【摘要】：鼠疫耶爾森氏菌(Yersinina pestis,以下簡稱鼠疫菌)曾引發(fā)世界范圍的三次鼠疫大爆發(fā),給人類帶來了災難。鼠疫之所以能夠如此猖獗的肆掠整個世界,主要是因為鼠疫作為自然疫源性疾病以其傳播速率極快,突發(fā)病急不可預測,死亡率極高和傳染性極強為特征的甲類烈性傳染病。本研究利用多種遺傳標記物對我國鼠疫東方型菌株進行分型,可以避免采用單一遺傳標記而導致的分辨率不足或偏性結(jié)果。主要研究結(jié)果如下： 1.建立了Luminex多重檢測SNP的技術(shù)平臺。鼠疫菌作為一種較為“年輕”且缺少基因重組的病原細菌,單核苷酸多態(tài)性(Single Nucleotide polymorphisms, SNP)可作為其非�？煽糠中秃拖到y(tǒng)發(fā)育分析指標。本研究建立基于Lumienx x-TAG微球(液態(tài)懸浮芯片)的鼠疫菌SNP分型技術(shù),利用多重PCR同時擴增鼠疫菌18個SNP位點片段,將擴增完成的產(chǎn)物進行多重ASPE (Allele Specific Primer Extension)反應以及Luminex技術(shù)多重SNP檢測,通過一次反應可以檢測出待測菌株中18個SNP位點的堿基特征。利用這一方法分析20株鼠疫東方型菌株,可以將其根據(jù)18個SNP位點的特征分為7個基因型,本方法具有判讀方便快速、高通量、成本低廉等特點,在病原菌的SNP分型方面具有廣闊的應用前景。 2.初步探討了我國鼠疫菌東方型的起源。目前國際上普遍認為,鼠疫的第三次大流行起源于中國云南,鼠疫菌東方型菌株也起源于云南。但這與我們前期所研究的結(jié)果在某些方面與這一輪點不相符,即我們目前所測定的云南東方型菌株都是比較“年輕”的菌株,而中國最古老的東方型菌株分離自福建。本研究將運用Luminex技術(shù)平臺檢測分析大量的云南東方型菌株,對云南東方型菌株有個初步的探討分型結(jié)果,我們對193株鼠疫東方型菌株進行分型,一共分為13個基因型,分別是a, b, c, d, e, f, g, h, i, j, k,l, m。53個SNP位點信息對后期期追溯東方型鼠疫菌的起源提供了重要的數(shù)據(jù)。
[Abstract]:Yersinia pestis (Yersinina pestis, (Yersinia pestis) has caused three outbreaks of Yersinia pestis worldwide and brought disaster to human beings. The reason why plague is rampant throughout the world is mainly because plague as a natural epidemic disease is characterized by its rapid transmission rate, sudden and unpredictable disease, very high mortality and highly infectious type A strong infectious disease. In this study, a variety of genetic markers were used to type the oriental strains of plague in China, which could avoid the lack of resolution or bias caused by the use of single genetic marker. The main results are as follows: 1. The technical platform of Luminex multiple detection SNP is established. Yersinia pestis as a relatively "young" and lack of gene recombination of pathogenic bacteria, single nucleotide polymorphism (Single Nucleotide polymorphisms, SNP) can be used as a very reliable typing and phylogenetic analysis index. In this study, the SNP typing technique of Yersinia pestis based on Lumienx x-TAG microspheres (liquid suspension chip) was established, and 18 SNP loci of Yersinia pestis were amplified simultaneously by multiple PCR. The amplified products were detected by multiple ASPE (Allele Specific Primer Extension) reaction and Luminex technique by multiple SNP detection. The base characteristics of 18 SNP sites in the tested strain could be detected by one reaction. By using this method, 20 strains of Yersinia pestis oriental strain can be divided into 7 genotypes according to the characteristics of 18 SNP loci. The method is characterized by easy and fast interpretation, high throughput and low cost. It has a broad application prospect in SNP typing of pathogenic bacteria. 2. The oriental origin of Yersinia pestis in China was preliminarily discussed. At present, it is widely believed that the third pandemic of plague originated in Yunnan, China, and the oriental strain of Yersinia pestis also originated in Yunnan. However, this does not accord with the results of our previous studies in some respects, that is, the oriental strains in Yunnan are relatively "young" strains, and the oldest oriental strains in China are isolated from Fujian Province. In this study, a large number of oriental strains of Yunnan Province were detected and analyzed by using Luminex technology platform, and a preliminary study on the results of typing of oriental strains of Yunnan Province was carried out. We typed 193 strains of Oriental Yersinia pestis into 13 genotypes. The information of SNP loci of a, b, c, d, e, f, g, h, i, j, kanll and m.53 provides important data for tracing the oriental Yersinia pestis origin in late stage.
【學位授予單位】：湖南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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