应用基因芯片分析结核分枝杆菌常见耐药基因型的研究  

Identification of drug-resistant gene type in Mycobacterium tuberculosis by PCR-oligonucleotide microchips

作　　者：吴雪琼[1] 张琼[2] 张俊仙[1] 梁建琴[1] 鲁红丽[2] 李洪敏[1] 张洪恩[2] 陆阳[1] 荣利[2] 吕翠环[3] 张广宇[3] 邢婉丽[2] 

机构地区：[1]解放军第309医院结核病中心研究室,北京100091 [2]生物芯片北京国家工程研究中心,北京102206 [3]河北省胸科医院,石家庄050041

出　　处：《中国防痨杂志》2006年第1期4-10,共7页Chinese Journal of Antituberculosis

基　　金："十五"国家重大科技专项"功能基因组和生物芯片"科研基金;军队医学杰出中青年人才科研基金项目(01J020)

摘　　要：目的应用基因芯片快速检测结核分枝杆菌耐药基因型,建立一种新的分子药敏试验方法。方法以传统药敏试验和PCR-直接测序方法为对照,应用基因芯片快速检测157株结核分枝杆菌临床分离株异烟肼(INH)、利福平(RFP)、链霉素(SM)和乙胺丁醇(EMB)耐药基因(katGr、poB、rp-sLr、rs和embB)的带荧光素标记的PCR产物。结果PCR-直接测序和基因芯片检测36株结核分枝杆菌药物敏感株的5种耐药基因均为野生型。121株结核分枝杆菌耐药分离株中,56株耐INH分离株,katG基因突变率为62.5%,其中katG缺失率为7.5%,315位密码子突变率为55.4%,279位密码子突变率为1.8%;104株耐RFP株,rpoB基因突变率为94.2%,最常见的突变位点为531位和526位密码子(突变率分别为60.6%、15.4%),双位点突变率为5.8%,还发现511、513、515、516、517、518和533位密码子突变;62株耐SM分离株,rpsL和rrs总突变率为88.7%(两者分别为82.3%、6.5%),rpsL突变位于43位和88位密码子(突变率分别为77.4%、4.8%),rrs突变位于513位和516位碱基(突变分别为4.8%、1.6%);57株为耐EMB株,embB基因突变率为61.4%,均为306位密码子突变,最常见的突变为ATG→GTG或ATA(突变率分别为35.1%、15.8%),还发现306位密码子ATG→ATC、ATT和CTG突变。通过基因芯片检出的突变与基因测序结果一致。结论应用基因芯片可分析大多数结核分枝杆菌耐药基因型,弥补传统药敏试验方法的不足,指导临床治疗。Objective To identify drug - resistant gene type in Mycobacterium tuberculosis isolates by PCR - oligonucleotide microchips, and to develop a new, rapid method for drug resistance. Method Using traditional drug susceptibility testing and PCR - DNA sequencing as control, katG, rpoB, rpsL, rrs, and embB genes from 157 M tuberculosis clinical isolates were analyzed by PCR- oligonucleotide microchip. Results The katG, rpoB, rpsL, rrs, and embB genes from 36 drug - sensitive M tuberculosis isolates analyzed were all wild - types. Of 121 drug - resistant M tuberculosis isolates, 56 isolates were resistant to INH, in which 62.5% had alterations in the katG, codon 315 is the most common site of gene mutation; 94.2% （98/104） rifarnpicin- resistant isolates had rpoB gene mutations. Codon 531 and 526 of the rpoB are the most common sites of nucleotide substitutions, the mutations at codon 511, 513, 515, 516, 517, 518 and 533 were also found. 88.7 % of 62 streptomycin-resistant isolates had mutations of the rpsL （82.3%） and rrs （6.5%） genes. There were mutations at codon 43, 88 of the rpsL and position 513, 516 of rrs. 61.4% of 57 ethambutol - resistant isolates had nucleotide substitutions （ATG→GTG or ATA , ATC, ATE CTG） at codon 306. The mutations found by gene chip were consistent with the results of DNA sequencing. Conclusion The drug - resistant genetypes in the most drug - resistant Mycobacterium tuberculosis isolates could be identified with PCR - oligonucleotide micmchip.

关 键 词：基因芯片 耐药基因型 聚合酶链反应 分枝杆菌 结核 

分 类 号：R450[医药卫生—治疗学]