A Polymerase Chain Reaction/Ligase Detection Reaction Fluorescent Microsphere Assay to Determine Plasmodium falciparum MSP-119 Haplotypes

摘要

The merozoite surface protein-1 (MSP-1) is a blood stage antigen currently being tested as a vaccine against Plasmodium falciparum malaria. Determining the MSP-1₁₉ haplotype(s) present during infection is essential for assessments of MSP-1 vaccine efficacy and studies of protective immunity in human populations. The C-terminal fragment (MSP-1₁₉) has four predominant haplotypes based on point mutations resulting in non-synonymous amino acid changes: E-TSR (PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (Wellcome type), and Q-TSR (Indo type). Current techniques using direct DNA sequencing are laborious and expensive. We present an MSP-1₁₉ allele-specific polymerase chain reaction (PCR)/ligase detection reaction–fluorescent microsphere assay (LDR-FMA) that allows simultaneous detection of the four predominant MSP-1₁₉ haplotypes with a sensitivity and specificity comparable with other molecular methods and a semi-quantitative determination of haplotype contribution in mixed infections. Application of this method is an inexpensive, accurate, and high-throughput alternative to distinguish the predominant MSP-1₁₉ haplotypes in epidemiologic studies.