Back to Journals » International Medical Case Reports Journal » Volume 14

Partial N Gene Sequencing for SARS-CoV-2 Verification and Pathway Tracing

Authors Lee SH, McGrath J, Connolly SP, Lambert J

Received 9 November 2020

Accepted for publication 25 December 2020

Published 11 January 2021 Volume 2021:14 Pages 1—10


Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Professor Ronald Prineas

Sin Hang Lee,1 Jonathan McGrath,2 Stephen P Connolly,2 John Lambert2

1Milford Molecular Diagnostics Laboratory, Milford, CT, USA; 2Department of Infectious Diseases, Mater Misericordiae University Hospital, Dublin, Ireland

Correspondence: Sin Hang Lee
Milford Molecular Diagnostics Laboratory, 2044 Bridgeport Avenue, Milford, CT 06460, USA
Tel +1 203-878-1438
Fax +1 203-878-0109

Abstract: When SARS-CoV-2 prevalence is low, many RT-qPCR-positive test results are false positives. Sequencing of a 398-bp cDNA PCR amplicon derived from a highly conserved segment with single nucleotide polymorphisms of the nucleocapsid (N) gene in presumptive positive samples can verify true positives and differentiate at least 27 phylogenetically distinct strains of SARS-CoV-2 for helping track virus strain movement between individuals and across geographical areas. We report using this partial N gene sequencing method to confirm a case of mild COVID-19 disease. The patient was first seen on March 15, 2020, in the emergency department of the university hospital in Dublin, Ireland. RT-qPCR test on a nasopharyngeal swab sample was positive for SARS-CoV-2. Partial sequencing of the N gene in the residue of the tested RNA extract showed a characteristic set of 3-consecutive GGG-to-AAC mutations at positions 28881, 28882, 28883, which is known to first appear in samples collected in Continental Europe in February 2020. Using this sequencing-based method to re-test 9 reference nasopharyngeal swab samples supplied by the Connecticut State Department of Public Health Microbiology Laboratory revealed that 2 of the 9 positive samples had a single nucleotide mutation in the 398-base segment of the SARS-CoV-2 N gene. One of the 2 mutant samples showed a mutation at position 28821, which was first reported in a sample recently collected in the neighboring New York state. The other sample showed a novel frameshift nucleotide “A” insertion between position 29051 and position 29057, which co-existed with its wildtype parental virus in one sample. Routine sequencing of RT-qPCR-positive samples can minimize or eliminate false-positive SARS-CoV-2 test results that may cause unnecessary anxiety among the population and prevent false-positive tests from shutting down schools and workplaces unnecessarily as businesses try to resume normal operations in the community.

Keywords: false-positive, Sanger sequencing, phylogenetically distinct strains, COVID-19, SARS-CoV-2 verification, single nucleotide mutation, partial N gene sequencing, virus strain tracing

Creative Commons License This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

Download Article [PDF]  View Full Text [HTML][Machine readable]