Ultraspecific One-Pot CRISPR-Based “Green-Yellow-Red” Multiplex Detection Strategy Integrated with Portable Cartridge for Point-of-Care Diagnosis

Versatile, informative, sensitive, and specific nucleic acid detection plays a crucial role in point-of-care pathogen testing, genotyping, and disease monitoring. In this study, we present a novel one-pot Cas12b-based method coupled with the "Green-Yellow-Red" strategy for multiplex detection. By integrating RT-LAMP amplification and Cas12b cleavage in a single tube, the entire detection process can be completed within 1 h. Our proposed method exhibits high specificity, enabling the discrimination of single-base mutations with detection sensitivity approaching single molecule levels. Additionally, the fluorescent results can be directly observed by the naked eye or automatically analyzed using our custom-designed software Result Analyzer. To realize point-of-care detection, we developed a portable cartridge capable of both heating and fluorescence excitation. In a clinical evaluation involving 20 potentially SARS-CoV-2-infected samples, our method achieved a 100% positive detection rate when compared to standard RT-PCR. Furthermore, the identification of SARS-CoV-2 variants using our method yielded results that were consistent with the sequencing results. Notably, our proposed method demonstrates excellent transferability, allowing for the simultaneous detection of various pathogens and the identification of mutations as low as 0.5% amidst a high background interference. These findings highlight the tremendous potential of our developed method for molecular diagnostics.