云南省边境地区疟原虫18S rRNA基因种类鉴定与序列分析

发布时间：2018-10-13 09:04

【摘要】：目的使用基于18S rRNA基因的巢式PCR方法对云南省边境地区镜检为恶性疟和间日疟的患者血样进行鉴定,分析该地区疟原虫18S rRNA基因序列之间的差异。方法 2004-2011年在云南省边境地区西双版纳勐腊、保山腾冲和德宏盈江,及缅甸那威、南卡江、芒东和拉咱等7个县(市)收集镜检为单一感染恶性疟原虫或间日疟原虫的全血或滤纸血样品。采用基于18S rRNA基因的巢式PCR方法对所有血样进行鉴定,阳性PCR产物进行测序。所获序列进行Blastn比对。应用MEGA 6.06软件采用邻接法构建系统进化树。结果 475份镜检为恶性疟原虫(256份)和间日疟原虫(219份)感染的血样中,经18S rRNA基因检测为恶性疟原虫感染的有242例,间日疟原虫感染176例和混合感染57例。镜检法和巢式PCR法检测结果一致的血样占81.7%(388/475)。两法检测不一致的血样发生频率与其原虫密度显著相关(Spearman’s r=-0.408,P0.05)。多序列比对分析结果显示,共计得到11条、10条恶性疟原虫、间日疟原虫18S rRNA基因同源序列,变异位点分别占2.9%(6/205)和22.5%(27/120)。所获恶性疟原虫18S rRNA基因序列与来自喀麦隆(Gen Bank登录号KC428742)等基因序列聚在一个大的分支,与来自荷兰和巴西的3个恶性疟原虫S型18S rRNA基因序列(GenBank登录号U36465、U36466和U36467)的亲缘关系较远。所获间日疟原虫序列与来自泰国的间日疟原虫A型小亚单位核糖体核糖核酸(SSU r RNA)基因序列(Gen Bank登录号U07367)等聚在一支,与来自泰国的间日疟原虫C型基因序列(Gen Bank登录号U07368)等参考序列亲缘关系较远。结论镜检为单一感染的血样中,巢式PCR检出57例混合感染。云南省边境地区7个县(市)疟原虫18S rRNA基因序列之间无明显差异。
[Abstract]:Objective to identify the blood samples of Plasmodium falciparum malaria and vivax malaria by nested PCR method based on 18s rRNA gene, and to analyze the difference of 18s rRNA gene sequence of Plasmodium falciparum in Yunnan border area. Methods in the border areas of Yunnan Province from 2004 to 2011, Mengla, Baoshan Tengchong and Dehong Yingjiang in Xishuangbanna, Myanmar, Nawei, Nanka River, Myanmar, were used. Whole blood or filter paper blood samples of single infected Plasmodium falciparum or Plasmodium vivax were collected from 7 counties (cities). All blood samples were identified by nested PCR based on 18s rRNA gene and the positive PCR products were sequenced. The sequence was compared with Blastn. MEGA 6.06 software was used to construct phylogenetic tree. Results two hundred and forty-five blood samples were infected with Plasmodium falciparum and Plasmodium vivax under microscope. Among them, 242cases were infected by 18s rRNA gene, 176 cases were infected by Plasmodium vivax and 57 cases were mixed infection. The results of microscopic examination and nested PCR were 81.7% (388 / 475). There was a significant correlation between the frequency of blood samples and the density of protozoa (Spearman's r-0.408). The results of multiple sequence alignment analysis showed that 11, 10 Plasmodium falciparum and Plasmodium vivax 18s rRNA genes were homologous, and the mutation sites were 2.9% (6 / 205) and 22.5% (27 / 120), respectively. The 18s rRNA gene sequence of Plasmodium falciparum and the sequence of (Gen Bank accession number KC428742 from Cameroon are clustered in a large branch. The phylogenetic relationship of 18s rRNA sequences of Plasmodium falciparum S (GenBank accession numbers U36465U36466 and U36467) from the Netherlands and Brazil is far away. The sequence of Plasmodium vivax and the small subunit ribosomal (SSU r RNA) gene sequence of Plasmodium vivax A from Thailand, (Gen Bank accession number U07367, were clustered in the same branch. The relationship was far from the reference sequence of Plasmodium vivax type C gene (Gen Bank accession number U07368) from Thailand. Conclusion among the blood samples with single infection detected by microscope, 57 cases of mixed infection were detected by nested PCR. There was no significant difference between the 18s rRNA gene sequence of Plasmodium falciparum in seven counties (cities) in the border area of Yunnan Province.
【作者单位】：云南省虫媒传染病防控研究重点实验室云南省疟疾研究中心云南省寄生虫病防治所;
【基金】：全球基金国家疟疾策略项目(No.CHN-S10-G13-M 20120103) 云南省科技计划项目(No.2010CD132)~~
【分类号】：R531.3

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