Modification of the rPLU5 Primer to Address Primer-Template Mismatch in a Plasmodium 2 Genus-Specific Nested PCR Assay

Abstract

A primer-template mismatch was identified at the second nucleotide from the 3′ end of the nest 1 reverse primer (rPLU5) used in the Plasmodium genus-specific nested PCR assay developed by Singh et al. (1999). The mismatches near the 3′ end of primer have a strong destabilizing effect on primer annealing. Therefore, rPLU5 was modified (C→T substitution) and nested PCR was optimized using annealing temperature gradients: 55–65 °C for nest 1 and 60–70 °C for nest 2. Optimal amplification was observed at 59–63 °C for nest 1 and 60–66 °C for nest 2, with the lowest temperatures recommended to maximize sensitivity and specificity. Furthermore, the performance of original and modified rPLU5 was compared using Plasmodium-positive DNA samples extracted from human clinical specimen and macaque specimen. The original rPLU5 amplified only the macaque sample, whereas the modified rPLU5 successfully amplified both samples, indicating improved detection. However, given the limited number of samples and absence of species identification, this modification should be interpreted as a precautionary optimization to enhance assay robustness rather than a performance limitation of the original rPLU5. Overall, this work presents an optimized nested PCR assay incorporating an improved primer design that minimizes mismatch-related amplification issues.