全基因组测序技术在口岸耐多药肺结核诊断中的应用探索  

Application of High Throughput Sequencing Technology in Diagnosis of Pulmonary Tuberculosis and Drug Resistance at Ports

作　　者：温颖 吴健 吴培丰 高颖龙 陈平 谭家辉 何洪涛 王向阳 张建明 赵卫[2] 谢茜 赵久波[2] 余健海 覃直然 Wen Ying;Wu Jian;Wu Peifeng;Gao Yinglong;Chen Ping;Tan Jiahui;He Hongtao;Wang Xiangyang;Zhang Jianming;Zhao Wei;Xie Qian;Zhao Jiubo;Yu Jianhai;Qin Zhiran(Guangzhou International Travel Healthcare Centre,Guangzhou,Guangdong,510635;School of Public Health,Southern Medical University,Guangzhou,Guangdong,510515)

机构地区：[1]广州国际旅行卫生保健中心,广东广州510635 [2]南方医科大学公卫学院,广东广州510515

出　　处：《口岸卫生控制》2021年第3期47-54,共8页Port Health Control

基　　金：原广东出入境检验检疫局科技计划项目(No.2018GDK28);广东省科技计划项目(No.2018B020207013);阳江市科技计划项目(No.2019010)。

摘　　要：目的探索全基因组测序(whole generation sequencing,WGS)技术在口岸耐多药(multidrug resistant,MDR)肺结核(pulmonary tuberculosis,PTB)诊断中的应用价值。方法自广州国际旅行卫生保健中心结核生物样品库中整理出单异烟肼耐药(isoniazid mono-resistant,HR)、单利福平耐药(rifampicin mono-resistant,RR)、MDR和广泛耐药(extensively drug resistant,XDR)4类耐药(drug resistant,DR)结核分离株92株,随机选取46株临床敏感菌株,菌种复苏后经液体比例法药物敏感性试验(drug resistant test,DST)和分子线性探针(line probe array,LPA)进行菌种鉴定、筛选和耐药性确认后,78株结核耐药菌株和43株全敏感菌株进行WGS检测,测序结果为非结核分枝杆菌(nontuberculosis mycobacteria,NTM)/结核分枝杆菌(mycobacterium tuberculosis,MTB)混合感染4株,最后可纳入耐药分析菌株117株。以DST结果作为参考标准,比较WGS和LPA在口岸肺结核及其耐药性检测中的效能。结果WGS SE50检测系统对单菌的NTM/MTB混合感染具有很好的鉴别作用,对常见基因突变katG_S315T1、rpoB_S531L等均可稳定检出,gyrA_S95T和katG_R463L双突变可能导致MDR-TB及以上耐药。结论单末端(single-end,SE)50 WGS-DNA纳米球(DNA nanoball,DNB)检测分析系统用于口岸MDR-TB的诊断具有良好的可行性。Objective To explore the application value of whole generation sequencing(WGS)in the diagnosis of multi drug resistant(MDR)pulmonary tuberculosis(PTB)at ports.Methods From the tuberculosis biobank of Guangzhou International Travel Health Care Centre,four types of drug resistance were sorted out:isoniazid mono-resistance(HR),rifampicin mono-resistance(RR),MDR and XDR,a total of 92 tuberculosis strains were isolated,and 46 clinically sensitive strains were randomly selected.After identification,screening and drug resistance confirmation by DST and LPA,78 strains of TB resistant strains and 43 strains of pan-sensitive strains were detected by WGS.The sequencing results showed that fourstrains were non tuberculosis mycobacteria(NTM)/Mycobacterium tuberculosis complex(MTBC)mixed infected,and 117 strains were included in the drug resistance analysis.The results of DST were used as reference standard to compare the efficacy of WGS and LPA in the detection of tuberculosis and its drug resistance at ports.Results The WGS SE50 detection system had a good identification effect on NTM/MTBC mixed infection of single bacteria,and could detect kat G gene mutation S315 T1、rpo Bgene mutation S531 L stably,gyr A gene mutation S95 T and kat G gene mutation R463 L double mutation may lead to MDR-TB resistance and above.Conclusion The single end(SE)50 bp WGS-DNB detection and analysis system is feasible for the diagnosis of MDR-TB at ports.

关 键 词：结核分枝杆菌 药物敏感性试验 点突变 诊断 

分 类 号：R521[医药卫生—内科学]