摘要
目的初步探讨寡核苷酸分型(Spoligotyping)和多位点数目可变串联重复序列分析(MLVA)用于浙江省结核分枝杆菌临床分离菌株的分型,以初步了解浙江省结核分枝杆菌的基因型特征。方法随机选取浙江省结核分枝杆菌临床分离菌株,常规培养,收集菌体,提取基因组DNA。采用聚合酶链反应(PCR)扩增整个DR区进行Spoligotyping分型,同时分别扩增15个VNTR位点进行MLVA分型。聚类分析采用BioNumerics软件。统计学分析采用卡方检验。结果对70株菌进行了基因分型,Spoligotyping结果显示,可分为2个基因群,即北京家族(Beijingfamily)和非北京家族(Non-Beijingfamily),分别占70和30,49株北京家族菌株中,89.8为典型北京家族;MLVA结果显示,可分4个基因群,分别为Ⅰ群占8.5,含5个基因型,Ⅱ群占18.3,含13个基因型,Ⅲ群占70.4,含38个基因型,Ⅳ群占2.8,含2个基因型。两种方法对菌株的基因分型结果非常吻合,Spoligotyping分型为北京家族的菌株均分布在MLVAⅢ型中,非北京家族菌株的基因多态性,分属于MLVAⅠ、Ⅱ和Ⅳ型。MLVA将70株菌分为58个基因型(含53个独特基因型),而Spoligotyping只分为18种基因型(含12个独特基因型)。结合对临床一线4种药物的耐药检测结果分析,两种方法的分型结果均显示北京家族菌株中表现为全敏感者71.4(35/49),表现为耐药者为28.6(14/49);而非北京家族菌株中表现为全敏感者为66.7,表现为耐药者为33.3,经卡方检验,两者间的差异无统计学意义(χ2=0.158,P>0.05)。结论初步证实浙江省结核分枝杆菌存在明显的基因多态性,主要流行型为北京家族。北京家族与耐药无明显相关性。MLVA株水平鉴定能力明显高于Spoligotyping,尤其是在鉴别北京家族菌株上具有明显的优势。
To analyze the genotypes of Mycobacteriurn tuberculosis clinical isolates in Zhejiang province with spacer oligonucleotide typing （spoligotyping） and multiple loci variable number tandem repeat （VNTR） analysis （MLVA）, 70 clinical strains of Mycobacterium tuberculosis isolated from Zhejiang province were selected randomly. After conventional cultivation, the bacterial DNA was extracted from these isolated strains and the PCR technology was used to amplify the total DR region with spoligotyping and to amplify simultaneously 15 loci of VNTR with MLVA. The clustering of genotypes was analyzed with BioNumberies （Version 5. 0）. Through spoligotyping, these clinical strains of T. tuberculosis could he categorized into two families： the Beijing family （70%） and non-Beijing family （30%）. In the Beijing family, 89.8% （44/49） belonged to the typical strains and these strains could be categorized into 4 genogroups with MLVA, i.e. Ⅰ,Ⅱ,Ⅲ, Ⅳ genogroups, in which Ⅰ genogroup （8.5 $ ） consisted of 5 genotypes; Ⅱ genogroup （18.3%） consisted of 13 genotypes Ⅲ genogroup （70.4%） consisted of 38 genotypes and Ⅳ genogroup （2.8 %） consisted of 2 genotypes. The results using these two methods of genoty- ping matched quite well, from which all the Beijing family strains were MLVA- Ⅲ and the non-Beijing family strains were distributed among MLVA-Ⅰ, Ⅱ, Ⅳ. All the 70 strains of M. tuberculosis could be divided into 58 genotypes including 53 unique genotypes by means of MLVA, while they could be divided into 18 genotypes including 12 unique genotypes by means of spoligotyping. Results of the susceptibility tests against the first-line drugs indicated that 71.4% （35/49） of the Beijing family strains were sensitive , while 28.6% （14/49） were resistant, but in the non-Beijing family strains, 66.7 % were sensitive and 33.3 were resistant. However, no significant difference existed between them as demonstrated by chi-square test （X^2 =0. 158, P〉0.05）. From these observation, it is evident that significant genetic polymorphism exists among the clinical strains of M. tuberculosis isolated from Zhejiang province with the predominantly prevalent strains of Beijing family and without obvious association on the drug-resistance. It is also demonstrated that MLVA should be superior to spoligotyping to analyze the genotypes of M. tuberculosis, especially the Beijing family strains.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2008年第12期1090-1094,共5页
Chinese Journal of Zoonoses
基金
浙江省医药卫生科技基金(No.2003B027)
国家自然科学基金“中国结核分枝杆菌基因多态性及其株水平鉴定技术研究”(No.30771853)
