期刊
ACS SENSORS
卷 -, 期 -, 页码 -出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.2c01104
关键词
parasite; CRISPR; Cas12; G-quadruplex DNAzyme; isothermal amplification; Leishmania donovani
资金
- National Natural Science Foundation of China [21904119, U2004197, 22122409]
- Programs for Science and Technology Innovation Talents in Universities of Henan Province [21HASTIT043]
- Outstanding Young Talents in Henan Province [ZYQR201912191]
- Science and Technology project of Henan Province [222102310011, 202102310092]
- Natural Science Foundation of Henan Province [222300420304]
- Research and Practice Project of Higher Education Teaching Reform in Henan Province [2021SJGLX056]
- Training Program for Young Key Teachers of Henan Province
In this study, a sensitive and label-free assay for diagnosing parasitic infections was developed using G-quadruplex DNAzyme as a reporter for CRISPR/Cas12. The assay showed a 5-fold increase in sensitivity for detecting L. donovani and eliminated the need for chemically labeled DNA probes.
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.
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