摘要
目的应用膜反向斑点杂交技术快速检测结核分支杆菌对异烟肼(INH)耐药性。方法设计与合成用于检测结核分支杆菌耐INHkat、inhA基因的寡核苷酸探针,点于硝酸纤维素膜上,与结核分枝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。结果20株INH敏感株中,仅9例出现野生型探针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→ACC;15株出现野生型探针inh1阳性杂交,另5株Inhal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Inhal探针阳性杂交,25株野生型探针inhl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%。结论膜反向斑点杂交技术可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 reverse dot blot hybridized technique. [Methods] The oligonucleotide probes of isoniazid-resistant genes （katG, inhA） were dropped on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were amplified and labeled with biotin by PCR, and them hybridized with oligonucleotide probes on membrane. [Results] Among 20 isoniazid-sensitive strains, 9 strains were positive hybridization with K1 as H37Rv, 10 sensitive strains were positive hybridization with probe K1b＇, PCR-DS showed the AGC→ACC mutation at codon 315 of katG gene, 1 strains were postive hybridization with K1c＇ PCR-DS showed the AGC→AAC mutation at codon 315; 15 isoniazid-sensitive strains were positive hybridization with probe inhl as H37Rv, 5 strains were positive hybridization with probe inhal, PCR-DS showed C→T mutation at - 15 site of inhA gene. Of 36 isoniazid-resistant strains, 17 strains were positive hybridization with probe K1 as H37 Rv; 18 strains were positive hybridization with probe Klb＇, 1 strains was negative hybridization, PCR-DS showed the GGC→AC mutation at codeon 279; 15 isoniazid-resistant strains were positive hybridization with probe inhl as H37Rv, 11 strains were positive hybridization with probe inhal. The mutation rate of katG and inhA gene was 50% and 30.65% by PCR-membrane reverse dot blot hybridization respectively. [Conclusion] The membrane-reverse dot blot hybridized technique is simple and rapid and can be used to detect isoniazid-resistance of partial mycobacterium tuberculosis clnical strains.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2006年第15期2272-2276,共5页
China Journal of Modern Medicine
