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[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 establish a genotyping method for Plasmodium vivax circumsporozoite protein (CSP) for identification of geographic strains. [Method] Chelex 10 0 was used to extract trace DNA in the blood of filter paper. Nested PCR and allele-specific PCR, agarose gel electrophoresis, dot blot and Southern blot hybridization were used to discriminate fragments Increase, analyze and appraise. [Results] Using the nested allele-specific PCR typing method described in this article, three discriminant fragments of different length ranges were amplified from a small amount of DNA extracted from a small piece of filter paper blood sample: 650-770 bp Species specific), 15 0 ~ 30bp (temperate-specific), 5 88 ~ 6 15bp (PV 2 type-specific). The method used to detect the reference strains appeared in the band type and length of the designed target sequence exactly match. Fifty-nine domestic blood samples were tested, of which 42 were identified as temperate family strains, 15 were tropical family members and 2 were PV 2 type strains. Of the 4 infected blood samples, two Pakistani isolates belonged to temperate family, and one strain from Burma and Laos belonged to the tropical family. [Conclusion] ① This method can distinguish PV 1 type, PV 2 type, temperate family and tropical family strains at the same time, which is more convenient and faster than the existing PCR (PCR probe hybridization) method. ② The preliminary test results of on- There are PV 1 type (temperate and tropical) insect strains and PV 2 type insect strains in our country, and there may also be strains of temperate Southeast Asia.