多位点可变串联重复序列技术用于西藏地区结核分枝杆菌基因分型的初步研究

Preliminary study on genotyping of Mycobacterium tuberculosis strains isolated in Tibet with multiple locus variable numbers of tandem repeats

目的 初步探讨多位点数目可变串联重复序列（MLVA）技术在西藏地区结核分枝杆菌基因分型中的应用和初步了解西藏地区结核分枝杆菌数目可变串联重复序列（VNTR）基因型种类及其分布。方法 在拉萨、山南、林芝、日喀则、那曲、昌都6个地区结核病防治中心收集结核分枝杆菌临床分离菌株,设计引物,采用PCR和琼脂糖凝胶电泳技术对结核分枝杆菌20个VNTR位点进行检测,并通过BioNumerics 4．5软件进行DNA指纹图谱多态性分析。结果 共收集到217株结核分枝杆菌,分为19个不同的VNTR基因型,其中以Ⅺ型为主要基因型占87．6%（属于北京家族）,其次Ⅵ型、ⅩⅤ型、ⅩⅥ型各占1．38%,Ⅰ型、Ⅶ型、ⅩⅢ型各占0．92%,12株结核分枝杆菌为单一的基因型。不同地区之间,以Ⅺ型为主要基因型,分别占各地区的86．8%、91．3%、78．95%、88．2%、95．05、89．3%。结论 西藏地区的结核分枝杆菌存在明显的基因多态性,主要流行型为VNTRⅪ型（即北京家族基因型）,初步研究结果显示北京家族基因型与卡介苗接种和耐药性无相关性。MLVA分型方法简单、快速,可以有效地用于结核分枝杆菌的基因分型和病原监测。

Objective To investigate the application of the multiple locus variable numbers of tandem repeats （MLVA） in genotype Mycobacterium tuberculosis strains isolated from Tibet, and to understand the characteristics of genotype and distribution. Methods 217 M. tuberculosis strains were collected from six regions of Tibet. Twenty tandem repeats loci in the total genome of M. tuberculosis （MTB） were analyzed by PCR and agarose gel electrophoresis method. The characteristics on polymorphism of DNA fingerprinting of 217 MTB strains were analyzed with BioNumerics 3.0 software. Results 217 M. tuberculosis strains detected with 20 MLVA loci were classified to 19 genotypes with 87.6% of the stains belonging to Beijing genotype and the other 18 genotypes were scattered, accounted for Ⅰ 38 % and 0.92% strains,respectively. Beijing genotype was not significantly associated with the resistance to all of the four drugs and BCG vaccination. Conclusion It is concluded that the strains of MTB isolated in Tibet present definite polymorphism and most of the epidemic strains belonged to Beijing family genotype and MTB genotyping. The Beijing genotype was not recognized as the one transferred from some of the drug resistance strains or from BCG vaccination. Being a fast and simple technique, MLVA method, seemed a better molecular typing method and could be used for genotyping in M. tuberculosis and monitoring pathogen.