中国莱姆病螺旋体MLVA分型方法的建立与研究
发布时间：2019-05-08 11:52
【摘要】：目的： 建立适合中国莱姆病螺旋体（又称伯氏疏螺旋体）的多位点可变数目串联重复序列分析（multi-locus variable number tandem repeat analysis, MLVA）基因分型方法，，对来自中国12个省45个地区的95株伯氏疏螺旋体进行MLVA分型鉴定和聚类分析，探讨中国伯氏疏螺旋体菌群特征，并为莱姆病的分子流行病学调查，疫情溯源及菌群进化分析提供实验基础和理论依据。 方法： 1. MLVA是一种以可变数目串联重复序列（variable number tandem repeat, VNTR）为基本单位的，根据每个位点VNTR在不同菌株中的多态性即拷贝数的差异，形成VNTR分辨率，通过VNTR分辨率的可叠加性以获得对菌株的最佳分辨效果的基因分型方法。 2.使用Tandem Repeats Finder基因扫描软件扫描中国优势菌种Borrelia garinii的国际参考菌株PBi的904.246kb染色体基因和质粒（CP26和LP54）基因，分析基因结构特点，筛选多个潜在的VNTR位点。 3.利用PCR（Polymerase Chain Reaction）技术检验潜在VNTR位点的普遍扩增性和特异性差异的分辨能力。 4.通过STR（Short Tandem Repeat）测序收集VNTR信息并建立菌株VNTR谱。 5.聚类分析，用BioNumerics5.1软件处理数据,用Categorical系数和UPGMA（Unweighted Pair-Group Method withArithmetic，非加权算术平均法）进行聚类分析，初步建立莱姆病螺旋体的MLVA分型方法，进而对95株中国菌株和6株国际参考株进行MLVA分型研究，并与多位点序列分析（multi-locus sequenceanalysis, MLSA）的基因分型结果进行比较。 结果： 1.筛选出5个VNTR位点进行组合，建立莱姆病螺旋体的MLVA方法。将101株莱姆病螺旋体分为51个独立的基因型，4个明显的基因簇，即Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana。分型结果与MLSA基因分型结果一致。 2.全部5个VNTR位点（VNTR-1, VNTR-2, VNTR-3, VNTR-4,和VNTR-5）对应的等位基因种类数分别为7,3,9,7和6；相应的位点多态性指数为0.79,0.22,0.77,0.71和0.67；5个VNTR位点分辨率叠加，多态性指数达0.96。 结论： 1.建立了一种适合中国莱姆病螺旋体菌株的ML VA基因分型的方法。MLVA分型方法操作简单，分型稳定，重复性好，分型结果准确可靠。 2.聚类分型结果表明中国莱姆病螺旋体表现出广泛的遗传异质性，存在Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana四个基因型，并且在南北方分布存在明显差异。
[Abstract]:Objective: to establish a multi-site variable number tandem repeat analysis (multi-locus variable number tandem repeat analysis, MLVA) genotyping method suitable for Lyme disease spirochetes in China. MLVA typing and cluster analysis of 95 strains of Borrelia burgdorferi from 45 regions of 12 provinces in China were carried out to explore the microflora characteristics of Borrelia burgdorferi in China, and to investigate the molecular epidemiology of Lyme disease. The analysis of the origin of the epidemic situation and the evolution of the flora provide experimental and theoretical basis. Methods: 1. MLVA is a kind of VNTR resolution, which is based on the variable number of tandem repeats (variable number tandem repeat, VNTR). According to the polymorphism of VNTR in different strains, that is, the number of copies, VNTR resolution is formed. The genotyping method of the best resolution of the strain was obtained by the superposition of VNTR resolution. 2. The Tandem Repeats Finder gene scanning software was used to scan the 904.246kb chromosome gene and plasmid (CP26 and LP54) genes of the international reference strain PBi of the dominant strain Borrelia garinii in China. The characteristics of the gene structure were analyzed and several potential VNTR sites were screened. 3. PCR (Polymerase Chain Reaction) technique was used to test the universal amplification and specific difference of potential VNTR loci. 4. The VNTR information was collected by STR (Short Tandem Repeat) sequencing and the VNTR spectrum of the strain was established. 5. Cluster analysis, BioNumerics5.1 software was used to process the data, Categorical coefficient and UPGMA (Unweighted Pair-Group Method withArithmetic, unweighted arithmetic average method were used for cluster analysis, and the MLVA classification method of Lyme disease spirochetes was established. Furthermore, 95 Chinese strains and 6 international reference strains were genotyped by MLVA, and the genotyping results were compared with those of multi-site sequence analysis (multi-locus sequenceanalysis, MLSA). Results: 1. Five VNTR loci were selected for combination to establish a MLVA method for Lyme disease spirochetes. 101 strains of Lyme disease spirochetes were divided into 51 independent genotypes and 4 obvious gene clusters, namely Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana.. The results of genotyping were consistent with those of MLSA genotyping. 2. The number of alleles corresponding to all five VNTR loci (VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5) was 7, 3, 9, 7 and 6, respectively, and the corresponding polymorphism index was 0.79, 0.22, 0.77, 0.71 and 0.67, respectively. The resolution of 5 VNTR loci was superimposed, and the polymorphism index was 0.96. Conclusions: 1. A method for MLVA genotyping of Lyme disease spirochetes in China was established. MLVA typing method is simple, stable, reproducibility and accurate and reliable. 2. The results of cluster typing showed that Lyme disease spirochetes in China showed extensive genetic heterogeneity, there were four Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana genotypes, and there were significant differences in the distribution of Lyme disease spirochetes in the north and south.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R514
本文编号：2471888
[Abstract]:Objective: to establish a multi-site variable number tandem repeat analysis (multi-locus variable number tandem repeat analysis, MLVA) genotyping method suitable for Lyme disease spirochetes in China. MLVA typing and cluster analysis of 95 strains of Borrelia burgdorferi from 45 regions of 12 provinces in China were carried out to explore the microflora characteristics of Borrelia burgdorferi in China, and to investigate the molecular epidemiology of Lyme disease. The analysis of the origin of the epidemic situation and the evolution of the flora provide experimental and theoretical basis. Methods: 1. MLVA is a kind of VNTR resolution, which is based on the variable number of tandem repeats (variable number tandem repeat, VNTR). According to the polymorphism of VNTR in different strains, that is, the number of copies, VNTR resolution is formed. The genotyping method of the best resolution of the strain was obtained by the superposition of VNTR resolution. 2. The Tandem Repeats Finder gene scanning software was used to scan the 904.246kb chromosome gene and plasmid (CP26 and LP54) genes of the international reference strain PBi of the dominant strain Borrelia garinii in China. The characteristics of the gene structure were analyzed and several potential VNTR sites were screened. 3. PCR (Polymerase Chain Reaction) technique was used to test the universal amplification and specific difference of potential VNTR loci. 4. The VNTR information was collected by STR (Short Tandem Repeat) sequencing and the VNTR spectrum of the strain was established. 5. Cluster analysis, BioNumerics5.1 software was used to process the data, Categorical coefficient and UPGMA (Unweighted Pair-Group Method withArithmetic, unweighted arithmetic average method were used for cluster analysis, and the MLVA classification method of Lyme disease spirochetes was established. Furthermore, 95 Chinese strains and 6 international reference strains were genotyped by MLVA, and the genotyping results were compared with those of multi-site sequence analysis (multi-locus sequenceanalysis, MLSA). Results: 1. Five VNTR loci were selected for combination to establish a MLVA method for Lyme disease spirochetes. 101 strains of Lyme disease spirochetes were divided into 51 independent genotypes and 4 obvious gene clusters, namely Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana.. The results of genotyping were consistent with those of MLSA genotyping. 2. The number of alleles corresponding to all five VNTR loci (VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5) was 7, 3, 9, 7 and 6, respectively, and the corresponding polymorphism index was 0.79, 0.22, 0.77, 0.71 and 0.67, respectively. The resolution of 5 VNTR loci was superimposed, and the polymorphism index was 0.96. Conclusions: 1. A method for MLVA genotyping of Lyme disease spirochetes in China was established. MLVA typing method is simple, stable, reproducibility and accurate and reliable. 2. The results of cluster typing showed that Lyme disease spirochetes in China showed extensive genetic heterogeneity, there were four Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana genotypes, and there were significant differences in the distribution of Lyme disease spirochetes in the north and south.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R514
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