G-Quadruplex DNAzyme-Substrated CRISPR/Cas12 Assay for Label-Free Detection of Single-Celled Parasitic Infection

Early diagnosis of parasitic diseases can dramatically alleviate medical, economic, and social burdens. Herein, we report a sensitive and label-free assay for diagnosing single-celled parasitic infections using G-quadruplex (G4) DNAzyme as a reporter for CRISPR/Cas12. The substitution of a fluorescent DNA reporter with G4 DNAzyme increased the sensitivity for detecting Leishmania donovani (L. donovani) by 5 times and obviated the need for using chemically labeled DNA probes. The G4 DNAzyme-substrated CRISPR/Cas12 (GsubCas12) assay yielded a limit of detection of 3.1 parasites in the detection of cultured L. donovani and was further applied to analyze L. donovani in infected mice. The results showed that the GsubCas12 assay could positively detect L. donovani in spleen samples from infected mice about 2 weeks after low-dose inoculation, nearly 2 weeks earlier than that of parasitological analysis. GsubCas12 assay is promising as a diagnostic tool for parasitic infection in resource-limited regions.