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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

DOI https://doi.org/10.2147/IMCRJ.S291166

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
Email shlee01@snet.net

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

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