基于颜色判定的环介导恒温扩增法快速检测结核分枝杆菌

Colorimetric detection of Mycobacterium tuberculosis by loop mediated isothermal amplification method

目的建立快速检测结核分枝杆菌环介导的恒温扩增法。方法针对结核分枝杆菌特异性保守靶序列IS6110,采用环介导的恒温扩增法(LAMP)引物设计原则设计6条引物,特异性识别靶基因序列上8个独立区域。LAMP反应过程中会产生焦磷酸镁白色沉淀,可以通过实时监测反应液浊度来判断反应结果。结果试验表明,在60～65℃恒温条件下,LAMP反应60 min内即可完成;如果在反应前添加钙黄绿素,可通过反应液颜色的变化来判断反应结果;LAMP方法的最低检出核酸浓度为101 pg/μl,其灵敏度是PCR方法最低检出浓度1.01 ng/μl的10倍,且具有良好的特异性。结论 LAMP方法检测结核分枝杆菌具有简单、快速、价廉、特异性强、灵敏度高等特点,适用于疾控工作及临床筛查或快速检测结核杆菌。

Objective To establish a loop-mediated isothermal amplification（LAMP） for rapid diagnosis for Mycobacterium tuberculosis.Methods Using the specific conserved sequence IS6110 of Mycobacterium tuberculosis,one set of primers was designed for recognizing eight independent sequence regions.LAMP reaction process will produce a white precipitate of magnesium pyrophosphate,and the reaction results can be judged by monitoring the turbidity.Results The realtime turbidity monitoring results show that LAMP reaction can be completed in a constant temperature of 60-65℃ in 60 min.If adding calcein before the reaction,green-positive results are significantly different from the yellow-negative results.The sensitivity of LAMP with detection limits of 101 pg / l is 10 times than that of PCR,and the primers designed had a good specificity too.Conclusion The LAMP method reported here demonstrates a potential and valuable means for detection of Mycobacterium tuberculosis,especially for its rapidity,simplicity,low cost,high specificity and sensitivity.The LAMP assay is suitable for clinical screening or on-site rapid diagnosis.