RPB0196

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 TGATGCCATGCGAAATGCTGGTATTGTTGG 30 0.505 46.7 63.35 9308.09 \
R TTAATGTAAGGCTTTGTTAAGTCAGTGTCA 30 0.505 33.3 55.53 9266.09 \

Gene Description

Target Gene GenBank ID
O-gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
\ RPA-CRISPR-Cas12a \ 30 37 Fluorometric detection (Fluorescent metal ion indicator (Calcein)) 10copies μl−1 \ \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2022 Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics Menglu Hu, Zhiqiang Qiu, Zirong Bi, Tian Tian, Yongzhong Jiang, and Xiaoming Zhou Proceedings of the National Academy of Sciences of the United States of America 35727982 10.1073/pnas.2202034119

Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics

Author(s):

Menglu Hu, Zhiqiang Qiu, Zirong Bi, Tian Tian, Yongzhong Jiang, and Xiaoming Zhou

Journal:

Proceedings of the National Academy of Sciences of the United States of America

Year:

2022

Abstract:

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.