间日疟原虫裂殖子表面蛋白的等位基因型检测

Genotype Detection of the Merozoite Surface Protein Alleles of Plasmodium vivax

目的 建立一种间日疟原虫裂殖子表面蛋白 1(PvMSP 1)基因型检测技术。 方法 设计针对PvMSP 1ICB5～ICB6多态区的引物进行PCR ,其产物用PvuⅡ内切酶消化、琼脂糖凝胶电泳检测 ,鉴别我国间日疟原虫现场分离株基因型。 结果 98份间日疟血样经套式PCR扩增均出现大小约为 40 0bp(Belem型 )或 470bp(Sal 1型 )的特异性片段。酶切消化后 ,45份 470bp样本出现 12 0bp和 3 5 0bp酶切片段 ,为Sal 1型 ;40份 40 0bp的样本中 3份仅出现 1条 40 0bp片段 ,为Belem型 ;3 5份出现 12 0bp和 2 80bp两种酶切片段 ,为Ⅲ重组型 ;2份 12 0bp和 2 40bp片段 ,为朝鲜型。结论 套式聚合酶链反应 限制性片段长度多态性 (PCR RFLP)技术可用于检测我国间日疟原虫的 3种PvMSP

Objective To develop a method for detecting the genotype of Plasmodium vivax merozoite surface protein 1 (PvMSP-1) alleles. Methods According to the sequence characteristic of PvMSP-1, nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to amplify the polymorphic region of ICB5-ICB6 which contains Q repeats and PvuII restriction site (Sal-1 type). The PCR product was digested by PvuII restriction endonuclease and the digested fragments were observed by 2% agarose gel electrophoresis. The allelic type was determined according to the banding pattern. Results Bands in size of 400 bp (Belem type ) and/or 470 bp (Sal-1 type ) appeared in all 98 P. vivax isolates, no band was found in negative control. After PvuII digestion, two Sal-1 type fragments (120 bp and 350 bp) were obtained from 45 samples of 470 bp. Single-band of 400 bp appeared in 3 of 40 samples with 400 bp as Belem type, two bands of 120 bp and 280 bp appeared from other 35 samples as recombination type III, and another 2 bands with 120 bp and 240 bp as Korean isolate. Conclusion The result showed that the nested PCR-RFLP may be applied in the detection and identification of the three PvMSP-1 allelic types in China.