Development and Optimization of PCR Protocol for the Amplification of Merozoite Thrombospondin-related Anonymous Protein (mTRAP) Gene from Falciparum- Positive Malaria Samples v1

Résumé

Plasmodium falciparum TRAP-like protein (PfTLP) is a transmembrane protein belonging to the TRAP/MIC2 family. It is expressed in salivary gland sporozoites and is linked to parasite motility and host cell invasion. Its isoform, merozoite thrombospondin-related anonymous protein (mTRAP), is expressed in merozoite and gametocyte stages. It plays important roles in parasite development, gamete egress, and mosquito transmission, making it a promising target for molecular and transmission-blocking malaria studies. This study developed and optimised PCR protocol for the amplification of the Plasmodium falciparum mTRAP gene from falciparum malaria-positive blood samples. Primers were designed from the Plasmodium falciparum 3D7 reference sequence XM_001347529.1. Five gradient PCR optimisation trials were conducted to establish specific and reproducible amplification conditions. The first four trials were performed with OneTaq Quick-Load 2X Master Mix across annealing temperature ranges of 50.8–60.8°C, 46.3–55.3°C, 43.1–47.7°C, and 45.8–50.4°C, produced non-specific amplification products. Re-optimisation using 2× EasyTaq‱ PCR SuperMix across a gradient range of 42.7–46.4°C generated a specific and reproducible amplification profile, with 45.1°C identified as the optimum annealing temperature. The optimised protocol successfully amplified the targeted mTRAP gene fragment from falciparum malaria-positive samples, and the amplicons were validated by sequencing and NCBI BLAST analysis. This validated PCR protocol provides a reliable method for mTRAP gene amplification and supports future molecular characterisation, transmission biology, and vaccine-related investigations involving Plasmodium falciparum mTRAP.

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