应用基因芯片技术检测耐利福平结核分枝杆菌基因型  

Rapid detection of rpoB mutations in mycobacterium tuberculosis by DNA chip

作　　者：张俊仙[1] 吴雪琼[1] 邢婉丽[2] 张琼[2] 梁建琴[1] 张洪恩[2] 陆阳[1] 张广宇[3] 李洪敏[1] 阳幼荣[1] 

机构地区：[1]解放军总医院第309临床部结研室,北京100091 [2]生物芯片北京国家工程研究中心,北京100084 [3]河北省胸科医院,河北石家庄050041

出　　处：《中国现代医学杂志》2007年第13期1572-1577,共6页China Journal of Modern Medicine

基　　金：973传染病专项科研基金(2005CB523102)

摘　　要：目的研制一种新型DNA芯片,用于快速检测结核分枝杆菌耐利福平rpoB基因突变。方法根据结核分枝杆菌rpoB基因序列设计探针并制作DNA芯片,用TAMRA标记的引物扩增结核分枝杆菌rpoB基因突变热点的片断,与DNA芯片杂交,同时以PCR-SSCP和DNA测序法为对照。结果240株结核分枝杆菌临床分离株中,55株全敏感株和66株耐其他药物的利福平敏感株的PCR-SSCP和DNA芯片杂交结果与结核分枝杆菌标准株完全相同;119株耐RFP临床分离株中DNA芯片杂交有117株检测到rpoB基因突变,其中111株单位点突变,78株为531位密码子TCG→TTG(Ser→Leu)、TCG→TGG(Ser→Trp)和TCG→TAC(Ser→Tyr)突变;24株为526位密码子CAC→TAC(His→Tyr)、CAC→GAC(His→Asp)、CAC→CCC(His→Pro)、CAC→CTC(His→Leu)和CAC→CGC(His→Arg)突变;4株为516位密码子GAC→GTC(Asp→Val)和GAC→GGC(Asp→Gly)突变;3株为533位密码子CTG→CCG(Leu→Pro)突变;1株为511位密码子CTG→CCG(Leu→Pro)突变;1株为513位密码子CAA→AAA(Gln→Lys)突变;6株为双位点突变,其中3株511和516位联合突变,2株533和515位联合突变,1株517位密码子CAG→CAC(Gln→His)和518位密码子AAC→GAC(Asn→Asp)联合突变。DNA测序结果与DNA芯片杂交结果完全一致。1株516位GAC-TAC(Asp→Tyr)突变的分离株及1株516位GAC-TAC(Asp→Tyr)和518位AAC-CAC(Asn→His)联合突变的分离株DNA芯片检测阴性,是因为芯片上无相应的探针。结论用DNA芯片可快速、特异地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变,可用于临床耐药性的检测,指导临床用药。[Objective] To develop a new method, DNA chip, which can be used for rapid detection of rpoB mutations in Mycobactefium tuberculosis. [Method] According to the rpoB gene sequence of Mycobacterium tuberculosis, the oligonucleotide probes were designed and synthesized, and then DNA chips were prepared. The DNA fragmerit which contains hot mutation sites of rpoB gene was amplified with TAMRA-lablled primers by PCR. Then it was hybridized with gene chip. PCR-SSCP technique and DNA sequencing were used as the control. [Results] Of 240 M. tuberculosis clinical, isolates, the results of DNA chips showed that the rpoB genes from 55 of drug-sensitive strains and 66 rifampicin-sensitive strains （but resistance to other anti-TB drug） were all wild types, similar to that of PCR-SSCP. Of 119 M. tuberculosis rifampicin-resistant isolates, 117 strains had rpoB gene mutations, 111 strains were single location mutations. 78 strains had a base mutations at codon 531, which were TCG→TTG （Ser→Leu）, TCG→TGG （Ser→Trp） and TCG→TAC （Ser→Tyr）. 24 strains had mutations of CAC→TAC （His-＋Tyr）, CAC→GAC （I-Iis→Asp）, CAC→CCC （His→Pro）, CAC→CTC （I-Iis→Leu）and CAC→CGC （His→Arg） at codon 526. 4 strains had GAC→GTC （Asp→Val） and GAC→CGC （Asp→Gly） mutations at codon 516. 3 strains had CTG→CCG （Leu→Pro） mutation at codon 533. 1 strain had CTG→CCG （Leu→Pro） mutation at codon 511.1 strain had CAA→AAA （Gln→ Lys） mutation at codon 513. 6 strains had a base mutation at two different codons, respectively. Of them, 3 strains had base mutations at codon 511 and 516, 2 strains at codon 533 and 515, 1 strain at codon 517 and 518. The resuits of DNA chips were consistent with that of DNA sequencing. 1 strain that had GAC-TAC （Asp→Tyr） mutation at codon 516 and 1 strain that had GAC-TAC （Asp→Tyr） mutations at codon 516 and AAC-CAC （Asn→His） at codon 518 showed wild type by DNA chips, which were because there were no relevant specific pro

关 键 词：结核分枝杆菌 利福平 RPOB基因 药物耐受性 DNA芯片 

分 类 号：R52[医药卫生—内科学]