摘要
目的应用寡核苷酸探针膜反向斑点杂交技术快速检测结核分支杆菌对异烟肼(isoniazid,INH)耐药性。方法设计与合成用于检测结核分支杆菌耐INH基因katGi、nhA的寡核苷酸探针,点于硝酸纤维素膜上,与结核分支杆菌临床分离株生物素标记的聚合酶链反应(PCR)产物进行反向斑点杂交,并与PCR-单链构象多态性(Polymerase chain reaction-Single stranded conformation polymorphism,PCR-SSCP)和PCR-直接测序(PCR-directsequencing,PCR-DS)结果比较。结果20株INH敏感株中,仅9株出现野生型探针K1阳性杂交,余11株中,10株K1b'杂交阳性,PCR-DS显示katG基因315位密码子AGC→ACC,1株K1c'杂交阳性,PCR-DS显示katG基因315位密码子AGC→AAC;15株出现野生型探针inh1阳性杂交,另5株Inha1探针杂交阳性,PCR-DS显示inhA基因-15位C→T突变。36株耐药株中,17株K1探针杂交阳性,18株K1b'杂交阳性,1株与所试探针不杂交,PCR-DS显示katG基因279位密码子GGC→GAC;11株与突变型Inha1探针阳性杂交,25株与野生型探针inh1杂交阳性。katG基因膜杂交突变检出率为50%,inhA基因膜杂交突变检出率为30.56%。结论寡核苷酸探针膜杂交技术可能成为检测部分结核分支杆菌耐异烟肼基因型简便、快速的方法。
Objective To establish the rapid detection of resistance to isoniazid in Mycobacterium tuberculosis by membrane oligonucleotide probes -reverse dot blot hybridized technique. Methods Prepared the oligonucleotide probes of isoniazid-resistant genes（katG, inhA） and drop it on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with oligonucleotide probes on membrane. Polymerase chain reaction-Single stranded conformation polymorphism （PCR-SSCP） and PCR-direct sequencing （PCR-DS） techniques were used as the control. Results Of 20 isoniazid-sensitive strains, 9 strains showed hybridization with probe K1 as H37Rv, 10 sensitive strains showed positive hybridization with K1b, PCR-DS showed the AGC→ACC mutation at codon 315 of katG gene, 1 strains showed positive hybridization with K1c＇, PCR-DS showed the AGC→AAC mutation at codon 315 of katG gene; 15 strains showed positive hybridization with probe inhl as H37Rv, 5 strains with Inhal, PCR-DS showed the C→T mutation at -15 site of inhA gene; Of 36 isoniazid-resistant strains, 17 strains showed positive hybridization with probe K1 as H37Rv; 18 strains with probe K1b＇, 1 strains showed negative hybridization, PCR-DS showed the GGC→GAC mutation at codon 279 of katG gene; 25 strains showed positive hybridization with probe inhl, 11 strains with Inhal. Mutation rate was 50 and 30.65 % respectively. Conclusion The membrane oligonucleotide probes -reverse dot blot hybridized technique was simple and rapid and could be used to detect isoniazid-resistance of partial Mycobacterium tuberculosis clinical strains.
出处
《河北医科大学学报》
CAS
2006年第4期241-245,共5页
Journal of Hebei Medical University
基金
军队医学杰出中青年人才科研基金项目(01J020)
关键词
分支杆菌
结核
异烟肼
药物耐受性
mycobacterium tuberculosis
isoniazid
drug tolerance
