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CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19.
Bustin, Stephen; Coward, Amy; Sadler, Garry; Teare, Louise; Nolan, Tania.
  • Bustin S; Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University, Chelmsford, Essex, CM1 1SQ, UK. Stephen.bustin@aru.ac.uk.
  • Coward A; Mid and South Essex NHS Foundation Trust, Broomfield Hospital, Chelmsford, Essex, CM1 7ET, UK.
  • Sadler G; Mid and South Essex NHS Foundation Trust, Broomfield Hospital, Chelmsford, Essex, CM1 7ET, UK.
  • Teare L; Mid and South Essex NHS Foundation Trust, Broomfield Hospital, Chelmsford, Essex, CM1 7ET, UK.
  • Nolan T; Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University, Chelmsford, Essex, CM1 1SQ, UK.
Sci Rep ; 10(1): 22214, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-989957
ABSTRACT
Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test's reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Reverse Transcriptase Polymerase Chain Reaction / Real-Time Polymerase Chain Reaction / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study Limits: Humans Country/Region as subject: Oceania Language: English Journal: Sci Rep Year: 2020 Document Type: Article Affiliation country: S41598-020-79233-x

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Reverse Transcriptase Polymerase Chain Reaction / Real-Time Polymerase Chain Reaction / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study Limits: Humans Country/Region as subject: Oceania Language: English Journal: Sci Rep Year: 2020 Document Type: Article Affiliation country: S41598-020-79233-x