【摘要】 目的采用微滴数字PCR技术(droplet digital PCR,dd PCR)检测全血中结核分枝杆菌特异性CFP10(10-k Da culture filtrate protein,CFP10)基因的拷贝数含量。方法收集10例活动性肺结核患者(active tuberculosis,a TB)、10例肺外结核患者(extrapulmonary tuberculosis,EPTB)和5例健康对照者(healthy donars,HDs)全血并提取DNA,优化引物最佳扩增条件并制定重组质粒标准曲线,对样本中CFP10基因的拷贝数含量分别用dd PCR与实时定量PCR(quantitative real-time PCR,q PCR)进行检测和比较。结果对重组质粒的灵敏度检测显示dd PCR技术明显优于q PCR技术。对样本的检测显示q PCR技术检测a TB、EPTB患者与健康人的全血中CFP10含量之间无统计学意义;但是dd PCR技术检测a TB、EPTB患者与健康人之间有显著统计学意义(P<0.0001);dd PCR检测a TB和EPTB患者(共20例)CFP10基因绝对定量的浓度约为3.4~94.0 copies/μL。结论本研究首次报道采用dd PCR技术能灵敏地用于TB患者全血的诊断。更多还原

【Abstract】 Objective To analyse content of mycobacterium tuberculosis( MTB) specific CFP10 gene copies from whole blood samples using droplet digital PCR( dd PCR) technique. Methods Whole blood samples were collected from 10 activetuberculosis( a TB) patients, 10extrapulmonarytuberculosis( EPTB) patients and 5 healthy donors( HDs),respectively. Total DNA from each blood samples were extracted. The optimal amplification conditions of the primers were optimized and the standard curves of CFP10 recombinant plasmids were established. 10-k Da culture filtrate protein( CFP10) gene copies from whole blood samples were determined and compared using dd PCR and quantitative real-time PCR( q PCR)techniques. Results Results showed that dd PCR had much higher sensitivity for measuring CFP gene than q PCR technique. Compared with HDs,there were significantly statistical differences between a TB and EPTB patients using dd PCR( P < 0. 0001),however there was no statistical significance using q PCR( P > 0. 05); The absolute quantitative concentration ranges of CFP10 gene in these 20 TB patients measured by dd PCR were about 3. 4 ~94. 0copies / μL. Conclusions This is the first report showing that dd PCR technique could used for diagnosis of TB patients from whole blood samples with high sensitive.更多还原