应用PCR-反向斑点杂交快速鉴定分枝杆菌菌种.docVIP

应用PCR-反向斑点杂交快速鉴定分枝杆菌菌种[摘要] 目的:应用聚合酶链反应-反向斑点杂交法(PCR-RDBHA)快速鉴定分枝杆菌至种的水平。方法:以分枝杆菌rpoβ基因编码序列为靶基因,应用PCR-RDBHA检测126株分枝杆菌临床分离株。以PCR-DNA测序为对照,对经PCR-RDBHA鉴定为非结核分枝杆菌(NTM)的临床分离株进行PCR-DNA测序。结果:126株临床分离株中,经鉴定115株(91.3%)为结核分枝杆菌复合群(MTC),11株(8.7%)为NTM。NTM中,4株胞内分枝杆菌,1株堪萨斯分枝杆菌,1株戈登分枝杆菌,2株瘰疬分枝杆菌,3株脓肿分枝杆菌。11株NTM PCR-RDBHA与PCR-DNA测序结果一致。结论:PCR-RDBHA能鉴定分枝杆菌临床分离株至种的水平,方法简便、快速,结果准确,具有良好的临床应用价值。 [关键词] 分枝杆菌;菌种鉴定;聚合酶链反应;反向斑点杂交 [中图分类号] R378.91[文献标识码]A [文章编号]1673-7210(2010)03(c)-035-02 PCR-reverse dot blot hybridization assay for rapid identification of Mycobacterium species ZHANG Xiaojuan (Thorax Disease Hospital of Shenyang, Shenyang 110044, China) [Abstract] Objective: To identify Mycobacterium species rapidly by polymerase chain reaction-reverse dot blot hybridization assay (PCR-RDBHA). Methods: 126 mycobacterial clinical isolates were detected by PCR-RDBHA based on the sequence of rpoβ gene. PCR-DNA sequencing of the nontuberculous mycobacteria (NTM) identified by PCR-RDBHA was performed as control. Results: Among 126 isolates, 115 (91.3%) were determined to be M.tuberculosis complex (MTC), 11 (8.7%) to be NTM which contained 4 M.intracellulare, 1 M.kansasii, 1 M.gordonae, 2 M.scrofulaceum and 3 M.abscessus. The results of 11 NTM identified by PCR-RDBHA were in agreement with PCR-DNA sequencing. Conclusion: It is simple, rapid, accurate and valuable in clinical application that PCR-RDBHA is applied to identify mycobacterial clinical isolates to species level. [Key words] Mycobacterium; Species identification; Polymerase chain reaction; Reverse dot blot hybridization assay 目前,常规的分枝杆菌菌种鉴定需采用分枝杆菌培养物进行生长特性试验(生长速度、生长温度、光产色试验、多种鉴别培养基上生长试验)及生化特性试验(耐热触酶试验、硝酸盐还原试验、烟酸试验、吐温-80水解试验、尿素酶水解试验、芳香硫酸酯酶试验、铁离子吸收试验、亚硝酸盐还原试验)等10余项试验,综合分析判定结果[1],方法复杂、费时,慢生长分枝杆菌需3～4周方可获得结果,且准确度较差,难以满足临床诊断和治疗的需要。本实验以分枝杆菌rpoβ基因为靶基因序列,应用聚合酶链反应-反向斑点杂交法(PCR-RDBHA)快速鉴定分枝杆菌临床分离株至种的水平,结果报道如下: 1 材料与方法 1.1 材料 1.1.1 分枝杆菌分离株经抗酸染色涂片检测阳性的分枝杆菌临床分离株126株,来自我院结核科和解放军第三?九医院。 1.1.2 引物及探针引物Ⅰ和Ⅱ及21种分枝杆菌寡核苷酸探针序列参照文献[