荧光定量PCR与焦磷酸测序联合应用检测异烟肼、利福平耐药性结核分枝杆菌

Test for isoniazid- and rifampicin-resistant Mycobacterium tuberculosis by fluorescent quantitative PCR and pyrosequencing technology

目的联合运用荧光定量PCR和焦磷酸测序技术快速检测异烟肼、利福平耐药性结核分枝杆菌。方法收集结核病疑似患者痰液样本,提取样本总DNA,运用Taqman荧光定量PCR对结核分枝杆菌的感染进行快速筛查。对检测结果为阳性的样本,采用焦磷酸测序法分别对异烟肼、利福平耐药结核分枝杆菌相关基因katG、rpoB的突变热点进行测序分析,与标准菌株序列比对,判断结核分支杆菌异烟肼、利福平的药敏性。采取Bactec 960药敏法鉴定结核分枝杆菌耐药性,并与焦磷酸测序法药敏检测结果进行比较,评估焦磷酸测序法的可靠性。结果荧光定量PCR技术检测痰液样本结核分枝杆菌的准确率达98.95%,检测时间约3 h;以Bactec 960药敏法鉴定结果为标准,焦磷酸测序法对结核分枝杆菌异烟肼、利福平的药敏鉴定结果准确性分别达93.75%和96.15%,其中,对耐药菌株判断,两种方法的一致性高达98%。结论采用荧光定量PCR检测技术可快速、准确地进行结核分枝杆菌感染筛查,运用焦磷酸测序技术则可快速准确地筛查出耐药样本,从痰液样本采集到结核分枝杆菌感染筛查以及药敏报告发布,全程仅需7 h;耐药菌株的检测可靠性达98%。

Objective To test for isoniazid- and rifampin-resistant Mycobacterium tuberculosis （MTB） rapidly by fluorescent quantitative PCR and pyrosequencing technology. Methods Sputum samples were collected from suspected patients with tubercnlosis, from which total DNA was extracted and used for rapid screening of MTB infection by Taqman fluorescent quantitative PCR. The positive samples were analyzed for hot spot mutation of katG and rpoB genes associated with isoniazid- and rifampin-resistant MTB by pyrosequencing, and the results were compared with that of standard strain sequence alignment, based on which the susceptibility of MTB to isoniazid and rifampiein were evaluated. The drug-resistance of MTB was tested by Bactee 960 method, and the results were compared with those by pyrosequencing technology to evaluate the reliability of pyrosequencing. Results The accuracy of fluorescent quantitative PCR for MTB in sputum samples was 98.95%, while the time-consuming was about 3 h. Using the result by Bactec 960 method as a standard, the accuracies of pyrosequencing for isoniazid- and rifampicin-resistant MTB were 93. 75% and 96. 15% respectively. The consistency of Bactec 960 method and pyrosequencing for judgment of drug-resistant MTB was 98%, while the time-consuming of pyrosequencing was about 4 h. Conclusion Fluorescent quantitative PCR may be used for rapid and accurate screening of MTB infection. However, drug-resistant MTB may be screened by pyrosequencing rapidly and accurately, by which the time-consuming from collection of sputum samples to screening of MTB infection and report of drug-sensitivity of MTB is only 7 h, and the reliability of test result is 98%.