结核分枝杆菌耐药相关基因扩增多重PCR体系的建立与评估

Establishment and evaluation of the multiplex PCR systems for amplifying Mycobacterium tuberculosis genes involved indrug resistance

目的建立结核分枝杆菌耐药相关基因多重PCR反应体系，评估该体系以临床分离株DNA和痰菌DNA为模板扩增耐药相关基因的成功率。方法常规提取结核分枝杆菌临床分离株（47株）及涂阳肺结核患者（21例）痰菌DNA。根据目前研究已发现的结核分枝杆菌耐药相关基因合成引物，建立2个多重PCR体系以扩增目的基因。扩增片段经测序证实后，计算该体系用于两种样本的扩增成功率。结果针对临床常用8种抗结核药物，选取结核分枝杆菌rpoB、katG、inhA、embB、gyrA、rpsL、rrs、eis共8个耐药相关基因设计合成引物。建立2个多重PCR体系扩增8个耐药基因，该体系扩增47株结核分枝杆菌临床分离株DNA和21例菌阳肺结核患者痰菌DNA的成功率分别为100．0％（47／47）和76．2％（16／21）。结论本研究建立了高效扩增8个结核分枝杆菌耐药基因的多重PCR体系，结果对进一步研发结核病耐药分子诊断产品具有重要意义。

Objective To establish multiplex PCR systems for amplifying Mycobacterium tuberculosis genes in- volved in drug resistance and evaluate the success rates of amplification using DNA templates from clinical isolates and sputum samples of pulmonary tuberculosis patients. Methods DNA was routinely extracted from 47 M. tuberculosis clinical isolates and 21 sputum samples of smear positive pulmonary tuberculosis patients. Primers for am- plifying genes involved in drug resistance were designed and synthesized, and 2 multiplex PCR systems were estab- lished. The amplified fragments were confirmed by sequencing, and the success rates of amplification by multiplex PCR were analyzed. Results We selected 8 gene sequences including rpoB, katG, inhA, embB, gyrA, rpsL, rrs and eis, which were involved in resistance to 8 commonly used anti-tuberculosis drugs. Two multiplex PCR systems were established to amplify 8 sequences, and the success rates of amplification were 100. 0% （47/47） for DNA from 47 clinical isolates and 76.2% （16/21） for DNA from 21 sputum samples of smear positive pulmonary tuberculosis patients. Conclusion Two multiplex PCR systems are established for amplifying eight M. tuberculosis gene sequences involved in resistance to anti-tuberculosis drugs used in clinical practice. The result has important value for developing new genotypic tests to diagnose drug-resistant tuberculosis.