RPB0413

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
S501RPAF1 TGTATAGATTGTTTAGGAAGTCTAATCTCAA 31 1.25 µM 29.03 52.31 9563.3 \
S501RPAR1 AGACTCAGTAAGAACACCTGTGCCTGTTAA 30 1.25 µM 43.33 60.66 9199.06 \
S478RPAF3 ACCAGATGATTTTACAGGCTGCGTTATAGCTTG 33 1.25 µM 42.42 61.51 10158.66 \
S478RPAR5 CAATTAAAACCTTTAACACCATTACAA 27 1.25 µM 25.93 49.26 8163.44 \

Gene Description

Target Gene GenBank ID
S gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
the detection sensitivity and operability, and reducing the turn-around time and cost. RT-RPA-Cas12a \ 15 min 37°C Cas12a 10 copies RNA/reaction \ 1

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2023 RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern Guiyue Tang,Zilong Zhang,Wei Tan,Fei Long,Jingxian Sun,Yingying Li,Siwei Zou,Yujiao Yang,Kezhu Cai,Shenwei Li,Zhiyi Wang,Jiakun Liu,Guobing Mao,Yingxin Ma,Guo-Ping Zhao,Zhen-Gan Tian,Wei Zhao Sens Actuators B Chem 36743821 10.1016/j.snb.2023.133433

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern

Author(s):

Guiyue Tang,Zilong Zhang,Wei Tan,Fei Long,Jingxian Sun,Yingying Li,Siwei Zou,Yujiao Yang,Kezhu Cai,Shenwei Li,Zhiyi Wang,Jiakun Liu,Guobing Mao,Yingxin Ma,Guo-Ping Zhao,Zhen-Gan Tian,Wei Zhao

Journal:

Sens Actuators B Chem

Year:

2023

Abstract:

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.