tetano
Editor, Senior Moderator
Biosensors (Basel)
. 2021 Dec 26;12(1):11.
doi: 10.3390/bios12010011.
Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities
Zhijian Yi[SUP] 1 [/SUP], Jean de Dieu Habimana[SUP] 2 3 [/SUP], Omar Mukama[SUP] 2 3 4 [/SUP], Zhiyuan Li[SUP] 2 3 [/SUP], Nelson Odiwuor[SUP] 3 5 6 [/SUP], Hanzhi Jing[SUP] 7 [/SUP], Chengrong Nie[SUP] 1 [/SUP], Mei Hu[SUP] 8 [/SUP], Zuoxian Lin[SUP] 2 3 [/SUP], Hongping Wei[SUP] 3 5 6 [/SUP], Lingwen Zeng[SUP] 1 9 [/SUP]
Affiliations
Abstract
Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.
Keywords: COVID-19; CRISPR/Cas12a; RT-LAMP; clinical nasopharyngeal samples; lateral flow strip; portable real-time fluorescence detector.
. 2021 Dec 26;12(1):11.
doi: 10.3390/bios12010011.
Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities
Zhijian Yi[SUP] 1 [/SUP], Jean de Dieu Habimana[SUP] 2 3 [/SUP], Omar Mukama[SUP] 2 3 4 [/SUP], Zhiyuan Li[SUP] 2 3 [/SUP], Nelson Odiwuor[SUP] 3 5 6 [/SUP], Hanzhi Jing[SUP] 7 [/SUP], Chengrong Nie[SUP] 1 [/SUP], Mei Hu[SUP] 8 [/SUP], Zuoxian Lin[SUP] 2 3 [/SUP], Hongping Wei[SUP] 3 5 6 [/SUP], Lingwen Zeng[SUP] 1 9 [/SUP]
Affiliations
- PMID: 35049639
- DOI: 10.3390/bios12010011
Abstract
Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.
Keywords: COVID-19; CRISPR/Cas12a; RT-LAMP; clinical nasopharyngeal samples; lateral flow strip; portable real-time fluorescence detector.