Rapid genotyping of Plasmodium vivax Pvs25 and Pv38 genes by using mismatch specific endonuclease

Abstract

Mismatch specific endonuclease (MSE) method was used to detect natural polymorphisms in Pvs25 and Pv38 genes of Plasmodium vivax. Eighty seven patients with P. vivax were recruited in the Republic of Korea (ROK). Pvs25 and Pv38 genes were amplified by polymerase chain reaction (PCR), and the PCR amplicons were mixed with reference DNA sequences. Following the denaturation and gradual annealing, the product mixtures were cleaved by the MSE. Heteroduplex types were readily detected by gel electrophoresis, where extra bands with shorter sizes would appear from the cleavage. After MSE cleavage of 657-bp product from Pvs25 mixtures, three genotypes were detected, while Pv38 mixtures with 1220-bp products presented two genotypes in ROK isolates. After the MSE cleavage, the mismatched samples of Pvs25 and Pv38 were completely sequenced, and the results were in complete agreement with the MSE analyses. In conclusion, genotyping of Pvs25 and Pv38 with MSE cleavage could be a potential method for the high-throughput screening of the large field samples.