结核分枝杆菌IS6110和IS1081微滴数字PCR检测体系的建立和应用

Establishment and apply of a droplet digital PCR system for detecting Mycobacterium tuberculosis IS6110 and IS1081

目的建立结核分枝杆菌IS6110和IS1081微滴数字PCR(droplet digital PCR,dd PCR)检测体系,并用于不同类型临床样本的检测。方法常规提取13株结核分枝杆菌和14株非结核分枝杆菌临床分离株DNA,9例结核分枝杆菌阳性肺结核患者和4例其他肺部疾病患者痰DNA,12例结核分枝杆菌阳性肺结核患者和7例健康者血浆DNA。设计合成IS6110和IS1081扩增引物和检测探针,建立dd PCR检测体系。应用该体系检测分枝杆菌、痰和血浆3种DNA样本中靶标拷贝数,进行统计学分析。结果建立了能同时检测IS6110和IS1081的dd PCR体系,该体系检测2个靶标的线性范围分别为0.5~8 733和0.2~2 893拷贝/μl反应体系。该体系具有较好的重复性(r>0.95),以非结核分枝杆菌为对照检测结核分枝杆菌的灵敏度和特异度均为100%。在痰和血浆DNA样本检测中,2个靶标拷贝数在肺结核组均显著高于对照组。结论结核分枝杆菌特异性核酸的dd PCR体系,可用于肺结核患者临床分离株、痰和血浆样本的微量核酸检测,对提高结核病病原学诊断能力有重要意义。

Objective To establish a droplet digital PCR （ddPCR） system for detecting Mycobacterium tuberculo- sis IS6110 and ISI081, and apply it to detect the two targets in different types of clinical specimens. Methods DNA was routinely extracted from 13 Mycobaeterium tuberculosis＇ clinical isolates, 14 nontuberculous mycobacteria （NTM） clinical isolates, 9 sputum samples of smear-positive pulmonary tuberculosis patients, 4 sputum samples of other pulmonary disease patients, 12 plasma samples of smear-positive pulmonary tuberculosis patients, and 7 plasma samples of healthy persons. Primers and probes for amplifying and detecting IS6110 and IS108! were designed and synthesized, and the ddPCR detection system was established. DNA templates from clinical isolates, sputum and plasma were detected using this system, and IS6110 and IS1081 copies were determined and analyzed. Results A ddPCR system to amplify and detect IS6110 and IS1081 simultaneously was established. It exhibited excellent linearity between the targets input and measured copies in a dynamic range of 0.5-8 733 （IS6110） and 0.2-2 893 （IS1081） copies/pal PCR reaction mixture. There was a high agreement between the results produced from two independent digital PCR assays （r 〉 0.95）. Using NTM as control, the sensitivity and specificity of the system for detecting M tuberculosis were 100%. For sputum and plasma DNA detection, both IS6110 and IS1081 had a significantly higher copy number in smear-positive pulmonary tuberculosis groups compared with the control group. Conclusion A ddPCR system to efficiently detect M. tuberculosis-specific nucleic acids is established successfully. The system can be widely used in trace DNA detection derived from clinical isolates, sputum and plasma. The result has important value for improving the pathogenic diagnosis of tuberculosis.