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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(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。
Objective To use nested PCR based on 18S rRNA gene to identify the blood samples of patients infected with P. falciparum and P. vivax in the border areas of Yunnan Province and to analyze the differences of 18S rRNA gene sequences in this area. Methods From 2004 to 2011, we collected microscopic examination of single-infected falciparum malaria in 7 counties (cities) in Mengla, Baoshan Tengchong and Dehong Yingjiang in Xishuangbanna border areas of Yunnan Province, and in Nawe, Nanchongjiang, Protozoal or Plasmodium vivax whole blood or filter paper blood samples. All blood samples were identified using the nested PCR method based on the 18S rRNA gene and the positive PCR products were sequenced. The obtained sequences were Blastn aligned. Using MEGA 6.06 software to construct phylogenetic tree by using adjacency method. Results Among the 475 blood samples examined for Plasmodium falciparum (256) and Plasmodium vivax (219), 242 were detected for Plasmodium falciparum by the 18S rRNA gene and 176 for Plasmodium vivax 57 cases of mixed infection. The blood samples which were consistent with the nested PCR method and the microscopic examination method accounted for 81.7% (388/475). The frequency of inconsistent blood samples detected by the two methods was significantly correlated with their parasite density (Spearman’s r = -0.408, P <0.05). The results of multiple sequence alignment showed that the sequences of 18S rRNA genes of Plasmodium vivax and Plasmodium vivax were 11, 10 and 2.9% (6/205) and 22.5% (27/120) respectively, . The 18S rRNA gene sequence of P. falciparum clustered with a gene sequence from Cameroon (GenBank accession number KC428742) and clustered in a large branch with the sequence of three S. Falciparum S 18S rRNA genes from the Netherlands and Brazil (GenBank accession number U36465, U36466 and U36467). The obtained P. vivax sequence was clustered with the sequence of the Soybean Plasmodium subtilis type A small subunit ribosomal RNA (GenBank accession number U07367) and so on from Thailand, The reference sequence of Plasmodium C-type gene (Gen Bank accession number U07368) and other reference sequences are distantly related. Conclusions Microscopic examination of a single infection in blood samples, nested PCR detected 57 cases of mixed infection. There was no significant difference in the sequence of 18S rRNA gene between malaria parasite in 7 counties (cities) in the border area of Yunnan Province.