344株结核分枝杆菌的基因分型研究

Study on genotyping of 344 strains of Mycobacterium tuberculosis

目的研究间隔区寡核苷酸序列基因分型技术、结核分枝杆菌散在分布重复单位（MIRU）,以及多位点串联重复序列（VNTR）在河南省结核病分枝杆菌基因分型中的应用,了解河南省局部地区MIRU-VNTR基因型种类与分布,以及北京家族基因型在河南省的分布特征。方法应用2007年在河南省的嵩县、新密县、扶沟县、中牟县等4个县及南阳市的结核病防治机构实验室分离培养的344株结核分枝杆菌,设计引物,应用PCR和琼脂糖凝胶电泳技术及间隔区寡核苷酸分型技术对结核分枝杆菌进行VNTR分型检测分析,基因分型聚类分析采用Bio-Numerics软件。结果对344株结核分枝杆菌临床分离株的间隔区寡核苷酸分型结果显示,有299株为北京家族基因型,占86.9%;12个MIRU位点检测结果显示明显的基因多态性;经基因聚类分析,可分5个基因群（Ⅰ~Ⅴ群）,292个基因型,Ⅰ~Ⅴ群分别占5.1%、67.8%、21.9%、1.4%、2.8%。结论河南省结核分枝杆菌MIRUs基因存在明显的多态性,存在≥5个MIRU型,主要流行型为Ⅱ型;北京家族基因型占主导地位,北京家族基因型与耐药无明显相关性。

OBJECTIVE To investigate the genotyping and distribution pattern of the variable number tandem repeats（VNTR） of Mycobacterium tuberculosis clinical isolates in Henan province with Multi-locus VNTR analysis preliminarily.METHODS M.tuberculosis clinical isolates selected from Henan province randomly were cultured with Lowenstein-Jensen medium.The genome DNA of the bacteria was extracted.The polymorphism of VNTR locus was tested in the DNA samples with PCR respectively.The clustering of genotype was analyzed with BioNumerics（Version5.0）.Chi-Square test was used for statistical analysis.RESULTS 86.9%of the strains belongs to Beijing genotype,Beijing genotype was not significantly associated with the resistance to all of the four first line drugs,but was significantly associated with the MDR.12 MIRUs loci of 344 M.tuberculosis clinical isolates were analyzed respectively.The results showed that there were obvious polymorphisms of VNTRs.The clustering of genotype showed that these strains could be categorized into gene clusters（Ⅰ,Ⅱ,Ⅲ,Ⅳ and Ⅴ）and 292 genotypes.5.1% were cluster Ⅰ including 15genotypes,67.8% were clusterⅡincluding 198 genotypes,21.9 % were cluster Ⅲ including 64 genotypes,1.4% were cluster Ⅳ including 4 genotypes,2.8% were cluster Ⅴ including 11 genotypes.CONCLUSION The result shows that there are obvious polymorphisms of MIRUs of M.tuberculosis clinical strains preliminarily.There are at least 5 clusters of MIRU genotype and cluster Ⅱ is the predominant prevalent strain in Henan province.There is no correlation exists between the drug resistant and beijing family Genotypes.