RPB0315

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
SARS-CoV-2 SARS-CoV-2, 2019-nCoV, COVID-19, COVID-19 virus, SARS2, Wuhan coronavirus, Human coronavirus 2019, COVID19, HCoV-19, SARS-2, SARS-CoV4 2697049 Nidovirales Coronaviridae Betacoronavirus Severe acute respiratory syndrome-related coronavirus virus

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
F CTTGAAATTCCACGTAGGAATGTGGCAACTTTAC 34 10 µM 41.18 60.71 10440.85 \
R GTATGCCAGGTATGTCAACACATAAACCTTCAG 33 10 μM 42.42 60.26 10105.65 \

Gene Description

Target Gene GenBank ID
orflab gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
an ultrasensitive, easy-to-perform and visual method for colorimetric analysis of nucleic acid and applied it to detect SARS-CoV-2 RNA. RPA-CRISPR-Cas12a system PA instruction booklet. 30 min 42°C CRISPR-Cas12a system 10 aM (∼6 copies per μL) 100fm \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2024 CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA Shaohua Gong,Kexin Song,Wei Pan,Na Li,Bo Tang Analyst 39011640 10.1039/d4an00479e

CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA

Author(s):

Shaohua Gong,Kexin Song,Wei Pan,Na Li,Bo Tang

Journal:

Analyst

Year:

2024

Abstract:

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.