中国寄生虫学与寄生虫病杂志

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云南省边境地区疟原虫18S rRNA基因种类鉴定与序列分析

张苍林,周红宁,聂仁华,刘慧,王剑,李春富,杨亚明*   

  1. 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,普洱 665099
  • 出版日期:2016-06-30 发布日期:2016-10-28

Species Identification and Sequence Analysis of Plasmodium spp. in Border Areas of Yunnan Province by 18S rRNA-based Nested PCR

ZHANG Cang-lin,ZHOU Hong-ning,NIE Ren-hua,LIU Hui,WANG Jian, LI Chun-fu,Yang Ya-ming*   

  1. Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research;Yunnan Provincial Center of Malaria Research;Yunnan Institute of Parasitic Diseases, Pu’er 665099, China
  • Online:2016-06-30 Published:2016-10-28

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,P<0.05)。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(GenBank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 rRNA)基因序列(GenBank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(GenBank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。

关键词: 恶性疟原虫, 间日疟原虫, 18S rRNA基因, 巢式PCR, 聚类分析

Abstract: Objective To analyze blood samples from patients with falciparum malaria and vivax malaria in border areas of Yunnan Province, using 18S rRNA-based nested PCR, and compare 18S rRNA sequences. Methods Blood or filter blood samples with positive microscopic results for Plasmodium falciparum or P. vivax infection were collected from Laza, Nankajiang, Mangdong and Nawei of Myanmar, and from Mengla, Tengchong and Yingjiang of Yunnan Province between 2004 and 2011. 18S rRNA-based nested PCR was conducted on the samples, and PCR products were sequenced and blasted. Phylogenetic tree was constructed using the neighbor-joining method with MEGA software(version 6.06). Results Microscopic examination revealed P. falciparum infection in 256 samples and P. vivax infection in 219 samples. The 18S rRNA-based PCR further confirmed P. falciparum infection in 242 samples, P. vivax infection in 176 samples, and mixed infection in 57 samples. The consistency rate was 81.7% (388/475) between microscopic and PCR results. The inconsistency rate significantly correlated with parasite density (Spearman’s r=-0.408, P<0.05). Sequence alignment revealed 11 and 10 homologous sequences for P. falciparum and P. vivax 18S rRNA gene, comprising 2.9%(6/205) and 22.5%(27/120) variable sites, respectively. The 18S rRNA of P. falciparum clustered with that from Cameroon(GenBank accession number KC428742), but was distantly related with the S-type 18S rRNA from the Netherlands (U36465) and Brazil (U36466 and U36467). The 18S rRNA of P. vivax clustered with A-type 18S rRNA from Thailand (U07367), but was distantly related with the C-type 18S rRNA from Thailand(U07368). Conclusion Nested PCR revealed mixed infection in 57 samples among those identified with single infection by microscopy. There is no significant difference in 18S rRNA sequence in seven counties/cities in Yunnan Province.

Key words: Plasmodium falciparum, Plasmodium vivax, 18S rRNA gene, Nested PCR, Phylogenetic analysis