RPB0203

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 CCTCAGATTCAACTGGCAGTAACCAGAATG 30 0.5 46.7 60.9 9184.05 \
R CTTCCTTGCCATGTTGAGTGAGAGCGGTGA 30 0.5 53.3 65.99 9269.05 \
P CAGTATTATTGGGTAAACCTTGGGGCCGACG/i6FAMdT/T/idSp/iBHQ1dT/TTTGATCGCGCCC 45 0.15 54.5 72.14 13554.81 \

Gene Description

Target Gene GenBank ID
SARS-Cov-2 N gene No. MN908947.3

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
\ RPA \ 6-12 min 37-42 Fluorometric detection (Fluorescent metal ion indicator (Calcein)) 0.42 copy/μL \ \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2022 Enhanced Isothermal Amplification for Ultrafast Sensing of SARS-CoV-2 in Microdroplets Mengyun Zhou , Chuan Fan , Lirong Wang , Tailin Xu , Xueji Zhang Analytical Chemistry 35234445 10.1021/acs.analchem.2c00008

Enhanced Isothermal Amplification for Ultrafast Sensing of SARS-CoV-2 in Microdroplets

Author(s):

Mengyun Zhou , Chuan Fan , Lirong Wang , Tailin Xu , Xueji Zhang

Journal:

Analytical Chemistry

Year:

2022

Abstract:

Rapid and high-throughput screening is critical to control the COVID-19 pandemic. Recombinase polymerase amplification (RPA) with highly accessible and sensitive nucleic acid amplification has been widely used for point-of-care infection diagnosis. Here, we report an integrated microdroplet array platform composed of an ultrasonic unit and minipillar array to enhance the RPA for ultrafast, high-sensitivity, and high-throughput detection of SARS-CoV-2. On such a platform, the independent microvolume reactions on individual minipillars greatly decrease the consumption of reagents. The microstreaming driven by ultrasound creates on-demand contactless microagitation in the microdroplets and promotes the interaction between RPA components, thus greatly accelerating the amplification. In the presence of microstreaming, the detection time is 6-12 min, which is 38.8-59.3% shorter than that of controls without microstreaming, and the end-point fluorescence intensity also increased 1.3-1.7 times. Furthermore, the microagitation-enhanced RPA also exhibits a lower detection limit (0.42 copy/μL) for SARS-CoV-2 in comparison to the controls. This integrated microdroplet array detection platform is expected to meet the needs for high-throughput nucleic acid testing (NAT) to improve the containment of viral transmission during the epidemic, as well as provide a potential platform for the timely detection of other pathogens or viruses.