Sanger sequencing for definitive detection of SARS-CoV-2

Under the best of circumstances, RT-qPCR assays granted emergency use authorization for presumptive detection of SARS-CoV-2 in clinical specimens generate at least 30% false-positive results and 20% false-negative results [32]. The World Health Organization (WHO) has acknowledged “In some circumstances, the distinction between background noise and actual presence of the target virus is difficult to ascertain” [33]. The WHO further advised that SARS-CoV-2 gene sequencing can be used for improved diagnostics [34]. However, most laboratories require specimens with high viral loads for sequencing [35]. Milford Molecular Diagnostics Laboratory has developed a routine sequencing assay for definitive detection of SARS-CoV-2 by partial sequencing of a 398-bp amplicon of the N gene cDNA and a 437-bp amplicon of the S gene for routine screening of 8 common amino acid mutations of concern. All isolates positive for at least one of these 8 amino acid mutations will be further sequenced for variant differentiation. Every report of positive sample will contain at least two electropherograms showing an N gene for detection (upper electropherogram) and an S gene for possible amino acid mutations (lower electropherogram) as follows.

Electropherogram (below) showing the ACE 2 receptor binding domain of the SARS-CoV-2 S gene with a solitary E484K amino acid mutation

This routine DNA sequence electropherogram covering the ACE 2 receptor binding domain shows 7 vertical black bars from left to right, pointing to 7 key nucleotides identified as A, G, T, G, A, T and A in the S gene of SARS-CoV-2 in a nasopharyngeal swab specimen taken from a patient with acute respiratory infection. The nucleotide A pointed by the 5^th bar is a mutated base, indicating an E484K amino acid mutation (G>A nucleotide mutation). All other 6 black bars point to wildtype bases, confirming the lack of K417N, K417T, L452R, S477N, E484Q, S494P and N501Y mutations in this virus isolate. All known newly emerging SARS-CoV-2 variants of concern are associated with at least one of the 8 amino acid mutations identified in this sequence. Specimens harboring any of these 8 amino acid mutations will be further sequenced for A67V, Δ69/70, T95I, Δ144Y, Y145del, H146del, W152C and V1176F mutations for B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617, B.1.168, P.1 and P.2 variant differentiation, said Dr. Sin Hang Lee, Director of Milford Molecular Diagnostics Laboratory