中国不同地区间日疟原虫种群结构及其在溯源检测中的应用

发布时间：2018-07-09 12:00

本文选题：间日疟 + 微卫星　；参考：《第二军医大学》2010年硕士论文

【摘要】： 疟疾是世界范围内危害最严重的一种蚊媒寄生虫病,尤其是对非洲、南美以及东南亚地区产生极大危害。目前,全球每年疟疾临床发病人数为3-5亿,其中100-300万人死于该病,90%的死亡数发生在非洲,绝大部分是5岁以下的儿童。间日疟原虫(Plasmodium vivax)是世界上分布最广的疟原虫,虽然其致病的严重程度不如恶性疟原虫高,但是由于其具有更广泛的适应性和较高的复发率,它仍然给人们的健康,尤其是儿童带来极大的危害。间日疟的流行和发展对全球的公共卫生安全带来重大的挑战。在中南美洲,中东地区,中亚、南亚和东南亚以及大洋洲和东非地区,每年约有26亿人受到间日疟的威胁,其中包括7至8千万的临床病例；在巴西亚马逊流域,间日疟原虫甚至已经超过恶性疟成为致死性疟疾的主要病原体,其传播和发病形式不容乐观。在我国,经过50多年不懈的努力,疟疾的发病从整体上已经得到有效控制,但近几年来,我国一些地区的疟疾疫情出现了回升趋势,特别是安徽等地区出现了间日疟的暴发流行。为应对当前疟疾流行的严峻形势,仍需要加强疟疾防治的基础研究,特别是研发疟疾防治相关的新技术,包括疟疾感染的溯源检测技术。在此项研究中,我们从云南、海南和华中三个不同流行地区采集间日疟疾患者血样本。云南地区样本采集地区按照地理分布主要分为三个区域：(1)怒江流域：流行区包括德宏自治州和中缅边界区域,样本来源为腾冲县、龙陵县、德宏自治州及与缅甸接壤地区包括境外的缅甸克钦邦拉咱地区。(2)澜沧江流域：流行区包括临沧地区、思茅地区及西双版纳傣族自治州,样本来源为西双版纳傣族自治州的景洪市和勐腊县。(3)红河流域：流行区包括红河自治州和文山自治州,样本来源为红河州红河县及元江县。在云南地区共采集疟疾患者血样本共310份。海南地区样本采集地包括三亚市,乐东县和东方市三个地区,采集样本数共计82份。华中地区样本采集地包括安徽、湖北和河南三省,采集样本数共计530份。所有样本均采用试剂盒提取基因组DNA。参考目前国际广泛应用于地理株种群结构和遗传多样性研究的工具和方法,选择间日疟基因组不同染色体上的23个微卫星位点(MSs)对我国间日疟原虫进行种群结构分析及遗传多样性研究,并通过一些地区特异性的位点初步建立溯源检测的体系。这些微卫星位点包括12.335、7.67、NA.1276、NA.2208、8.332、6.34、2.21、10.29、3.35、3.27、1.501、3.502、9-AT、14-AT、1-TCA、4-GAA、5-TCT-1、5-TCT-2、6-TAC、11-CAA、11-TGA、13-CTT、u2.6, d4,其中前12个为引子文献的多态性位点,后12个为自行筛选的位点,核心重复单位为2至8个碱基不等。每个MS位点设计3条引物,其中1条为荧光标记引物。采用半巢氏PCR方法扩增每个微卫星位点,第二轮PCR采用荧光标记引物,将PCR产物进行GENESCAN检测。检测得到不同的片段长度,代表微卫星等位基因的不同类型。我们还选择了两个SNPs位点作为溯源检测的核心位点,这两个位点与疟原虫抗药性基因相关,实验证明它们具有地区特异性,使得初步建立溯源检测系统成为可能。我们对所有样本微卫星检测结果进行整理,并利用相应的生物信息学的软件进行统计学的分析。我们用GenALEx软件进行平均等位基因,期望杂合度以及方差分析,用在线分析软件LIAN计算种群的连锁不平衡性,Mega软件用于绘制遗传进化树图,并进行溯源相关的判别分析。我们发现所选择的大部分MS位点在不同地区的样本中具有高度的变异性,平均每个位点有5-33个等位基因,云南地区样本的平均等位基因数目为14.27±1.63,期望杂合度为0.768±0.035,海南地区平均等位基因数目为8.53±0.98,期望杂合度为0.707±0.042,华中地区平均等位基因数目为10.27±1.33,期望杂合度为0.638±0.056。华中地区的样本具有显著的连锁不平衡性(p0.001)。云南和海南地区样本之间的遗传距离更近为0.097,而华中地区的样本和这两个地区相距较远分别为0.161和0.197。在以上研究结果的基础上,我们尝试用判别分析的方法初步探讨旨在建立间日疟疾溯源检测技术的判别系统。结果发现两个微卫星位点在此系统中有良好的区分性,可作为中国这三大地区的特异性的分子标记。另外,本实验其它研究发现的两个SNPs和一个MS具有较好的区分性。采用三个MS和两个SNP作为我国间日疟溯源检测的分子标志,对我国三个间日疟疾主要流行区样本进行溯源分析,结果显示：海南地区的33个样本均被正确判定为该地区样本,正确率达到100%。华中地区181个样本有179个被正确判定,其余2个样本被判定为海南地区样本,正确率达到98.9%。云南地区92个样本中有78个样本被正确判定为该地区样本,而分别有3个和11个样本被判定为海南和华中地区样本,判定的正确率为84.8%。三个地区总体结果有94.8%的样本能够正确地被判断为来源地区的样本,交叉验证的结果相同,有较高的判别符合率。最后,我们从华中地区选择了未参与系统建立的20个样本对此系统进行验证,结果发现华中地区验证结果均符合实际情况,正确率达到100%。综合本研究结果表明,中国各个地区间日疟原虫基因组具有较高的变异度,其种群结构有显著特点,从进化上来看,云南和海南地区样本遗传关系更近,它们与华中地区样本差异较大。而华中地区样本在我们选择的微卫星位点上体现出显著的连锁不平衡性,提示该地区间日疟原虫的高近亲繁殖率和低的基因组有效重组率。我们初步建立的溯源监测系统具有良好的溯源检测能力,判别符合率和验证正确率均达到90%以上。随着样本数的增加,此系统的准确性和稳定性会进一步提高,以期能为我国的疟疾防治和疟疾消除计划提供技术支撑。
[Abstract]:Malaria is the most dangerous mosquito borne parasitic disease in the world, especially in Africa, South America and South East Asia. At present, the number of malaria cases in the world is 3-5 billion, of which 100-300 million people die from the disease, 90% of the deaths occur in Africa, and most of them are children under 5 years of age. Plasmodium vivax) is the most widely distributed malaria parasite in the world, although its severity is not as high as that of Plasmodium falciparum, but because of its wider adaptability and higher recurrence rate, it still brings great harm to people's health, especially for children. The prevalence and development of Plasmodium falciparum to the global public health safety belt In central and South America, the Middle East, Central Asia, South and South East Asia, and Oceania and East Africa, about 2 billion 600 million people are threatened with Plasmodium vivax each year, including 7 to 80 million of clinical cases; and in the Amazon basin of Brazil, Plasmodium vivax has even exceeded falciparum malaria as the main pathogen of fatal malaria, The form of its spread and disease is not optimistic. In China, after 50 years of unremitting efforts, the incidence of malaria has been effectively controlled. However, in recent years, the epidemic situation of malaria in some areas of our country has been rising, especially in Anhui and other areas, the outbreak of vivax malaria. Basic research on malaria control still needs to be strengthened, especially the new technologies related to the development of malaria control, including the traceability and detection technology of malaria infection.
In this study, we collect blood samples from three different epidemic areas in Yunnan, Hainan and central China. The area of sample collection in Yunnan region is divided into three regions according to the geographical distribution: (1) the Nu River basin: the popular areas include Dehong Autonomous Prefecture and the Sino Burma border region, and the samples are from Tengchong County, Long Ling County, Dehong and Dehong. Zhi Zhou and the border area with Burma include the Burma Kachin State LA Zan area abroad. (2) the Lancang River Basin: the popular areas include Lincang, Simao and Xishuangbanna Dai Autonomous Prefecture. The samples are from Jinghong and Mengla counties of the Xishuangbanna Dai Autonomous Prefecture. (3) the Red River Basin: the popular areas include the Honghe autonomous state and Wenshan Autonomous Prefecture, The sample source is Honghe County and Yuanjiang County, Honghe Prefecture. A total of 310 samples of blood samples were collected in Yunnan area. The samples collected in Hainan area include Sanya, Le Dong county and Dongfang City, with a total of 82 samples. The sampling plots in Central China include Anhui, Hubei and Henan provinces with a total of 530 samples. Samples were used to extract genomic DNA. with a reference kit for reference to the research tools and methods widely used in the population structure and genetic diversity of geographical strains. 23 microsatellite loci on different chromosomes of Plasmodium vivax (MSs) were selected to analyze the population structure and genetic diversity of Plasmodium vivax in China. These microsatellite loci include 12.335,7.67, NA.1276, NA.2208,8.332,6.34,2.21,10.29,3.35,3.27,1.501,3.502,9-AT, 14-AT, 1-TCA, 4-GAA, 5-TCT-1,5-TCT-2,6-TAC, 11-CAA, 11-TGA, 13-CTT, u2.6, D4, and the first 12 are polymorphic loci in the introductory literature, and the last 12 are from the source. The core repeats were 2 to 8 bases. 3 primers were designed for each MS site, 1 of which were fluorescent marker primers. The microsatellite loci were amplified by the semi nest PCR method, and the second rounds of PCR were used to detect the PCR products by GENESCAN. The length of the fragment was detected, and the microsatellite was detected. We also selected two SNPs loci as the core loci of traceability detection. These two loci are related to the resistance genes of Plasmodium. Experiments show that they have regional specificity, which makes it possible to establish a preliminary traceability detection system. All of these microsatellite detection results are collated and utilized. The software of bioinformatics should be analyzed statistically. We use GenALEx software to carry out average alleles, expect heterozygosity and analysis of variance, and use online analysis software LIAN to calculate the linkage disequilibrium of the population. Mega software is used to map genetic evolution tree and carry out traceability related discriminant analysis.
We found that most of the selected MS loci have high variability in the samples of different regions, with 5-33 alleles per locus on average. The average number of alleles in the Yunnan region is 14.27 + 1.63, the expected heterozygosity is 0.768 + 0.035, the average number of alleles in Hainan is 8.53 + 0.98, and the expected heterozygosity is 0.707. The average number of alleles in Central China was 10.27 + 1.33, and the expected heterozygosity was 0.638 + 0.056. in Central China, and there was significant linkage disequilibrium (p0.001). The genetic distance between Yunnan and Hainan regions was closer to 0.097, while the samples from central China and the two regions were 0.161 and 0.197. respectively, respectively. On the basis of the results of the study, we try to use discriminant analysis to preliminarily discuss the discriminant system aimed at establishing the detection techniques for the date of the day of malaria. The results show that two microsatellite loci have good distinctiveness in this system and can be used as specific molecular markers in the three regions of China. In addition, other studies found in this experiment Two SNPs and one MS have good distinctiveness. Using three MS and two SNP as the molecular markers of the traceability detection of Plasmodium vivax in China, it traced the samples of the main epidemic areas of three days of malaria in China. The results showed that 33 samples in Hainan were correctly judged to be the samples in the region, and the correct rate reached 1 in Central China. 179 of the 81 samples were correctly judged, the remaining 2 samples were judged to be Hainan samples, and 78 of the 92 samples in the 98.9%. Yunnan region were correctly judged to be the sample in the region, while 3 and 11 samples were judged to be Hainan and central China, and the correct rate of determination was the overall results of the three regions. 94.8% of the samples can be correctly judged as the samples from the source area. The results of the cross validation are the same, and there is a higher rate of discrimination. Finally, we have selected 20 samples from central China to verify the system. The results found that the results of the central China certification were all in line with the actual situation, and the correct rate reached 100%..
The results of this study show that the genome of Plasmodium vivax has a high variation in China and its population structure has significant characteristics. From the evolutionary point of view, the genetic relationship between Yunnan and Hainan regions is closer, and they are different from those in Central China. The significant linkage disequilibrium indicates the high near breeding rate of Plasmodium vivax and the low effective recombination rate of the genome. Our preliminary traceability monitoring system has good traceability detection ability, and the accuracy and accuracy of the discrimination and validation are above 90%. With the increase of the sample number, the accuracy and stability of the system will be increased. We will further improve the technology in order to provide technical support for malaria control and malaria elimination programs in China.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R382

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