应用聚合酶链反应-膜芯片技术快速鉴定分支杆菌菌种

目的建立一种简便、快速、灵敏、特异的分支杆菌菌种鉴定方法。方法通过 1 6SrDNA聚合酶链反应 单链构象多态性 (polymerasechainreaction singlestrandedconformationpolymorphism ,PCR SSCP)分析鉴定 1 4 0株分支杆菌临床分离株 ;设计与合成用于鉴定分支杆菌菌种的 1 6SrDNA寡核苷酸探针 ,制作膜芯片 ,与待测菌株生物素标记的 1 6SrDNA基因PCR产物进行反向斑点杂交。结果 1 4 0株分支杆菌临床分离株中 ,经 1 6SrDNAPCR SSCP初步鉴定 ,1 33株为结核分支杆菌复合群 ,7株为非结核分支杆菌。经膜芯片杂交分析 ,1 33株结核分支杆菌分离株鉴定为结核分支杆菌复合群 ;7株非结核分支杆菌中 ,1株鉴定为戈登分支杆菌 ,1株为土分支杆菌 ,1株为偶发分支杆菌 ,1株为胞内分支杆菌 ,另 3株与分析探针杂交阴性。分析 2 8种分支杆菌标准菌株和 9种非分支杆菌菌株 ,结果显示寡核苷酸探针是特异的。结论 1 6SrDNAPCR 膜芯片技术灵敏度高、特异性强、简便、快速 ,可用于鉴定分支杆菌菌种

RAPID IDENTIFICATION OF MYCOBACTERIUM SPECIES BY POLYMERASE CHAIN REACTION-GENE MEMBRANE CHIP

ObjectiveTo establish a simple,rapid,sensitive and specific method for identification of mycobacterium species. MethodsOne hundred and forty clinical isolates of mycobacteria were identified by 16S rDNA polymerase chain reaction single stranded conformation polymorphism(PCR SSCP) analysis.Oligonucleotide probe and gene membrane chip of 16S rDNA gene which identifid mycobacterium species were prepared. The 16S rDNA target DNA fragment from detected strains was labeled with biotin by PCR amplification, and then reverse dot blot hybridized with oligonucleotide probe on gene membrane chip. ResultsOne hundred and forty clinical isolates of mycobacteria by PCR SSCP were analyzed, 133 strains were mycobacterium tuberculosis complex, 7 strains were non tuberculous mycobacterium, 133 clinical isolates of mycobacteria were identified as mycobacterium tuberculosis complex by reverse dot blot hybridization. Of 7 clinical isolates of non tuberculous mycobacterium, 1 strain was M.gordonae, 1 strain was M.terra, 1 strain was M.fortuitum, 1 strain was M.intracellular, another 3 strains were negative hybridization with all probes. The standard strains of 28 mycobacteria and 9 nonmycobacteria by gene membrane chip were analyzed, the result showed that the oligonucleotide probe was specific. ConclusionThe 16S rRNA PCR gene membrane chip technique is simple and rapid with higher sensitivity, specificity, and could be used for identification of mycobacterium species.