探针熔解分析法快速检测结核分枝杆菌链霉素耐药突变  

Probe melting analysis for rapid detection of streptomycin-resistant mutations in Mycobacterium tuberculosis clinical isolates

作　　者：张向东[1] 张婷 牛建军[1] 温慧欣[1] 胡思玉[2] 李庆阁[2] 

机构地区：[1]厦门市疾病预防控制中心,厦门36102l [2]分子诊断教育部工程研究中心,厦门大学生命科学学院生物医学科学系,厦门361005

出　　处：《中国人兽共患病学报》2011年第1期53-57,共5页Chinese Journal of Zoonoses

基　　金：厦门市第二批重大疾病科研攻关项目(XMWZK0601);“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项(2008ZX10003-004)资助

摘　　要：目的评价探针熔解分析法快速检测结核分枝杆菌链霉素耐药突变的应用价值,为临床应用提供依据。方法首先用中国药品生物制品检定所提供的含37份标准非结核分枝杆菌的标准盘进行特异性评价,然后将野生型结核分枝杆菌H37Rv基因组DNA梯度稀释进行灵敏度考察,最后对906株结核分枝杆菌临床分离株(其中37份有药物敏感性试验结果)的链霉素耐药相关基因进行检测,并对突变菌株、有药敏结果的菌株以及疑似杂合的菌株进行测序分析。结果标准盘评价结果证实该体系特异检测H37Rv结核分枝杆菌。分析灵敏度为5～50拷贝每反应。在所有的906份结核分枝杆菌培养标本中,103份标本发生突变且与测序结果一致,18份标本存在杂合信号,17份标本无信号。37份有药敏结果的标本中,敏感的标本用探针熔解分析法所得结果均为野生型,耐药的16份标本仅检测到7份突变,另有9份耐药的标本实时检测为野生型,与测序结果相符。结论探针熔解分析技术特异性好,可以快速、准确地检测结核分枝杆菌链霉素耐药常见突变,有望直接用于临床标本链霉素耐药性检测。The purpose of the present study was to evaluate the utility of probe melting analysis（PMA） in rapid detection of streptomycin-resistant mutations in Mycobacterium tuberculosis（MTB）.The method was first evaluated by analyzing a reference panel containing 37 standard non-tuberculous mycobacterium（NTM） isolates.The results showed that PMA could specifically detect TB except one NTM strain,whereas this had no impact on its specificity.The analytical sensitivity was studied by purified genomic DNA of a reference strain H37Rv and the results showed that down to 5-50 copies per reaction of MTB genomic DNA could be repeatedly detected.The clinical performance was evaluated by analyzing 906 clinical isolates for rpsL and rrs mutations.The results showed 103 samples gave mutant results,which were in concordance with sequencing analysis.Of 18 samples gave heterozygous results and 17 samples failed to give signal.Among the 37 isolates with drug susceptibility results,all the susceptible samples were found to harbor no mutations,while 7 of 16 resistant samples were mutation positive.These results demonstrated that PMA was specific,and could detect all common rpsL and rrs mutations causing streptomycin-resistance.This technique has the potential to be used for rapidly screening streptomycin-resistant mutations in Mycobacterium tuberculosis clinical isolates.

关 键 词：探针熔解分析 结核分枝杆菌 链霉素 耐药突变 

分 类 号：R115[医药卫生—公共卫生与预防医学]