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| 荧光PCR熔解曲线法在耐多药结核病诊治中的应用及分析 |
| Application and analysis of fluorescence PCR melting curve in the diagnosis and treatment of multi drug resistant tuberculosis |
| 投稿时间：2023-05-24 |
| DOI：10.3969/j.issn.1000-0399.2024.02.006 |
| 中文关键词: 熔解曲线法 微孔板法 耐多药结核病 耐药基因检测 药物筛选 |
| 英文关键词: Dissolution curve method Microplate plate method Multidrug resistant tuberculosis Drug resistance detection Drug screening treatment adjustment scheme |
| 基金项目:2022年贵州省卫生健康委科学技术基金项目（编号：gzwkj2022-174） |
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| 中文摘要: |
| 目的 探讨荧光聚合酶链反应(PCR)熔解曲线法在耐多药结核病(MDR-TB)的快速诊断和有效药物快速筛选中的应用价值。方法 以利福平(RFP)、异烟肼(INH)、乙胺丁醇(EMB)、氟喹诺酮类(FQs)为检测药物,应用熔解曲线法和微孔板药敏法,对比检测2021年1~7月贵阳市公共卫生救治中心罗氏固体培养阳性的84株菌株。两种方法药敏结果不一致的菌株用基因测序法作验证。结果 以微孔板法为标准,熔解曲线法对MDR-TB (微孔板诊断)的检出符合率、灵敏度、特异度、阳性预测值、阴性预测值和Kappa值依次为89.3%(75/84)、91.5%(43/47)、86.5%(32/37)、89.6%(43/48)、88.9%(32/36)、0.78。溶解曲线法对RFP、INH、EMB、FQs的检出符合率、灵敏度、特异度、阳性预测值、阴性预测值和Kappa值分别为76.2%~97.6%、86.7%~95.7%、73.9%~100%、41.9%~100%、94.1%~98.6%和0.45~0.95。对22份(26.19%,22/84)微孔板法敏感、熔解曲线法耐药的菌株进行测序验证,结果均显示含有耐药突变;而微孔板法耐药、熔解曲线法敏感的6份(0.71%,6/84)菌株在测序区域均为野生型。结论 荧光PCR熔解曲线法对微孔板药敏法检测结果的灵敏度、特异度、符合率、一致性均较高,可为耐多药结核病的初筛诊断及治疗方案制定提供实验室依据。 |
| 英文摘要: |
| Objective To explore the application of fluorescent PCR melting curve analysis in rapid diagnosis and rapid screening of effective drugs for multidrug-resistant tuberculosis (MDR-TB).Methods Melting curve analysis and microplate drug susceptibility method were used for rifampicin (RFP),isoniazid (INH),ethambutol (EMB),and fluoroquinolones (FQs) drug resistant detection.From January 2021 to July 2021,84 strains with positive culture in Lowenstein-Jensen (L-J) solid medium from Guiyang Public Health Center were collected.The strains with inconsistent drug results between the two methods were verified by Sanger sequencing.Results Using the microplate method as the standard,the coincidence rate,sensitivity,specificity,positive predictive value,negative predictive value and Kappa value of melting curve analysis for MDR-TB（Microplate diagnosis）was 89.3% (75/84),91.5% (43/47),86.5% (32/37),89.6(43/48),88.9% (32/36),and 0.78,respectively.The coincidence rate,sensitivity,specificity,positive predictive value,negative predictive value and Kappa value of RFP,INH,EMB and FQs was 76.2%~97.6%,86.7%~95.7%,73.9%~100%,41.9%~100%,94.1%~98.6% and 0.45~0.95 with dissolution curve method,respectively.Twenty-two strains (26.19%,22/84) that were sensitive to microplate method and resistant to melting curve method were sequenced,and the results showed that all the strains contained drug-resistant mutations.Six strains (0.71%,6/84) were found to be resistant by microplate method and sensitive by melting curve analysis.Sequencing results showed that they were all wild-type in the detection region.Conclusions The fluorescent PCR melting curve analysis has high sensitivity,specificity,coincidence rate and consistency for the detection results of microplate drug sensitivity method,which can provide laboratory basis for the primary screening diagnosis and treatment plan formulation of multidrug-resistant drug-resistant tuberculosis. |
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