PCR-SSCP技术在检测煤工尘肺并发肺结核患者感染的结核分枝杆菌耐药基因突变中的应用

Application of PCR-SSCP Technique to Detection of Drug Tolerant Genetic Mutation in M.Tuberculosis among Coal Worker's Pneumoconiosis Patients Complicated with Tuberculosis

目的探索尘肺结核患者感染的结核分枝杆菌(M.tuberculosis)耐药基因突变与耐药性的关系,及聚合酶链反应-单链构象多态性分析(PCR-SSCP)的临床应用价值。方法在102例淮南矿区煤工尘肺并发结核患者中分离出M.tuberculosis菌株32株,采用PCR-SSCP法检测katG、rpoB和rpsL基因突变,与采用常规药敏试验(AST)法检测的耐药结果进行对比分析。结果采用PCR-SSCP法检测katG、rpoB和rpsL基因,突变率分别为43.75%(14/32),53.13%(17/32)和31.25%(10/32);采用AST法检测INH、RFP和SM,耐药率分别为53.13%(17/32),65.63%(21/32)和40.63%(13/32),其中多耐药株19例(59.38%),包括耐3种药物的11例(42.31%),耐2种药的8例(30.77%),单耐药株7例(26.92%),敏感株6例,耐药率81.25%。PCR-SSCP法检出3个基因联合突变的有7株(7/26,26.92%),与AST法的符合率为63.64%(7/11);2个基因突变的6株(6/26,23.08%),符合率为50.00%(4/8);单基因突变的8株(8/26,30.77%),符合率为57.14%(4/7)。结论PCR-SSCP技术适用于淮南矿区煤工尘肺并发结核患者感染的M.tuberculosis katG、rpoB和rpsL耐药基因突变的筛选,将其与AST法联合应用,在指导临床用药方面具有着重要的意义。

Objective To study the relationship of drug tolerant genetic mutation and drug tolerance in M. tuberculosis and to explore the value of clinical application of PCR-SSCP. Methods A total of 32 clinical isolated strains of M. tuberculosis were collected among 102 coal worker＇s pneumoconiosis patients complicated with tuberculosis. The genetic mutation of katG, rpoB and rpsL were detected by PCR-SSCP, and the results were compared with those by traditional antimicrobial susceptibility test（AST）. Results The genetic mutation ratesofkatG, rpoB and rpsL by PCR-SSCP were 43.75%（14/32）, 53.13%（17/32）and 31.25% （10/32）respectively. The tolerance results of INH, RFP and SM by AST were 53.13% （17/32）, 65.63% （21/32）and 40.63%（13/32）. Results showed that there were 19（59.38%）multi-drug tolerant strains in 32 clinical isolated strains, including 11 （42. 31%）three-drug tolerant strains, 8（30. 77%）two-drug tolerant strains, and 7（26.92%）one drug tolerant strains. The rate of total drug tolerance was 81.25%. By PCR- SSCP, 7 strains of allied mutation of three genes, 6 of two mutations and 8 of only one were detected. The compliance rates with AST method were 63.64% （7/11）, 50.00% （4/8）and 57.14% （4/7）respectively. Conclusions PCR-SSCP technique is applicable to the screen of drug tolerant genetic mutation of katG, rpoB and rpsL in M. tuberculosis among the pneumoconiosis patients complicated with tuberculosis. When the technique is applied in combination with AST method, it would be of great significance in the clinical prescription.