Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
Establishment and Evaluation of a Recombinase-Aided Isothermal Amplification Assay for the Detection of Plasmodium—Shao Lei
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Abstract: Objective To establish a recombinase-mediated isothermal amplification (RAA) assay for the detection of Plasmodium parasites. Methods Specific primers targeting the conserved 18S small-subunit ribosomal RNA (I8S rRNA) gene of Plasmodium were designed, and the optimal primer pair was screened and identified. An RAA system for nucleic acid amplification mediated by recombinase was then established. Using this RAA system, we detected standard Plasmodium plasmids as well as four Plasmodium species [Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium malariae (P. malariae), and Plasmodium ovale (P. ovale)] and six other blood-transmitted parasites (Leishmania infantum, Leishmania donovani, Toxoplasma gondii, Entamoeba histolytica, Babesia canis, and Babesia rodhaini). The sensitivity and specificity of the assay were evaluated. Results A set of primers with good amplification performance was selected. The optimized RAA system using this primer pair can complete amplification in 20 minutes at 37°C. The detection limit for standard plasmids was 10 copies/μL; for P. falciparum, P. vivax, and P. ovale, it was 10 copies/μL; and for P. malariae, it was 10 copies/μL. The RAA assay showed no cross-reactivity with the other blood-transmitted parasites, demonstrating excellent specificity. When applied to samples from foreign student blood donors, the RAA results were consistent with those obtained by fluorescence quantitative PCR. Conclusion We have successfully developed a rapid, simple, and specific RAA assay for the detection of Plasmodium parasites, which will greatly facilitate blood screening and clinical testing in primary and remote hospitals.
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