Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR detects B.1.1.7 lineage in Washington State

Abstract

Real-time epidemiological tracking of variants of concern (VOCs) can help limit the spread of more contagious forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as those containing the N501Y mutation. Typically, genetic sequencing is required to be able to track VOCs in real-time. However, sequencing can take time and may not be accessible in all laboratories. Genotyping by RT-ddPCR offers an alternative to rapidly detect VOCs through discrimination of specific alleles such as N501Y, which is associated with increased transmissibility and virulence. Here we describe the first cases of the B.1.1.7 lineage of SARS-CoV-2 detected in Washington State by using a combination of reverse-transcription polymerase chain reaction (RT-PCR), RT-ddPCR, and next-generation sequencing. We initially screened 1035 samples positive for SARS-CoV-2 by our CDC-based laboratory-developed assay using ThermoFisher's multiplex RT-PCR COVID-19 assay over four weeks from late December 2020 to early January 2021. S gene target failures (SGTF) were subsequently assayed by RT-ddPCR to confirm four mutations within the S gene associated with the B.1.1.7 lineage: a deletion at amino acid (AA) 69-70 (ACATGT), deletion at AA 145, (TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were detected in two specimens; follow-up sequencing revealed a total of 9 mutations in the S gene and phylogenetic clustering within the B.1.1.7 lineage. Next, we continued screening samples for SGTF detecting 23 additional B.1.1.7 variants by RT-ddPCR and confirmed by sequencing. As VOCs become increasingly prevalent, molecular diagnostic tools like RT-ddPCR can be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of SARS-CoV-2.

Keywords: B.1.1.7; COVID-19; N501Y; SARS-CoV-2; SGTF; ddPCR.

Figure 1

RT‐PCR, RT‐ddPCR, and sequencing surveillance effort for initial detection of B.1.1.7 lineage. Samples sent to UW Virology for SARS‐CoV‐2 molecular detection were initially screened for S gene transcript failure (SGTF) by multiplex RT‐PCR as a proxy for variant detection. SARS‐CoV‐2 positive samples with SGTF were subsequently genotyped for specific allelic discrimination by RT‐ddPCR. Both SGTF samples that genotyped as B.1.1.7 lineage by RT‐ddPCR were also sequenced and clustered with N501Y.V1 clades in the United States. RT‐ddPCR, droplet digital RT‐PCR; RT‐PCR, reverse‐transcription polymerase chain reaction; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2

Figure 2

Multiplexed qRT‐PCR fluorescence of S gene dropout in SARS‐CoV‐2 positive samples. Multicomponent amplification plots are shown for (A) U.K. Variant #1 (55538) and (B) U.K. Variant #2 (55545). PCR cycle is plotted on the X axis, with the quantity of fluorescence detected in real‐time on the Y axis. Two out of three fluorophores are detected by qRT‐PCR for both samples on the 7500 Real‐Time PCR System (Life Technologies) and analyzed on 7500 software v.2.3 (Life Technologies). VIC and FAM are reporters for the N gene and ORF1ab, respectively, and are seen here with robust amplification. ABY, the probe for the S gene, is present in the reaction mix but is not detected by fluorescence, indicating a potential B.1.1.7 SARS‐CoV‐2 variant. FAM, 6‐carboxyfluorescein; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; VIC, 2ʹ‐chloro‐7ʹphenyl‐1,4‐dichloro‐6‐carboxy‐fluorescein

Figure 3

RT‐ddPCR amplification results for SARS‐CoV‐2 B.1.1.7 lineage. (A) U.K. Variant Sample #1 (55538) and (C) U.K. Variant Sample #2 (55545) have amplification for AA69‐70del and N501Y mutation. (E) Positive control demonstrates amplification for AA69‐70del and N501Y mutation and (G) B.1.1.7 Negative control (Non‐B.1.1.7 SARS‐CoV‐2 and water) shows no amplification for AA69‐70del and N501Y mutation for RT‐ddPCR amplicon set 1. (B) U.K. Variant Sample #1 (55538) and (D) U.K. Variant Sample #2 (55545) have amplification for AA145 deletion and S982A mutation. (F) Positive control demonstrates amplification for AA145 deletion and S982A mutation and (H) B.1.1.7 Negative control (Non‐B.1.1.7 SARS‐CoV‐2 and water) shows no amplification for AA145 deletion and S982A mutation for RT‐ddPCR amplicon set 2. RT‐ddPCR, droplet digital RT‐PCR; RT‐PCR, reverse‐transcription polymerase chain reaction; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2

Figure 4

Phylogenetic tree focused on Washington State SARS‐CoV‐2 samples collected from November 2020 through February 2021. In (A) the phylogenetic tree is filtered to only show Washington samples; 501Y.V1 (B.1.1.7) samples are shown in orange (arrow). (B) 501Y.V2 clade showing Washington samples (red X) in context of global SARS‐CoV‐2, selected by genetic proximity to the Washington samples. UW‐55538 and UW‐55545 separately cluster at the bottom of the clade. SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2