Optimization of Nested-PCR for the detection of Plasmodium malariae in laboratory settings in Southern Vietnam.

UNLABELLED: Nested Polymerase Chain Reaction is a highly effective technique for detecting infectious diseases, particularly in cases involving low level of parasitemia infections such as malaria.

OBJECTIVE

This study aimed to optimize a Nested-PCR protocol for the specific detection of Plasmodium malariae, with a defined scope focused on refining cycling conditions, annealing parameters, and procedural steps to ensure reliable performance in laboratory settings in Southern Vietnam.

METHODS

The assay targeted the P. malariae 18S rRNA gene using PLU5/PLU6 and MAL1/MAL2 primers. Cycle numbers were optimized at 25, 30, and 35 in the first round, and 25 and 30 in the second round. The annealing temperature for MAL1/MAL2 primers were evaluated from 54 to 60°C, and PCR products were visualized using the ChemiDoc-It2 system.

RESULTS

The MAL1/MAL2 primer pair demonstrated high specificity for P. malariae, with no amplification observed for other Plasmodium species (including: P. falciparum, P. vivax, P. ovale and P. knowlesi) or common microbial contaminants. The optimal conditions were determined to be 35 cycles in the first PCR round, 25 cycles in the second round, with an annealing temperature of 54°C for the MAL1/MAL2 primers. The protocol achieved a limit of detection as low as 0.5 parasites/μL.

CONCLUSIONS

The optimized protocol is well-suited for laboratories in Southern Vietnam.