Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

A Routine Sanger Sequencing Target Specific Mutation Assay For SARS-CoV-2 Variants Of Concern And Interest

Version 1 : Received: 9 September 2021 / Approved: 23 September 2021 / Online: 23 September 2021 (18:58:26 CEST)

How to cite: Lee, S.H. A Routine Sanger Sequencing Target Specific Mutation Assay For SARS-CoV-2 Variants Of Concern And Interest. Preprints 2021, 2021090415 (doi: 10.20944/preprints202109.0415.v1). Lee, S.H. A Routine Sanger Sequencing Target Specific Mutation Assay For SARS-CoV-2 Variants Of Concern And Interest. Preprints 2021, 2021090415 (doi: 10.20944/preprints202109.0415.v1).

Abstract

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein along with certain mutations in other regions within the S protein as specific variants in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.

Keywords

Delta variant; Variants of concern; Variants of interest; SARS-CoV-2; Spike protein; Nested RT-PCR; Sanger sequencing; Amino acid mutations; ACE2 RBD; N-terminal domain (NTD)

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