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Study record
Chaintoutis 2021First Published: 2021 Jul 21Updated Date: 2021 Jul 21

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions (preprint)

  1. Study Type
  2. Observational
  1. Study Aim
  2. Diagnostic/Prognostic
  3. Epidemiology
  1. Study Design
  2. Cross-sectional
  1. Intervention Assignment
  2. Not Applicable
Reference record

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions

Chaintoutis SC, Chassalevris T, Tsiolas G, Balaska S, Vlatakis I, Mouchtaropoulou E, Siarkou VI, Tychala A, Koutsioulis D, Skoura L, Argiriou A, Dovas CI
Journal article
Report Results
The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94% and a linear range of over 5 log10 copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs
Reference record

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions (preprint)

Chaintoutis SC, Chassalevris T, Tsiolas G, Balaska S, Vlatakis I, Mouchtaropoulou E, Siarkou VI, Tychala A, Koutsioulis D, Skoura L, Argiriou A, Dovas CI
Unpublished article (preprint)
Report Results
The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94% and a linear range of over 5 log10 copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.Competing Interest StatementThe authors have declared no competing interest.Funding StatementN/AAuthor DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The entire study has been approved by the AUTH Research Ethics Committee, with reference number 126293/2021. The members of the AUTH Research Ethics Committee are: A/Prof. Dimitrios Stamovlasis (chair, School of Philosophy and Education, stadi@edlit.auth.gr), Prof. Ioannis Stefanidis (vice-chair, School of Law, AUTH, ids@law.auth.gr), Prof. Victoria Samanidou (School of Chemistry, AUTH, samanidu@chem.auth.gr), A/Prof Andreana Assimopoulou (Dept. of Chemical Engineering, AUTH, andreana@auth.gr), A/Prof Anastasia Tsingotjidou (School of Veterinary Medicine, AUTH, astsing@vet.auth.gr). All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesN/A [Preprints are preliminary reports of work that have not been peer reviewed. Refer to the original preprint or preprint server for specific information about the individual preprint.]