Genotyping of Plasmodium falciparum by allele-specific amplification of the merozoite surface protein-1 locus

Abstract

Problem/background : Allele-specific amplification is an important tool for a large scale study of geographic distribution of pathogens harboring polymorphic genes. In this study, we have developed a simple method to differentiate strains of Plasmodium falciparum based on polymorphic block 2 located at the 5’ portion of the merozoite surface protein-1 gene (PfMsp-1). Block 2 of PfMsp-1, a strong candidate for asexual blood-stage vaccine, contains 3 basic sequence types with MAD20 type, K1 type and RO33 type as representative alleles.

Objective : To develop a polymerase chain reaction (PCR) method capable of detecting PfMsp-1 genotypes.

Design : Experimental study.

Setting : Department of Parasitology, Faculty of Medicine, Chulalongkorn University.

Materials and Methods : The method deployed semi-nested PCR targeting allelespecific sequences of each representative type of blocks 2 of PfMsp-1. The performance of the method was evaluated with 50 P. falciparum blood samples collected from patients in Chanthaburi, Tak and Yala provinces.

Results : The method is sensitive to detect as few as 5 parasites in the samples whereas specific alleles of block 2 could be unequivocally determined. Minor parasite populations in the isolates containing multiple clone infections were detected by the method. Of the 50 P. falciparum isolates examined, MAD20, K1 and RO33 allelic types were identified in 28, 14 and 8 samples by the PCR genotyping method, respectively. Multiple clone infections were observed in 4 of these isolates.

Conclusion : The PCR genotyping method developed in this study is applicable for large-scale population genetic analysis of P. falciparum population based on the PfMsp-1 locus.