Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR
1
detects B.1.1.7 lineage in Washington State
2
3
Garrett A. Perchetti
1,a
, Haiying Zhu
1,a
, Margaret G. Mills
1
, Lasata Shrestha
1
, Cassia
4
Wagner
2,3
, Shah Mohamed Bakhash
1
, Michelle Lin
1
, Hong Xie
1
, Meei-Li Huang
1
, Patrick
5
Mathias
1,4
, Trevor Bedford
2,3
, Keith R. Jerome
1,3
, Alexander L. Greninger
1,3,*,#
, Pavitra
6
Roychoudhury
1,3,*,#
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8
1
Department of Laboratory Medicine and Pathology, Virology Division, University of
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Washington, Seattle, WA, United States
10
2
Department of Genome Sciences, University of Washington, Seattle, WA, United
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States
12
3
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center,
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Seattle, WA, United States
14
4
Department of Biomedical Informatics and Medical Education, University of
15
Washington School of Medicine, Seattle, WA, United States
16
17
a
These authors contributed equally
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*Co-senior authors
19
20
#
Corresponding authors
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Pavitra Roychoudhury, proychou@uw.edu
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1616 Eastlake Ave E, Suite 320, Seattle, WA 98102
23
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
Box 358080
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Phone: 206 667 7801
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26
Alexander L. Greninger, agrening@uw.edu
27
1616 Eastlake Ave E, Suite 320, Seattle, WA 98102
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Phone: 415 439 3448
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Fax: 206 616 4340
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Running Title: ddPCR assay for SARS-CoV-2 N501Y mutation
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Keywords: ddPCR, N501Y, B.1.1.7, SARS-CoV-2, Spike protein, COVID-19
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Abstract Word Count: 234
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Manuscript Word Count: 2,370
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ABSTRACT
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Real-time epidemiological tracking of variants of interest can help limit the spread
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of more contagious forms of SARS-CoV-2, such as those containing the N501Y
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mutation. Typically, genetic sequencing is required to be able to track variants of
41
interest in real-time. However, sequencing can take time and may not be accessible in
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all laboratories. Genotyping by RT-ddPCR offers an alternative to sequencing to rapidly
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detect variants of concern through discrimination of specific mutations such as N501Y
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that is associated with increased transmissibility. Here we describe the first cases of the
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B.1.1.7 lineage of SARS-CoV-2 detected in Washington State by using a combination of
46
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.10.21253321doi: medRxiv preprint
RT-PCR, RT-ddPCR, and next-generation sequencing. We screened 1,035 samples
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positive for SARS-CoV-2 by our CDC-based laboratory developed assay using
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ThermoFisher’s multiplex RT-PCR COVID-19 assay over four weeks from late
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December 2020 to early January 2021. S gene dropout candidates were subsequently
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assayed by RT-ddPCR to confirm four mutations within the S gene associated with the
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B.1.1.7 lineage: a deletion at amino acid (AA) 69-70 (ACATGT), deletion at AA 145,
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(TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were
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detected in two specimens, and follow-up sequencing revealed a total of 10 mutations in
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the S gene and phylogenetic clustering within the B.1.1.7 lineage. As variants of
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concern become increasingly prevalent, molecular diagnostic tools like RT-ddPCR can
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be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of
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SARS-CoV-2.
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BACKGROUND
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The first known case of the SARS-CoV-2 B.1.1.7 variant in the United States was
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reported in Colorado on December 29
th
2020 and the next day, it was confirmed in
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California (1, 2). University of Washington (UW) Virology had begun surveillance a few
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days prior using PCR to screen SARS-CoV-2 positive samples for the absence, or
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“dropout” of the S gene, which encodes the spike (S) protein on the surface of the viral
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particle. The B.1.1.7 variant is characterized by 17 mutations, eight of which occur
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within the S gene domain (3, 4). This region of the SARS-CoV-2 genome is of interest
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due to the B.1.1.7 lineage being associated with increased transmissibility, but also
69
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.10.21253321doi: medRxiv preprint
because the FDA emergency use authorization for COVID-19 vaccines in the United
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States target the S protein (5, 6).
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Over the course of four weeks, we screened more than a thousand SARS-CoV-2
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positive samples for the S gene target failure (SGTF). We selected random clinical
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specimens that were positive using a CDC-based laboratory developed test (LDT) for
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SARS-CoV-2, and typically had a cycle threshold (C
T
) under 35 (7–11). These SARS-
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CoV-2 positive samples were then amplified with the TaqPath COVID-19 assay
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(ThermoFisher Scientific, Waltham, MA, USA), a multiplex RT-PCR targeting the S
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gene, as well as regions within the N gene and ORF1ab (12). Candidates for the B.1.1.7
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variant have a positive detection for the N gene and ORF1ab, with a negative for the S
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gene. SGTFs are candidates for the B.1.1.7 lineage, but the TaqPath assay is not
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necessarily specific for that variant exclusively due to other signature mutations, so
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genetic sequencing is used to confirm the B.1.1.7. lineage (13). However, sequencing
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can be a time-consuming and resource-intensive process that not all laboratories have
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integrated into their clinical workflow. Analysis of publicly available sequencing data by
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the Broad Institute revealed that it takes a median 85 days to get from sample to
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publicly available sequence in the United States (14, 15).
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Here we describe a novel droplet reverse-transcription digital-PCR (RT-ddPCR)
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assay that specifically detects four mutations associated with the B.1.1.7 variant,
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particularly the N501Y mutation. This mutation in the S gene is shared by the U.K. and
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the South African variant (B.1.351) (16). Preliminary data has indicated B.1.1.7 to be
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more transmissible, while B.1.351 is considered to be less well neutralized by
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antibodies induced by certain vaccines, such as the AstraZeneca and Novavax
92
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.10.21253321doi: medRxiv preprint
vaccines (3, 16–18). This RT-ddPCR assay can distinguish SARS-CoV-2 positive
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samples that carry this important N501Y mutation, as well specific allelic discrimination
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of the B.1.1.7 lineage, without genetic sequencing.
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METHODS
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Specimen Selection Criteria
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From December 25
th
2020 to January 20
th
2021, 1,035 specimens positive for
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SARS-CoV-2 by our CDC-based LDT were screened for SGTFs (Fig.1) (19–21).
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Approximately 50% of these samples came from King County, followed by Pierce
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County with around 15%, Benton and Franklin Counties at 10%, and the remainder from
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other counties in Washington State. We selected samples with C
T
s<35 when available
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to reduce the effect of assay stochasticity, with consideration of initial data showing that
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the B.1.1.7 variant has been associated with higher viral loads (i.e. lower C
T
s) (22).
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Extracted nucleic acids were stored at 4
o
C or -20
o
C prior to amplification. This work was
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approved under a waiver of consent by the University of Washington institutional review
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board (STUDY00000408).
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PCR
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PCR was performed using TaqPath COVID-19 Combo Kit (ThermoFisher,
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Waltham, MA, USA) with 10 µL of extracted nucleic acid used as template per 25µL
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reaction. This multiplex real-time RT-PCR assay targets the S gene, N gene, and
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ORF1ab of SARS-CoV-2. Reactions utilized a kit-provided positive control (1x10
4
114
copies/µL) diluted with TaqPath COVID-19 Control Dilution Buffer, and dH
2
O as a
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.10.21253321doi: medRxiv preprint
