuberculosis, the specificity of real-time fluorescence LAMP method was 96.55% （28/29）, while the specificity of real-time fluorescence PCR method was 96.55% （28/29）. Conclusion The method of real-time fluorescence loop-mediated isothermal amplification （LAMP） for detection of Mycobaeterium tuberculosis with ESE-Quant tube scanner platform was established, which has strong specificity, high sensitivity, good stability and can be used for rapid detection of Myeobacterium tuberculosis in sputum.