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Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.
Rowan, Aileen G; May, Philippa; Badhan, Anjna; Herrera, Carolina; Watber, Patricia; Penn, Rebecca; Crone, Michael A; Storch, Marko; Garson, Jeremy A; McClure, Myra; Freemont, Paul S; Madona, Pinglawathee; Randell, Paul; Taylor, Graham P.
  • Rowan AG; Section of Virology, Department of Infectious Disease, Imperial College London, United Kingdom; Centre for Haematology, Department of Infection and Inflammation, Imperial College London, United Kingdom. Electronic address: a.rowan@imperial.ac.uk.
  • May P; Centre for Haematology, Department of Infection and Inflammation, Imperial College London, United Kingdom; Developmental Disorders, South East Genomics Laboratory Hub, Guy's and St Thomas' NHS Trust, United Kingdom.
  • Badhan A; Section of Virology, Department of Infectious Disease, Imperial College London, United Kingdom.
  • Herrera C; Section of Immunology of Infection, Department of Infectious Disease, Imperial College London, United Kingdom.
  • Watber P; Section of Virology, Department of Infectious Disease, Imperial College London, United Kingdom.
  • Penn R; Section of Virology, Department of Infectious Disease, Imperial College London, United Kingdom.
  • Crone MA; London Biofoundry, Imperial College Translation and Innovation Hub, Imperial College London, United Kingdom; Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, United Kingdom; UK Dementia Research Institute Care Research and Technology Centre, Imp
  • Storch M; London Biofoundry, Imperial College Translation and Innovation Hub, Imperial College London, United Kingdom; Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, United Kingdom.
  • Garson JA; Division of Infection and Immunity, University College London, United Kingdom.
  • McClure M; Section of Virology, Department of Infectious Disease, Imperial College London, United Kingdom.
  • Freemont PS; London Biofoundry, Imperial College Translation and Innovation Hub, Imperial College London, United Kingdom; Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, United Kingdom; UK Dementia Research Institute Care Research and Technology Centre, Imp
  • Madona P; North West London Pathology, Charing Cross Hospital, Imperial College Healthcare NHS Trust, United Kingdom.
  • Randell P; North West London Pathology, Charing Cross Hospital, Imperial College Healthcare NHS Trust, United Kingdom.
  • Taylor GP; Section of Virology, Department of Infectious Disease, Imperial College London, United Kingdom. Electronic address: g.p.taylor@imperial.ac.uk.
J Virol Methods ; 294: 114174, 2021 08.
Article in English | MEDLINE | ID: covidwho-1226316
ABSTRACT
There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Specimen Handling / RNA, Viral / COVID-19 Nucleic Acid Testing / SARS-CoV-2 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: J Virol Methods Year: 2021 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Specimen Handling / RNA, Viral / COVID-19 Nucleic Acid Testing / SARS-CoV-2 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: J Virol Methods Year: 2021 Document Type: Article