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.
Highlights
The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to global public health with a widespread infection of 236 million cases and 4.8 million deaths as of 5 October 2021 [1]
CRICOLAP was established by integrating the CRISPR/Cas12a coupled with reverse transcription loop-mediated isothermal amplification (RT-LAMP) to boost the sensitivity and two independent parallel systems (LFS and real-time fluorescence) for result readout
From various attempts (Table S4), we realized that a biotinylated-single-stranded DNA (ssDNA) reporter of 11 bases (BioTTTTTTTTATT/bio-8TA2T) could strongly hybridize with a 32-base ssDNA probe sequence (Table S5) on the lateral flow strip (LFS) test line; it was undetectable once cleaved by Cas12a (Figure S1)
Summary
The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to global public health with a widespread infection of 236 million cases and 4.8 million deaths as of 5 October 2021 [1]. There is no proven treatment for COVID-19; several vaccines have been approved for emergency use, and others are still under clinical trials [3,4]. Given that vaccinated individuals remains infectious while asymptomatic, it is still vital to develop highly sensitive and user-friendly detection tools to counter pandemic transmission [5,6]. Several diagnostic techniques, including molecular techniques (polymerase chain reaction, isothermal amplification, and CRISPR/Cas-based techniques) and serological tests, have been developed for SARS-CoV-2 detection, several of them have not yet been approved for clinical use. Various parts of the globe are still relying on laboratory-centered instruments—PCR, clinical symptom assessment, and body temperature monitoring, which are far beyond meeting the requirements of epidemiological surveillance [7,8]
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