基因芯片技术检测结核分支杆菌耐利福平基因突变的研究

目的建立基因芯片检测结核分支杆菌耐利福平基因突变，结合基因测序和常规药物敏感性试验进行比较，探讨其实用价值。方法根据结核菌rpoβ基因突变特点，设计不同的寡核苷酸探针，点样制备可检测rpoβ基因突变的芯片，检测利福平（Rifompicin，RFP）株的rpoβ变异情况。结果137株临床分离结核菌，49株耐RFP，占35．8％，平均MIC为179．6±135．0ug／mL。Rpop突变率为95．7％（47／49），主要为点突变。突变点依次为531 Ser（TCG）→Leu（TTG）（40．8％）、531 Ser（TCG）→Trp（TGG）（12．2％）、526 His（CAC）→Tyr（TAC）（6．1％）、526His（CAC）→Asp（GAC）（6．1％）。526 His（CAC）→Asn（AAC）（6．1％）、526 His（CAC）→Pro（CCC）（8．2％）、516Asp（GAC）→Val（GTC）（12．2％）、533 Leu（CTG）→Arg（CGG）（4．1％）和513 GIn（CAA）→Pro（CCA）（2．1％）。结论本研究建立的检测结核菌rpoβ基因突变的基因芯片技术敏感性高，特异性强，可应用于结核菌耐RFP基因的检测。

Genetic microarray for detection of rpoβmutation associated with Mycobacterium tuberculosis resistance to rifampicin

Objective To establish the DNA probe microarray for detection M.tuberculosis rpoβ gene mutations associated with rifampicin(RFP)resistance,and evaluate its applicable value.Methods According to the characteristic of M tuberculosis rpoβ gene mutation related to RFP resistante.oligonucleotide probes were designed and spotted to prepare DNA chip.The detection efficacy was compared with DNA direct sequencing of the rpoβ and drug susceptibility test.Results 49 out of 1 37(35.8%)M.tuberculosis isolales were resistant to RFP with the average minimal inhibitory concentration of(179.6±135.0)μg/mL.The mutation rate of rpoβwas 95.7%(47/49),mainly point mutation.The mutationaI sites in order were 531Ser(TCG)→Leu(TTG)(40.8%),531 Set(TCG)→Trp(TGG)(12.2%),526His(CAC)→Tyr(TAC)(6.1%),526His(CAC)→Asp(GAC)(6.1%),526His(CAC)→Asn(AAC)(6.1%),526His(CAC)→Pro(CCC)(8.2%).516Asp(GAC)→Val(GTC)(12.2%),533Leu(CTG)→Arg(CGG)(4.1%),and 513GIn(CAA)→Pro(CCA)(2.1%).Conclusion The established DNA probe microarray is specific and sensitive for determination of M.tuberculosis resistant to RFP,and may be an effective tool for rapid determination of M.tuberculosis resistant to RFP in clinicalIaboratories.