MLVA和Spoligotyping用于西藏地区216株结核分枝杆菌临床分离株的基因分型研究

Genotyping study of 216 Mycobacterium tuberculosis strains isolated from the patients in Tibet with MLVA and Spoligotyping

目的评价间隔区寡核苷酸分型(Spoligotyping)及多位点可变数量串联重复序列分析(MLVA)两种分型方法在西藏地区结核病分子流行病学中的应用。方法收集西藏地区结核分枝杆菌(Mycobacterium tuberculosis)临床分离株,应用Spoligotyping及MLVA两种分型方法进行比较分析。结果共在西藏地区收集到216株结核分枝杆菌临床分离株,采用Spoligotyping分型方法,216株菌可分为3个基因群13种基因型,其中最大的1个基因群即北京家族(Beijing family)含有195株菌,占90.28%。北京家族菌株中,有BCG接种史者占45.64%(89/195),无BCG接种史者占54.36%(106/ 1195),两者间的差异无统计学意义(x^2=0.059,P>0.05)。采用MLVA分型方法,216株菌可分成19个基因群108种基因型,其中80种基因型只有1株菌,占37.03%(80/216),另有136株菌表现出28种基因型,成簇数为28,占62.96%(136/216)。在20个VNTR位点的等位基因多态性发现Miru31位点的多态性最高,多态性指数(h)达到0.77,而Mtub29、Mtub12位点的多态性较差,都低于0.05。其中Mtub02位点可鉴别北京家族和非北京家族,它鉴别的北京家族与Spoligotyping鉴别的北京家族符合率达到100%。结论西藏地区结核分枝杆菌具有明显的接引多态性,其主要流行型为北京家族。北京家族菌株与BCG接种无相关性。应用Spoligotyping和MLVA两种分型方法进行结核病流行病学研究,将提高结核病的流行病学调查和病原学监测效果。

Objective To evaluate the application of spacer oligonucleotide typing （Spoligotyping） and multiple loci VNTRs （variable number of tandem repeats） analysis （MLVA） in molecular epidemiology study of tuberculosis and to discuss the characteristics of M. tubercuiosis strains in Tibet. Methods M. tubercuiosis strains isolated from the patients in Tibet, and typed by Spoligotyping and MLVA respectively to compare the results of the both methods and access the application of both methods in epidemiological studies of tuberculosis. Results Two hundred and sixteen clinical isolates of M. tuberculosis were collected in Tibet. By means of Spoligotyping, 216 clinical isolates were typed into 3 gene groups, displaying 13 genutypes. In which the biggest group was M. tuberculosis Beijing family including 195 strains （90.28%）. In Beijing family strains, there were 89 strains （45.64%） from the patients with BCG vaccine, while 106 strains （54.36%） from the patients without BCG vaccine. There was no significant difference in statistics （X^2 = 0.059, P 〉 0.05）. With MLVA, 216 clinical isolates were typed into 19 gene groups, displaying 108 genutypes, in which 80 strains （37.03%） were typed 80 genotypes respectively, and 136 strains （62.96%） were typed 28 genotypes which showed 28 clusters respectively. According to the results in the 20 VNTR loci, Miru31 showed the highest diversity, Its h was 0.77, while Mtub29 and Mtubl2 showed the lowest diversity, their h was less than 0.05. The Mtuh02 could identify the Beijing family strains and non-Beijing family strains, of which the results completely correspond to that by Spoligotyping. Conclusion In the study it is preliminarily confirmed that there is high polymorphism of M . tuberculosis and Beijing family is the main genotype and main prevalent strain in Tibet. According to the resuhs we could not confirm that there is a relationship between Beijing family strains and BCG vaccine. The results of this study also indicated that Spoligotyping and MLVA are useful for molecular epidmiological studies on tuberculosis and improve the effection of epidemiological investigation and pathological surveillance of tuberculosis.