RPB0007

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
HCoV-OC43 Human coronavirus OC43, Human coronavirus (strain OC43), Human coronavirus strain OC43 31631 Nidovirales Coronaviridae Betacoronavirus Betacoronavirus 1 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 TGCTGGCATTGGTTTACATTTAAAAGTTAATT 32 0.42 28.1 55.04 9858.49 \
R AATCTTTTACACGCTCATAGCATTTCATCT 30 0.42 33.3 55.86 9065.97 \
P TCAGCGTGTTGATGAGAACGGTGATAAATT[TAMRA-DT]A[THF]A[BHQ-DT]TCAGTTCTTTGTTGT 47 0.12 34.7 63.54 15176.9 \

Gene Description

Target Gene GenBank ID
\ \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
a MddRPA assay for multiplexed quantitative detection of infectious diseases. MddRPA \ 20 39 fluorescence microscope 2.23 copies per μL \ >96%

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2023 Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus Ji Wook Choi, Won Ho Seo, Taejoon Kang, Taewook Kang, Bong Geun Chung Lab On A Chip 37083004 10.1039/d3lc00025g

Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus

Author(s):

Ji Wook Choi, Won Ho Seo, Taejoon Kang, Taewook Kang, Bong Geun Chung

Journal:

Lab On A Chip

Year:

2023

Abstract:

Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per μL to 2500 copies per μL could be detected with high linearity (R2 > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.