本文選題：間日瘧 + 微衛(wèi)星��； 參考：《第二軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 瘧疾是世界范圍內(nèi)危害最嚴(yán)重的一種蚊媒寄生蟲病,尤其是對非洲、南美以及東南亞地區(qū)產(chǎn)生極大危害。目前,全球每年瘧疾臨床發(fā)病人數(shù)為3-5億,其中100-300萬人死于該病,90%的死亡數(shù)發(fā)生在非洲,絕大部分是5歲以下的兒童。間日瘧原蟲(Plasmodium vivax)是世界上分布最廣的瘧原蟲,雖然其致病的嚴(yán)重程度不如惡性瘧原蟲高,但是由于其具有更廣泛的適應(yīng)性和較高的復(fù)發(fā)率,它仍然給人們的健康,尤其是兒童帶來極大的危害。間日瘧的流行和發(fā)展對全球的公共衛(wèi)生安全帶來重大的挑戰(zhàn)。在中南美洲,中東地區(qū),中亞、南亞和東南亞以及大洋洲和東非地區(qū),每年約有26億人受到間日瘧的威脅,其中包括7至8千萬的臨床病例；在巴西亞馬遜流域,間日瘧原蟲甚至已經(jīng)超過惡性瘧成為致死性瘧疾的主要病原體,其傳播和發(fā)病形式不容樂觀。在我國,經(jīng)過50多年不懈的努力,瘧疾的發(fā)病從整體上已經(jīng)得到有效控制,但近幾年來,我國一些地區(qū)的瘧疾疫情出現(xiàn)了回升趨勢,特別是安徽等地區(qū)出現(xiàn)了間日瘧的暴發(fā)流行。為應(yīng)對當(dāng)前瘧疾流行的嚴(yán)峻形勢,仍需要加強(qiáng)瘧疾防治的基礎(chǔ)研究,特別是研發(fā)瘧疾防治相關(guān)的新技術(shù),包括瘧疾感染的溯源檢測技術(shù)。 在此項研究中,我們從云南、海南和華中三個不同流行地區(qū)采集間日瘧疾患者血樣本。云南地區(qū)樣本采集地區(qū)按照地理分布主要分為三個區(qū)域：(1)怒江流域：流行區(qū)包括德宏自治州和中緬邊界區(qū)域,樣本來源為騰沖縣、龍陵縣、德宏自治州及與緬甸接壤地區(qū)包括境外的緬甸克欽邦拉咱地區(qū)。(2)瀾滄江流域：流行區(qū)包括臨滄地區(qū)、思茅地區(qū)及西雙版納傣族自治州,樣本來源為西雙版納傣族自治州的景洪市和勐臘縣。(3)紅河流域：流行區(qū)包括紅河自治州和文山自治州,樣本來源為紅河州紅河縣及元江縣。在云南地區(qū)共采集瘧疾患者血樣本共310份。海南地區(qū)樣本采集地包括三亞市,樂東縣和東方市三個地區(qū),采集樣本數(shù)共計82份。華中地區(qū)樣本采集地包括安徽、湖北和河南三省,采集樣本數(shù)共計530份。所有樣本均采用試劑盒提取基因組DNA。參考目前國際廣泛應(yīng)用于地理株種群結(jié)構(gòu)和遺傳多樣性研究的工具和方法,選擇間日瘧基因組不同染色體上的23個微衛(wèi)星位點(MSs)對我國間日瘧原蟲進(jìn)行種群結(jié)構(gòu)分析及遺傳多樣性研究,并通過一些地區(qū)特異性的位點初步建立溯源檢測的體系。這些微衛(wèi)星位點包括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個為引子文獻(xiàn)的多態(tài)性位點,后12個為自行篩選的位點,核心重復(fù)單位為2至8個堿基不等。每個MS位點設(shè)計3條引物,其中1條為熒光標(biāo)記引物。采用半巢氏PCR方法擴(kuò)增每個微衛(wèi)星位點,第二輪PCR采用熒光標(biāo)記引物,將PCR產(chǎn)物進(jìn)行GENESCAN檢測。檢測得到不同的片段長度,代表微衛(wèi)星等位基因的不同類型。我們還選擇了兩個SNPs位點作為溯源檢測的核心位點,這兩個位點與瘧原蟲抗藥性基因相關(guān),實驗證明它們具有地區(qū)特異性,使得初步建立溯源檢測系統(tǒng)成為可能。我們對所有樣本微衛(wèi)星檢測結(jié)果進(jìn)行整理,并利用相應(yīng)的生物信息學(xué)的軟件進(jìn)行統(tǒng)計學(xué)的分析。我們用GenALEx軟件進(jìn)行平均等位基因,期望雜合度以及方差分析,用在線分析軟件LIAN計算種群的連鎖不平衡性,Mega軟件用于繪制遺傳進(jìn)化樹圖,并進(jìn)行溯源相關(guān)的判別分析。 我們發(fā)現(xiàn)所選擇的大部分MS位點在不同地區(qū)的樣本中具有高度的變異性,平均每個位點有5-33個等位基因,云南地區(qū)樣本的平均等位基因數(shù)目為14.27±1.63,期望雜合度為0.768±0.035,海南地區(qū)平均等位基因數(shù)目為8.53±0.98,期望雜合度為0.707±0.042,華中地區(qū)平均等位基因數(shù)目為10.27±1.33,期望雜合度為0.638±0.056。華中地區(qū)的樣本具有顯著的連鎖不平衡性(p0.001)。云南和海南地區(qū)樣本之間的遺傳距離更近為0.097,而華中地區(qū)的樣本和這兩個地區(qū)相距較遠(yuǎn)分別為0.161和0.197。在以上研究結(jié)果的基礎(chǔ)上,我們嘗試用判別分析的方法初步探討旨在建立間日瘧疾溯源檢測技術(shù)的判別系統(tǒng)。結(jié)果發(fā)現(xiàn)兩個微衛(wèi)星位點在此系統(tǒng)中有良好的區(qū)分性,可作為中國這三大地區(qū)的特異性的分子標(biāo)記。另外,本實驗其它研究發(fā)現(xiàn)的兩個SNPs和一個MS具有較好的區(qū)分性。采用三個MS和兩個SNP作為我國間日瘧溯源檢測的分子標(biāo)志,對我國三個間日瘧疾主要流行區(qū)樣本進(jìn)行溯源分析,結(jié)果顯示：海南地區(qū)的33個樣本均被正確判定為該地區(qū)樣本,正確率達(dá)到100%。華中地區(qū)181個樣本有179個被正確判定,其余2個樣本被判定為海南地區(qū)樣本,正確率達(dá)到98.9%。云南地區(qū)92個樣本中有78個樣本被正確判定為該地區(qū)樣本,而分別有3個和11個樣本被判定為海南和華中地區(qū)樣本,判定的正確率為84.8%。三個地區(qū)總體結(jié)果有94.8%的樣本能夠正確地被判斷為來源地區(qū)的樣本,交叉驗證的結(jié)果相同,有較高的判別符合率。最后,我們從華中地區(qū)選擇了未參與系統(tǒng)建立的20個樣本對此系統(tǒng)進(jìn)行驗證,結(jié)果發(fā)現(xiàn)華中地區(qū)驗證結(jié)果均符合實際情況,正確率達(dá)到100%。 綜合本研究結(jié)果表明,中國各個地區(qū)間日瘧原蟲基因組具有較高的變異度,其種群結(jié)構(gòu)有顯著特點,從進(jìn)化上來看,云南和海南地區(qū)樣本遺傳關(guān)系更近,它們與華中地區(qū)樣本差異較大。而華中地區(qū)樣本在我們選擇的微衛(wèi)星位點上體現(xiàn)出顯著的連鎖不平衡性,提示該地區(qū)間日瘧原蟲的高近親繁殖率和低的基因組有效重組率。我們初步建立的溯源監(jiān)測系統(tǒng)具有良好的溯源檢測能力,判別符合率和驗證正確率均達(dá)到90%以上。隨著樣本數(shù)的增加,此系統(tǒng)的準(zhǔn)確性和穩(wěn)定性會進(jìn)一步提高,以期能為我國的瘧疾防治和瘧疾消除計劃提供技術(shù)支撐。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R382

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