荧光PCR熔解曲线法检测结核分枝杆菌对利福平和异烟肼耐药性的价值

The value of the fluorescence PCR melting curve method in detecting the resistance of Mycobacterium tuberculosis to rifampicin and isoniazid

目的分析南京市结核分枝杆菌(MTB)感染患者的耐药情况,评价荧光PCR熔解曲线法检测结核分枝杆菌对利福平(RFP)和异烟肼(INH)耐药性的临床价值,探究RFP和INH耐药相关基因突变的特征。方法对鉴定为MTB的780株菌株同时进行绝对浓度法药物敏感性试验和荧光PCR熔解曲线法检测对RFP和INH的耐药性。以绝对浓度法药物敏感性试验结果为标准,分析熔解曲线法检测RFP和INH的灵敏度、特异度、符合率和一致性(Kappa检验)。结果780株MTB中,22株(2.82%)为RFP单耐药,62株(7.95%)为INH单耐药,143株(18.33%)为耐多药。以绝对浓度法药敏试验结果为标准,荧光PCR熔解曲线法检测MTB对RFP和INH耐药性的灵敏度分别为98.18%和85.85%,特异度分别为95.28%和97.39%,符合率分别为95.90%和94.36%,Kappa值分别为0.88和0.85。RFP分子耐药菌株中全部检测出rpoB基因位点突变,其中以rpoB 529~533位点为最常见的突变(63.87%,122/191),突变位点为rpoB 507~512的20株菌株中只有7株表型耐药,rpoB基因双位点突变的18株菌株全部表型耐药;INH分子耐药菌株中最常见的突变为katG 315位点(66.49%,127/191),其次是inhA启动子区(17.80%,34/191),90%以上的INH分子耐药菌株为表型耐药。结论南京市结核耐药情况严重,以耐多药结核病为主。荧光PCR熔解曲线法与药敏试验结果高度一致,且弥补了药敏试验对于低度耐药突变株菌检测的局限性。耐药相关基因突变位点的检测有利于结核病的及时诊断和个体化治疗的实施。

This study was aimed at analyzing the drug resistance of Mycobacterium tuberculosis(MTB)infected patients in Nanjing;evaluating the clinical value of the fluorescence PCR melting curve method in detecting the resistance of Mycobacterium tuberculosis to rifampicin(RFP)and isoniazid(INH);and exploring the characteristics of gene mutations related to RFP and INH resistance.The drug resistance of MTB to RFP and INH was detected with absolute concentration drug sensitivity tests and the fluorescence PCR melting curve method.The sensitivity,specificity,coincidence rate and consistency(kappa test)of resistance to RFP and INH,detected with the melting curve method,were analyzed with absolute concentration drug sensitivity tests as standard.Phenotypic resistance results indicated that 22(2.82%)of 780 MTB strains were RFP single drug resistant,62(7.95%)were INH single drug resistant,and 143(18.33%)were multidrug resistant.Molecular resistance results indicated that 44(5.64%)were RFP single drug resistant,44(5.64%)were INH single drug resistant,and 147(18.85%)were multidrug resistant.With absolute concentration drug sensitivity tests as standard,the sensitivity,specificity and coincidence rate of MTB with RFP and INH resistance,detected with the fluorescence PCR melting curve method,were 98.18%and 85.85%,95.28%and 97.39%,95.90%and 94.36%,respectively.The kappa values were 0.88 and 0.85,respectively.rpoB gene locus mutations were detected in all RFP resistant strains,and the rpoB locus 529-533 was the most common mutation(63.87%,122/191).Only 7 of 20 strains with the rpoB locus 507-512 mutation had phenotypic drug resistance.All 18 strains with double site mutation of the rpoB gene had a resistance phenotype.The most common mutation in INH resistant strains was the katG 315 site(66.49%,127/191),followed by the inhA promoter region(17.80%,34/191).More than 90%of INH resistant strains had a resistance phenotype.The fluorescence PCR melting curve method was highly consistent with the results of drug sensitivity testing,and overcame the limitations of drug sensitivity tests in the detection of mutant strains with low drug resistance.Detection of drug resistant gene mutation sites aids in timely diagnosis and individualized treatment of tuberculosis.