结核菌L型的形成及其分离与鉴定  

Isolation and determination for L-form variation of Mycobacterium tuberculosis

作　　者：王和[1] 梁菁苹[1] 徐艳[1] 罗振华[1] 

机构地区：[1]贵阳医学院微生物学教研室,550004

出　　处：《贵州医药》2005年第12期1059-1062,共4页Guizhou Medical Journal

摘　　要：目的探讨结核分枝杆茵L型的形成规律及其检测与鉴定方法。方法将结核分枝杆菌分别接种于苏通液体培养基以及含最低抑菌浓度(MIC)的利福平、异烟肼、乙胺丁醇的苏通液体培养基内培养,L型自发形成或被诱导形成后过滤和传代培养获得L型纯培养物。观察L型纯培养物的形态与生长特性以及用结核分枝杆茵特异性基因片段的引物对L型进行菌种的PCR检测与鉴定。结果结核分枝杆菌在非高渗透压培养基内培养3天后可自发形成L型,最低押菌浓度的利福平、异烟肼、乙胺丁醇能够迅速诱导结核分枝杆菌大量形成L型。培养物经0.22μm孔径滤菌器过滤后传代培养可获得L型纯培养物,结核分枝杆菌特异性基因片段的PCR反应能够检测结核分枝杆菌L型并且对L型进行菌种的快速鉴定。结论结核分枝杆菌能够自发形成和在抗结核药物的诱导下形成L型,从而导致病原学检查的漏诊或误诊以及影响治疗效果。采用非高渗培养法能够有效检出结核分枝杆菌L型,用结核分枝杆茵特异性基因片段的PCR反应能够对结核分枝杆菌稳定L型进行菌种快速鉴定。Objective To probe the regulatory of L-form variation of M. tuberculosis（MT） and the methods of isolation and determination. Methods The MT were inoculated separately into the Sauton liquid media without any inducement and the liquid media with minimal bactericidal concentration of RFP, INH or EMB for culturing at 37℃. After the L-forms were generated spontaneously or induced in the media, the cultures were filtered with filter of 0.22um aperture and then sub-cultured by the medium without any inducement to isolate the pure cultures of the L-form. The properties of morphology and growth of pure cultures of the L-form were determined by the PCR reaction with the specific primer for the gene of MT. Results After 3 day of culturing, the L-form cells generated spontaneously would be found in the non-high osmotic cultures of MT, and the L-form would be induced rapidly so that the variable cells were generated with a great quantity in the media with minimal bactericidal concentration of RFP, INH or EMB. The pure cultures of L-form would be isolated from the original cultures by the method of filtering, and then the species of the L-form would be determined rapidly by PCR with the primer for specific gene of the MT. Conclusion By the non-high osmotic culturing, the L-form derived from MT will be isolated effectively and the species of the L-forms will be determined rapidly by the PCR for specific gene of MT.

关 键 词：结核分枝杆菌 细菌L型 鉴定 

分 类 号：R378.911[医药卫生—病原生物学]