Development of an Enzymatic Recombinase Amplification Assay for the Rapid Detection of Plasmodium Nucleic Acids

Abstract

Background

Malaria remains a global health challenge, with 249 million cases and 608,000 deaths reported in 2022. While China achieved malaria elimination, imported cases surged by 194.4% in 2023, underscoring the need for rapid diagnostics. Traditional methods like microscopy and rapid diagnostic tests (RDTs) face limitations in sensitivity and infrastructure requirements. This study aimed to establish and optimize a “one-pot” enzymatic recombinase amplification (ERA) assay for the molecular detection of Plasmodium falciparum and Plasmodium vivax, and to evaluate the efficacy of this assay through methodological verification and clinical performance.

Methods

We designed a specific ERA assay targeting the conserved regions of P. falciparum and P. vivax genetic material. We evaluated the sensitivity and specificity of this assay using synthetic plasmids and genomic material. Additionally, we tested the stability of the reaction by incorporating potential interfering substances into the reaction system. Finally, we analyzed the detection performance of the ERA method against real-time fluorescent quantitative PCR and rapid diagnostic tests using clinical samples.

Results

The detection process could be completed within 25 min at 35°C–40°C, and the results could be interpreted either under UV light or using a GeneScope instrument. The detection limit of the ERA assay was 250 copies/mL, which was 40 times more sensitive than fluorescent quantitative PCR. When evaluating the clinical performance using 75 clinical specimens, the detection rate of the ERA method was 94.54% compared with 89.09% for fluorescent quantitative PCR. The ERA assay and fluorescent quantitative PCR can achieve positive detection when blood samples were diluted 1024 times or even 4096 times. Comparatively, the detection capabilities of rapid diagnostic tests were significantly lower than that of the ERA assay.

Conclusion

The ERA method shows good performance in the detection of P. falciparum and P. vivax, and can be used as a complementary tool for malaria screening and clinical diagnosis.