间日疟原虫环子孢子蛋白基因型的鉴别研究  

STUDIES ON IDENTIFICATION OF CIRCUMSPOROZOITE PROTEIN GENOTYPING OF PLASMODIUM VIVAX

作　　者：黄天谊 王小力 黎学铭 黄亚铭 曾风秀 车莹 张思淼 付伟忠 张再兴[3] 张国森[3] 蔡贤铮[4] 王善青[4] 王光泽[4] 

机构地区：[1]贵州省寄生虫病研究所,贵阳550001 [2]广西壮族自治区寄生虫病研究所,南宁530021 [3]云南省疟疾防治研究所,思茅665000 [4]海南省热带病防治研究所,海口570203

出　　处：《中国寄生虫学与寄生虫病杂志》2000年第5期272-276,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金!资助项目 (No .396 6 0 0 72 )&&

摘　　要：[目的 ]建立间日疟原虫环子孢子蛋白 (CSP)基因分型法用于地理株的鉴定。[方法 ]Chelex 10 0离子交换法提取滤纸血样中的微量DNA ;套式PCR和等位特异PCR技术、琼脂糖电泳分析法、斑点印迹和Southern印迹 探针杂交技术分别用于判别性片段的扩增、分析和鉴定。[结果 ]用本文描述的套式 等位 特异PCR分型法 ,从一小片滤纸血样提取的微量DNA中 ,扩增出不同长度范围的 3种判别性片段 :6 5 0～ 770bp(间日疟种特异 ) ,15 0～2 30bp(温带族特异 ) ,5 88～ 6 15bp(PV 2型特异 )。用本法检测参考虫株出现的带型和长度与设计的靶序列完全吻合。检测 5 9份国内感染血样 ,42份鉴定为温带族虫株 ,15份为热带族虫株 ,2份为PV 2型虫株。 4份境外感染血样中 ,2个巴基斯坦分离株均为温带族 ,缅甸和老挝各 1株均为热带族虫株。 [结论 ]①本法能同时区分PV 1型 ,PV 2型以及温带族与热带族虫株 ,且较现有 (PCR 探针杂交 )法更为简便、快捷 ;②现场血样初步检测结果显示 :我国存在PV 1型 (温带族与热带族 )虫株和PV 2型虫株 ,也可能存在温带族东南亚组虫株。Objective] To develop a new method of genotyping circumsporozoite protein (CSP) gene for identification of field isolates of Plasmodium vivax . [Methods] Improved Chelex 100 ion exchange method was used to extract DNA from blood filter paper samples, nested PCR and allele specific PCR techniques, agarose gel electrophoresis analysis and dot/southern blotting probe hybridization were employed for amplification, resolution and identification of the diagnostic fragments. [Results] Using the nest allele specific PCR assay reported here, small amounts of DNA extracted from a piece of blood filter paper sample were amplified which produced three different size ranges of diagnostic bands: 650～770 bp PV species specific band, 170～230 bp diagnostic band for temperate zone family and 588 bp band for PV type 2. The sizes and patterns of the bands produced by the reference strains were consistent with those of designed target sequences. Of 59 examined isolates from 6 provinces of China, 42 temperate zone family strains, 15 tropical zone family strains and two PV type 2 strains were identified. [Conclusion] 1, Three genotype strains of P.vivax mentioned above could be identified by this method with only two rounds of PCR and without probe hybridization. 2, The preliminary results showed that PV type 1 including temperate zone family strains and tropical zone family strains as well as PV type 2 strains are present in China. In addition, another CSP genotype with both sequence characteristics of temperate zone and tropical zone family might also be present in China.

关 键 词：间日疟原虫 环子孢子蛋白 基因型 鉴别 PCR 

分 类 号：R382.31[医药卫生—医学寄生虫学]