發(fā)布時間：2018-04-08 23:03

  本文選題：間日瘧原蟲　切入點：PCR鑒定　出處：《中國疾病預(yù)防控制中心》2008年碩士論文

【摘要】： 目的鑒定西藏林芝地區(qū)瘧原蟲蟲種并計算當(dāng)?shù)鼐用駧x率,分析其瘧區(qū)性質(zhì)以及瘧疾流行程度;檢測林芝地區(qū)間日瘧原蟲裂殖子表面蛋白1基因(PνMSP-1)和環(huán)子孢子蛋白基因(PνCSP)的多態(tài)性,揭示當(dāng)?shù)亻g日瘧原蟲主要分子標(biāo)志特點,為科學(xué)制定瘧疾防治策略和措施提供有關(guān)依據(jù)和基線數(shù)據(jù)。 方法(1)整理收集于2006年林芝地區(qū)背崩鄉(xiāng)居民濾紙干血滴,經(jīng)Chelex-100煮沸法提取其DNA,采用多重PCR方法對樣本DNA進(jìn)行鑒定。對檢測為陽性的樣本,再采用套式PCR方法作鑒定驗證;(2)計算居民帶蟲率;(3)采用Chelex-100煮沸方法提取收集于2007年的林芝地區(qū)間日瘧患者濾紙干血滴DNA和安徽懷遠(yuǎn)間日瘧患者的濾紙干血滴DNA;(4)以PνMSP-1和PνCsp的特異性引物,分別對上述鑒定為間日瘧原蟲陽性的樣本進(jìn)行套式PCR擴(kuò)增,并對擴(kuò)增產(chǎn)物測序;(5)將PνMSP-1和PνCSP的測序結(jié)果在GeBank數(shù)據(jù)庫中作blastn和blastx比對,建立N-J進(jìn)化樹,并將林芝地區(qū)PνMSP-1基因型構(gòu)成比與國內(nèi)數(shù)據(jù)作Fisher's確切統(tǒng)計。 結(jié)果(1)共整理得林芝地區(qū)背崩鄉(xiāng)居民濾紙干血滴388份,檢測出3份間日瘧原蟲感染樣本,計算得背崩鄉(xiāng)居民帶蟲率為0.77%;(2)收集到林芝地區(qū)間日瘧患者濾紙干血滴35份和安徽懷遠(yuǎn)間日瘧患者濾紙干血滴1份;(3)對上述39份間日瘧原蟲DNA作PνMSP-1和PνCSP特異性擴(kuò)增,其中PνMSP-1的DNA序列在N-J進(jìn)化樹中聚為A(Sal-1類)、B、C和D(Belem類)四類,四類序列分別翻譯為氨基酸后經(jīng)比對后分為3組,A、B類對應(yīng)Sal-1型,C類對應(yīng)第三型(ⅡPR型),D類對應(yīng)Belem型;各組的構(gòu)成比與云南、海南和安徽三省的構(gòu)成比例存在明顯差異(Fisher統(tǒng)計P值分別為:0.0163,0.1752和0.0037);林芝地區(qū)PνCSP序列在N-J進(jìn)化樹中聚為一類,其翻譯的氨基酸序列均與VK210株高度相似。 結(jié)論(1)林芝地區(qū)的陽性濾紙干血滴均為間日瘧原蟲感染樣本,未發(fā)現(xiàn)惡性瘧原蟲感染或惡性瘧原蟲和間日瘧原蟲混合感染樣本。說明間日瘧原蟲為當(dāng)?shù)氐膬?yōu)勢蟲種,但是否判斷間日瘧原蟲為當(dāng)?shù)氐奈ㄒ涣餍邢x種及其流行程度仍需要長期監(jiān)測以下結(jié)論。(2)本研究首次系統(tǒng)分析了林芝地區(qū)間日瘧原蟲PνMSP-1和PνCSP基因多態(tài)性,對當(dāng)?shù)亻g日瘧原蟲進(jìn)行分子水平上的分類,為瘧疾防治工作提供有力的分子生物學(xué)數(shù)據(jù)。
[Abstract]:Objective to identify the Plasmodium species in Linzhi region of Tibet and calculate the rate of carrying parasites among local residents, and to analyze the nature of malaria and the prevalence of malaria.To detect the polymorphism of Plasmodium vivax merozoite surface protein-1 gene (P 謂 MSP-1) and cyclospore protein gene (P 謂 CSP) in Linzhi area, and to reveal the characteristics of the main molecular markers of Plasmodium vivax.To provide relevant basis and baseline data for scientific formulation of malaria control strategies and measures.Methods 1) the dry blood drops of filter paper were collected from the residents of Ringzhi area in 2006, and their DNA was extracted by Chelex-100 boiling method. The DNA samples were identified by multiplex PCR method.For samples tested positive,Then the nested PCR method was used to verify the identification and validation) the rate of infection was calculated by using Chelex-100 boiling method. The filter paper dripping DNA of vivax malaria patients collected in Linzhi area in 2007 and the filter paper dry blood dripping DNA of vivax malaria patients in Huaiyuan of Anhui Province were extracted by Chelex-100 boiling method.The specific primer for MSP-1 and Csp,Nested PCR amplification was carried out on the samples identified as Plasmodium vivax positive, and the amplified products were sequenced. (5) the results of blastn and blastx were compared with blastn and blastx in the GeBank database to establish N-J evolutionary tree.The genotypic ratio of P 謂 MSP-1 in Linzhi area was compared with the domestic data for Fisher's.Results (1) 388 dry blood drops of filter paper were collected from the residents of Lichi County, and 3 samples of Plasmodium vivax infection were detected.A total of 35 samples of filter paper dry blood dripping from vivax malaria patients in Linzhi area and 1 sample from Anhui Huaiyuan vivax malaria patients were collected. The specific amplification of P. vivax DNA was performed on 39 Plasmodium vivax DNA samples mentioned above.The DNA sequences of P v MSP-1 are grouped into four classes in N-J evolutionary tree: A(Sal-1 class B C and D(Belem class. After being translated into amino acids, the four kinds of sequences are divided into three groups: Sal-1 type B class corresponding to Sal-1 type C type corresponding to the third type (type 鈪，

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