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中国防痨杂志 ›› 2006, Vol. 28 ›› Issue (1): 4-10.

• 论著 • 上一篇    下一篇

应用基因芯片分析结核分枝杆菌常见耐药基因型的研究

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；   

  1. 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；
  • 出版日期:2006-01-10 发布日期:2006-11-03
  • 基金资助:
    本课题为“十五”国家重大科技专项“功能基因组和生物芯片”科研基金和军队医学杰出中青年人才科研基金项目(01J020)

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

Wu Xueqiong1,Zhang Qiong,Zhang Junxian,et al   

  1. 1.Tuberculosis Research Laboratory,Tuberculosis Center,the 309th Hospital of PLA,Beijing 100091,China
  • Online:2006-01-10 Published:2006-11-03

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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。

关键词: 基因芯片, 耐药基因型, 聚合酶链反应, 分枝杆菌,结核

Abstract: 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 drugsensitive M tuberculosis isolates analyzed were all wild-types.Of 121 drugresistant 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) rifampicin-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,ATT,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 microchip.

Key words: oligonucleotide microchip, drug-resistant genetype, PCR, Mycobacterium tuberculosis

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