Rapid and Specific Detection of Active SARS-CoV-2 With CRISPR/Cas12a

Abstract

Rapid and sensitive nucleic acid detection of SARS-CoV-2 has contributed to the clinical diagnosis and control of COVID-19. Although detection of virus genomic RNA (gRNA) has been commonly used in clinical diagnosis, SARS-CoV-2 gRNA detection could not discriminate between active infectious virus with remnant viral RNA. In contrast to genomic RNA, subgenomic RNAs (sgRNAs) are only produced when the virus is actively replicating and transcription, detection of sgRNA could be an indication to evaluate infectivity. CRISPR/Cas-based nucleic acid detection methods have been considered potential diagnostic tools due to their intrinsic sensitivity, specificity and simplicity. In this study, to specifically detect active virus replication, we developed a CRISPR-based active SARS-CoV-2 (CRISPR-actCoV) detection strategy by detecting sgRNAs of SARS-CoV-2. CRISPR-actCoV with CRISPR Cas12a-assisted fluorescence reporter system enables detection of sgRNAs at 10 copies in 35 min with high specificity and can be read out with naked eyes. Further, we performed CRISPR-actCoV mediated sgRNA detection in 30 SARS-CoV-2 potentially infected clinical samples, and 21 samples were SARS-CoV-2 sgRNA positive. A quantitative RT-PCR assay was also performed to detect gRNA of SARS-CoV-2 in parallel. Among the 30 clinical samples, 27 samples were gRNA positive. Taken together, CRISPR-actCoV provides an alternative for rapid and accurate detection of active SARS-CoV-2 and has great significance in better response of coronavirus causing epidemic disease.

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a “Booster” for SARS-CoV-2 Point-of-Care Testing

“…According to the 3σ rule, the determined limit of detection (LOD) of the portable BDCHA-based biosensor was 291.48 aM, showing good sensitivity of the proposed method. The proposed sensor outperformed recently reported methods − (Table S4) in detection time and sensitivity.…”

A rapid point-of-care population-scale dipstick assay to identify and differentiate SARS-CoV-2 variants in COVID-19-positive patients

“…In the current work, a quick point-of-care test for the early detection of the SARS-CoV-2 virus was developed. In contrast to the traditional real-time-based approach, which is complex and time-consuming (taking about 2- [20], use of thermal incubators for isothermal ampli cation [21], use of CRISPR-Cas technology [22], potentiometric biosensors [23] and nal readout using a colorimetric based assay with mobile application [24] or the dipstick based results [21]. The rapid point-of-care test this study outperforms the others since it aids in variant identi cation (Delta, Omicron-BA.1 & BA.2) while also being accurate, robust, inexpensive, and quick.…”

Mesophilic Argonaute-based isothermal detection of SARS-CoV-2

“…Consequently, the target nucleic acid leaves a strong signature that allows detection through a fluorescence instrument or paper test. For example, several reports have demonstrated a rapid, portable CRISPR-based test for SARS-CoV-2 detection (Aman et al, 2020 ; Broughton et al, 2020 ; Ding et al, 2020 ; Liu et al, 2021b ). According to these reports, there are still some limitations, such as PAM/PFS sequence restriction of the target sequence, potential degradation and high cost of long RNA guides, and multiplex detection requiring multiple Cas enzymes (Li et al, 2019 ).…”