Cheng Zhou-Hua, Luo Xi-Yan, Yu Sheng-Song, Min Di, Zhang Shu-Xia, Li Xiao-Fan, Chen Jie-Jie, Liu Dong-Feng, Yu Han-Qing
CAS Key Laboratory of Urban Pollutant Conversion, Department of Environmental Science and Engineering, University of Science and Technology of China, 230026, Hefei, China.
Fujian Institute of Hematology, Fujian Provincial Key Laboratory on Hematology, Fujian Medical University Union Hospital, 350001, Fujian, China.
Nat Commun. 2025 Jan 30;16(1):1166. doi: 10.1038/s41467-025-56516-3.
The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing.
基于CRISPR的检测方法已得到广泛应用，但仍受一锅法诊断方法非通用性的限制。在此，我们报告一种用于检测流行性病原体的通用一锅法荧光方法，可在15至20分钟内得出结果。该方法使用肝素钠通过干扰Cas12a-crRNA结合过程精确调节Cas12的顺式切割能力，从而由于等温扩增产物的积累产生显著荧光。此外，这种通用检测方法适用于经典和次优PAM，以及各种Cas12a亚型，如LbCas12a、AsCas12a和AapCas12b。与qPCR或RT-qPCR相比，这种强大的方法在从唾液或废水样本中检测猴痘假病毒、甲型流感病毒和SARS-CoV-2时，灵敏度和特异性超过95%。此外，肝素钠每千次使用的成本仅为0.01至0.04美元。总体而言，这种基于肝素钠的通用快速一锅法为即时检测提供了潜在可能性。
