某高级中学结核病爆发分离菌株的实验室鉴定

Identification of strains isolated from tuberculosis outbreak in a high school

目的鉴定从某中学结核病爆发分离菌株的种属。方法2006年5—8月辽宁省某高级中学部分学生出现活动性结核病的临床表现,从10例涂片阳性患者的痰标本中分离得到3份菌株经表型鉴定方法,传统生化方法,扩增hupB基因、dnaA-dnaN和NTF-1区;Spoligotyping以及MIRU基因分型;16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析进行鉴定。结果临床分离株经生化方法初步鉴定1株为结核分枝杆菌复合群和2株为非结核分枝杆菌,随后经表型鉴定和扩增hupB基因、dnaA-dnaN和NTF-1区;Spoligotyping以及MIRU基因分型,说明属于结核分枝杆菌复合群的菌株为结核分枝杆菌北京基因型现代株,MIRU基因型为223325173533。经16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析说明属于非结核分枝杆菌的菌株为猪分枝杆菌。结论从结核病爆发累及病人的标本中分离出结核分枝杆菌北京基因型现代株和猪分枝杆菌,猪分枝杆菌在暴发流行中的意义尚需进一步研究。

Objective To identify the bacterial species and strain types of isolates from tuberculosis outbreak in a high school. Methods From May to August 2006, some students developed clinical symptoms of active tuberculosis in a high school, Liaoning Province. Three clinical strains isolated from sputa of 10 patients with smear positive were identified by phenotypic tests; conventional biochemical methods; amplification of hupB gene, dnaA-dnaN region, NTF-1 region, Spoligotyping and micobacterial interspersed repetitive unit （MIRU） typing; partial sequencing of 16S rRNA, 16S-23S internal transcribed spacer （ITS） region, hsp65 gene （441bp） and PCR restriction enzyme analysis of the hsp65 gene. Results Clinical isolates were identified as oneMycobacterium tuberculosis complex and two non-tuberculous mycobacteria （NTM） by conventional biochemical methods. Subsequent analyzed by phenotypic tests and amplification of hupB gene, dnaA-dnaN region, NTF-1 region, Spoligotyping and MIRU genotyping, these results indicated that the species identification of Mycobacterium tuberculosis complex as Mycobacterium tuberculosis Beijing genotype modern strain, the genotype profile of MIRU was 223325173533. By partial sequencing of 16S rRNA, 16S-23S ITS region, hsp65 gene （441 bp） and PCR restriction enzyme analysis of the hsp65 gene supported the species identification of MOTT as M. porcinum. Conclusion The mycobacterium species isolated from patients involved in tuberculosis outbreak are mycobacterium tuberculosis Beijing genotype modern strain and M. porcinum, the significance of M. porcinum in tuberculosis outbreak should be further studied.