西藏林芝地区疟原虫虫种分子生物学鉴定

发布时间：2018-04-08 23:03

  本文选题：间日疟原虫　切入点：PCR鉴定　出处：《中国疾病预防控制中心》2008年硕士论文

【摘要】： 目的鉴定西藏林芝地区疟原虫虫种并计算当地居民带虫率,分析其疟区性质以及疟疾流行程度;检测林芝地区间日疟原虫裂殖子表面蛋白1基因(PνMSP-1)和环子孢子蛋白基因(PνCSP)的多态性,揭示当地间日疟原虫主要分子标志特点,为科学制定疟疾防治策略和措施提供有关依据和基线数据。 方法(1)整理收集于2006年林芝地区背崩乡居民滤纸干血滴,经Chelex-100煮沸法提取其DNA,采用多重PCR方法对样本DNA进行鉴定。对检测为阳性的样本,再采用套式PCR方法作鉴定验证;(2)计算居民带虫率;(3)采用Chelex-100煮沸方法提取收集于2007年的林芝地区间日疟患者滤纸干血滴DNA和安徽怀远间日疟患者的滤纸干血滴DNA;(4)以PνMSP-1和PνCsp的特异性引物,分别对上述鉴定为间日疟原虫阳性的样本进行套式PCR扩增,并对扩增产物测序;(5)将PνMSP-1和PνCSP的测序结果在GeBank数据库中作blastn和blastx比对,建立N-J进化树,并将林芝地区PνMSP-1基因型构成比与国内数据作Fisher's确切统计。 结果(1)共整理得林芝地区背崩乡居民滤纸干血滴388份,检测出3份间日疟原虫感染样本,计算得背崩乡居民带虫率为0.77%;(2)收集到林芝地区间日疟患者滤纸干血滴35份和安徽怀远间日疟患者滤纸干血滴1份;(3)对上述39份间日疟原虫DNA作PνMSP-1和PνCSP特异性扩增,其中PνMSP-1的DNA序列在N-J进化树中聚为A(Sal-1类)、B、C和D(Belem类)四类,四类序列分别翻译为氨基酸后经比对后分为3组,A、B类对应Sal-1型,C类对应第三型(ⅡPR型),D类对应Belem型;各组的构成比与云南、海南和安徽三省的构成比例存在明显差异(Fisher统计P值分别为:0.0163,0.1752和0.0037);林芝地区PνCSP序列在N-J进化树中聚为一类,其翻译的氨基酸序列均与VK210株高度相似。 结论(1)林芝地区的阳性滤纸干血滴均为间日疟原虫感染样本,未发现恶性疟原虫感染或恶性疟原虫和间日疟原虫混合感染样本。说明间日疟原虫为当地的优势虫种,但是否判断间日疟原虫为当地的唯一流行虫种及其流行程度仍需要长期监测以下结论。(2)本研究首次系统分析了林芝地区间日疟原虫PνMSP-1和PνCSP基因多态性,对当地间日疟原虫进行分子水平上的分类,为疟疾防治工作提供有力的分子生物学数据。
[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 谓 MSP-1) and cyclospore protein gene (P 谓 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 谓 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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