tetano
Editor, Senior Moderator
Int J Mol Sci. 2020 Apr 8;21(7). pii: E2574. doi: 10.3390/ijms21072574.
Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens.
Yip CC[SUP]1[/SUP], Ho CC[SUP]2[/SUP], Chan JF[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP], To KK[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP], Chan HS[SUP]7[/SUP], Wong SC[SUP]8[/SUP], Leung KH[SUP]4[/SUP], Fung AY[SUP]4[/SUP], Ng AC[SUP]4[/SUP], Zou Z[SUP]4[/SUP], Tam AR[SUP]9[/SUP], Chung TW[SUP]1[/SUP], Chan KH[SUP]3,[/SUP][SUP]4,[/SUP][SUP]6[/SUP], **** IF[SUP]10[/SUP], Cheng VC[SUP]1[/SUP], Tsang OT[SUP]11[/SUP], Tsui SKW[SUP]12[/SUP], Yuen KY[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP].
Author information
Abstract
The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID[SUB]50[/SUB]/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.
KEYWORDS:
COVID-19; COVID-19-nsp2 assay; GolayMetaMiner; SARS-CoV-2; clinical evaluation; genome subtraction; nsp2; real-time RT-PCR; sensitivity; specificity
PMID:32276333DOI:10.3390/ijms21072574
Free full text
Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens.
Yip CC[SUP]1[/SUP], Ho CC[SUP]2[/SUP], Chan JF[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP], To KK[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP], Chan HS[SUP]7[/SUP], Wong SC[SUP]8[/SUP], Leung KH[SUP]4[/SUP], Fung AY[SUP]4[/SUP], Ng AC[SUP]4[/SUP], Zou Z[SUP]4[/SUP], Tam AR[SUP]9[/SUP], Chung TW[SUP]1[/SUP], Chan KH[SUP]3,[/SUP][SUP]4,[/SUP][SUP]6[/SUP], **** IF[SUP]10[/SUP], Cheng VC[SUP]1[/SUP], Tsang OT[SUP]11[/SUP], Tsui SKW[SUP]12[/SUP], Yuen KY[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP].
Author information
Abstract
The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID[SUB]50[/SUB]/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.
KEYWORDS:
COVID-19; COVID-19-nsp2 assay; GolayMetaMiner; SARS-CoV-2; clinical evaluation; genome subtraction; nsp2; real-time RT-PCR; sensitivity; specificity
PMID:32276333DOI:10.3390/ijms21072574
Free full text