posted on 2024-02-07, 21:15authored byShuaiwei Chen, Cui Wu, Chunyan Qian, Yanan Pang, Kaiming Guo, Ting Wang, Linlin Bai, Feng Qian, Zunzhong Ye, Zhenping Liu, Zhaohui Qiao, Yongming Wang, Rui Wang
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.