应用荧光PCR熔解曲线法分析结核分枝杆菌耐药反应的可行性探究

Feasibility on Drug Resistance Analysis of Mycobacterium Tuberculosis by Fluorescence PCR Melting Curve Method

目的:分析荧光聚合酶链反应(PCR)熔解曲线法(MC)检测结核分枝杆菌(MTB)耐药反应的可行性。方法:选取本院2021年9月至2023年1月本院收治的结核病患者120例,收集患者痰标本进行菌株培养和鉴定,最终纳入102例(株)为本研究观察对象。每份样本均进行罗氏固体药敏试验和荧光PCR-MC检测MTB菌株耐药性。对于耐药性检测结果不一致标本,采用基因芯片法进行验证。结果:102株MTB,经罗氏固体药敏试验检出异烟肼耐药菌株18株(17.65%),检出利福平耐药菌株33株(32.35%);荧光PCR-MC法检测异烟肼耐药菌株16株(15.69%),检测利福平耐药菌株37例(36.27%)。以罗氏固体药敏试验检测结果作为金标准,荧光PCR-MC法检测异烟肼耐药性的Kappa值为0.718,敏感度为72.22%,特异度为92.16%,一致性分析基本一致;荧光PCR-MC法检测利福平耐药性的Kappa值为0.783,敏感度为90.91%,特异度为89.96%,一致性分析基本一致。荧光PCR-MC法检测结果显示,对异烟肼耐药的2例MTB菌株均为katG 315位密码子突变;对利福平耐药的7例中,1例ropB 511位密码子、1例ropB 526位密码子和5例ropB 531位密码子突变。基因芯片法耐药基因检测结果与荧光PCR-MC检测结果一致。结论:荧光PCR-MC检测MTB菌株耐药性(异烟肼和利福平)检测具有良好的效能。

Objective:To analyze the feasibility of fluorescence polymerase chain reaction(PCR)melting curve(MC)method to detect drug resistance of Mycobacterium tuberculosis(MTB).Methods:A total of 120 patients with tuberculosis admitted to the hospital were enrolled between September 2021 and January 2023.The sputum samples of patients were collected for culture and identification of pathogens,and there were 102 cases(strains)included as the observation objects finally.Each sample underwent the detection of MTB strain resistance by drug sensitivity test of Lowenstein solid culture and fluorescence PCR-MC method,and then the samples with inconsistent results in drug sensitivity test were given the detection of drug resistance genes by gene chip method to verified.Results:Among 102 MTB strains,the drug sensitivity test of Lowenstein solid culture showed that there were 18 strains(17.65%)with isoniazid resistance and 33 strains(32.35%)with rifampicin resistance.Fluorescence PCR-MC method showed that there were 16 strains(15.69%)with isoniazid resistance and 37 strains(36.27%)with rifampicin resistance.Taking the results of drug sensitivity test of Lowenstein solid culture as the golden standard,the Kappa value,sensitivity and specificity of detecting isoniazid resistance by fluorescence PCR-MC method were 0.718,72.22%and 92.16%,and the consistency analysis was basically consistent.Taking the results of drug sensitivity test of Lowenstein solid culture as the golden standard,the Kappa value,sensitivity and specificity of detecting rifampicin resistance by fluorescence PCR-MC method were 0.783,90.91%and 89.96%,and the consistency analysis was basically consistent.The results of fluorescence PCR-MC method showed that in the 2 cases with isoniazid resistance,both were codon mutation at katG315 locus.In the 7 cases with rifampicin resistance,there was 1 case with codon mutation at ropB 511 locus,1 cases at ropB 526 locus and 5 cases at ropB 531 locus,respectively.The results of gene chip method were consistent with the results of fluorescence PCR-MC method.Conclusion:Fluorescence PCR-MC method has good detection efficiency for drug resistance(isoniazid,rifampicin)of MTB.