Extract-free one-pot ambient RPA-CRISPR detection of Plasmodium in whole blood

Malaria caused by Plasmodium remains a major global health burden, especially in resource-limited settings where rapid and accurate diagnosis is essential. Most molecular diagnostic methods require DNA extraction, thermal cycling, and specialized laboratory infrastructure, limiting field applicability. We aimed to develop an extraction-free, one-pot CRISPR-based platform for rapid Plasmodium detection directly from whole blood under ambient conditions.

Crude blood lysates were directly applied to a one-pot recombinase polymerase amplification (RPA)-CRISPR assay coupled with lateral flow detection. Analytical sensitivity and specificity were evaluated using purified DNA and whole-blood lysates. Diagnostic performance was evaluated using 116 malaria-positive specimens and 109 malaria-negative controls for the detection of Plasmodium falciparum and Plasmodium vivax, the two species responsible for most global malaria cases.

The extract-free RPA-CRISPR assay achieved a detection limit of 100 copies/μL from crude blood lysates, corresponding to approximately 12-20 parasites/μL. The assay successfully detected both P. falciparum and P. vivax without cross-reactivity. Clinical evaluation showed 93.1% sensitivity and 100% specificity compared with reference qPCR. Sensitivity reached 97.8% for high-density infections, 95.1% for moderate-density infections, and 82.8% for low-density infections. The workflow was completed within 40 min at room temperature without specialized instrumentation.

This extraction-free, one-pot ambient-temperature platform enables rapid and sensitive detection of Plasmodium directly from whole blood. Although validated only for P. falciparum and P. vivax, the assay targets conserved Plasmodium 18S rRNA regions and may have broader applicability following further validation.