应用基因芯片技术检测结核分枝杆菌链霉素耐药性的研究

目的研制一种新型DNA芯片,用于快速检测结核分枝杆菌耐链霉素rpsl和rrs基因突变。方法根据结核分枝杆菌rpsl和rrs基因序列设计探针并制作DNA芯片,用TAMRA(四甲基罗丹明)标记的引物扩增结核分枝杆菌rpsl和rrs基因突变热点的片段,与DNA芯片杂交,同时以聚合酶链反应-单链构象多态性(polymerase chain reaction-single stranded conformation polymorphism,PCR-SSCP)和DNA测序法为对照。结果144株结核分枝杆菌临床分离株中,42株链霉素敏感株的PCR-SSCP和DNA芯片杂交结果与结核分枝杆菌标准株完全相同;102株链霉素耐药株中有79株检测到rpsl基因突变77.5%(79/102),其中74株为43位密码子AAG→AGG突变,5株为88位密码子AAG→AGG突变;rrs基因突变5%(5/102),4株为513位密码子A→C突变,1株为516位密码子C→T突变,均与PCR-SSCP和DNA芯片杂交结果一致,余18株未检测到突变。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链霉素分离株的rpsl和rrs基因突变,可用于临床耐药性的检测,指导临床用药。

Rapid detection of rpsl and rrs mutations in Mycobacterium tuberculosis by DNA chip

To develop a new method,DNA chip,which can be used for rapid detection of rpsl and rrs mutations in Mycobacterium tuberculosis.Aaccording to the rpsl and rrs gene sequence of Mycobacterium tuberculosis.The oligonucleotide probes were designed and synthesized,and DNA chips were prepared.The DNA fragment containing the hot mutation sites of rpsl and rrs genes were amplified with TAMRA-labeled primers by PCR,and then it was hybridized with gene chip.PCR-SSCP technique and DNA sequencing were used as the control.Of 144 M.tuberculosis clinical isolates,the results of DNA chips showed that the rpsl and rrs genes from 42 of Streptomycin-sensitive strains were all wild types,similar to that of PCR-SSCP.Of 102 M.tuberculosis Streptomycin-resistant isolates,79 strains had rpsl gene mutations,74 strains were AAG→AGG at codon 43;5 strains were AAG→AGG at codon 88.the other 18 isolates had no mutations in DNA chip.It is evident that DNA chip might be a rapid and specific method for the detection of rpsl and rrs gene mutation in the majority of Streptomycin-resistant Mycobacterium tuberculosis.It could be used for clinical detection of Streptomycin-resistant isolates.