中國萊姆病螺旋體MLVA分型方法的建立與研究
發(fā)布時間：2019-05-08 11:52
【摘要】：目的： 建立適合中國萊姆病螺旋體（又稱伯氏疏螺旋體）的多位點可變數(shù)目串聯(lián)重復(fù)序列分析（multi-locus variable number tandem repeat analysis, MLVA）基因分型方法，，對來自中國12個省45個地區(qū)的95株伯氏疏螺旋體進行MLVA分型鑒定和聚類分析，探討中國伯氏疏螺旋體菌群特征，并為萊姆病的分子流行病學(xué)調(diào)查，疫情溯源及菌群進化分析提供實驗基礎(chǔ)和理論依據(jù)。 方法： 1. MLVA是一種以可變數(shù)目串聯(lián)重復(fù)序列（variable number tandem repeat, VNTR）為基本單位的，根據(jù)每個位點VNTR在不同菌株中的多態(tài)性即拷貝數(shù)的差異，形成VNTR分辨率，通過VNTR分辨率的可疊加性以獲得對菌株的最佳分辨效果的基因分型方法。 2.使用Tandem Repeats Finder基因掃描軟件掃描中國優(yōu)勢菌種Borrelia garinii的國際參考菌株P(guān)Bi的904.246kb染色體基因和質(zhì)粒（CP26和LP54）基因，分析基因結(jié)構(gòu)特點，篩選多個潛在的VNTR位點。 3.利用PCR（Polymerase Chain Reaction）技術(shù)檢驗潛在VNTR位點的普遍擴增性和特異性差異的分辨能力。 4.通過STR（Short Tandem Repeat）測序收集VNTR信息并建立菌株VNTR譜。 5.聚類分析，用BioNumerics5.1軟件處理數(shù)據(jù),用Categorical系數(shù)和UPGMA（Unweighted Pair-Group Method withArithmetic，非加權(quán)算術(shù)平均法）進行聚類分析，初步建立萊姆病螺旋體的MLVA分型方法，進而對95株中國菌株和6株國際參考株進行MLVA分型研究，并與多位點序列分析（multi-locus sequenceanalysis, MLSA）的基因分型結(jié)果進行比較。 結(jié)果： 1.篩選出5個VNTR位點進行組合，建立萊姆病螺旋體的MLVA方法。將101株萊姆病螺旋體分為51個獨立的基因型，4個明顯的基因簇，即Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana。分型結(jié)果與MLSA基因分型結(jié)果一致。 2.全部5個VNTR位點（VNTR-1, VNTR-2, VNTR-3, VNTR-4,和VNTR-5）對應(yīng)的等位基因種類數(shù)分別為7,3,9,7和6；相應(yīng)的位點多態(tài)性指數(shù)為0.79,0.22,0.77,0.71和0.67；5個VNTR位點分辨率疊加，多態(tài)性指數(shù)達0.96。 結(jié)論： 1.建立了一種適合中國萊姆病螺旋體菌株的ML VA基因分型的方法。MLVA分型方法操作簡單，分型穩(wěn)定，重復(fù)性好，分型結(jié)果準確可靠。 2.聚類分型結(jié)果表明中國萊姆病螺旋體表現(xiàn)出廣泛的遺傳異質(zhì)性，存在Borreliaburgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana四個基因型，并且在南北方分布存在明顯差異。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R514
本文編號：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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R514
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本文編號：2471888
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