SARS-CoV-2 (COVID-19 Virus) Variant of Concern (VoC) Surveillance

SARS-COV-2 N501Y/E484K MUTATION TESTING BY REAL-TIME PCR

As of March 22, 2021, all COVID-19 PCR-positive specimens with Ct value ≤35* in Ontario will be tested for the N501Y and E484K mutations using a multiplex real-time PCR assay. This test replaces the N501Y single target test that was implemented February 3, 2021. These two mutations are present in the more commonly described Variants of Concern (VOCs) as follows:

Variant of Concern

Mutation(s)1

B.1.1.7 (202012/01) (originally identified in the UK)

N501Y

B.1.351 (501Y.V2) (originally identified in South Africa)

N501Y, E484K

P.1 (originally identified in Brazil)

N501Y, E484K

1Only mutations detected by the VOC multiplex assay are listed.

All mutation positive results (and subsequent sequencing results where applicable, see below) will be reported to the ordering healthcare provider and to the public health unit.

Ongoing universal VOC mutation testing of all PCR-positive specimens will be revisited as needed.

* The specimen’s SARS-CoV-2 cycle threshold (Ct) value must be 35 to ensure successful N501Y/E484K mutation testing.  A subset of samples with a CT ≤ 35 may not be successfully tested for VOC mutations likely due to a low level of virus present or RNA degradation.


SARS-CoV-2 VOC Genome Sequencing for Surveillance

As of March 22, 2021, only specimens positive for the E484K mutation (with or without the N501Y mutation) with Ct value ≤30* will be sequenced and reported for surveillance purposes. Specimens that are N501Y positive and E484K negative are presumed to be VOCs (likely B.1.1.7) based on current epidemiology, and will no longer be routinely sequenced to report the lineage.

Ongoing sequencing of all E484K-positive specimens will be revisited as needed.

A subset of SARS-CoV-2-positive specimens in which no mutations were detected by the real-time PCR will be selected for genome sequencing for surveillance purposes. For these, only aggregate results will be reported in PHO COVID-19 data and surveillance products to support ongoing monitoring of circulating lineages.

* The specimen’s VOC mutation testing PCR cycle threshold (Ct) value must be ≤30 for successful genome sequencing.  A subset of samples with a CT ≤ 30 may not be successfully sequenced likely due to a low level of virus present, RNA degradation, or sequencing technical issues.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

SARS-COV-2 VARIANT SCREENING

SARS-CoV-2 PCR-positive specimen

1 ml preferred (min 500 µl)

N/A

Submission and Collection Notes

1

SARS-CoV-2 VOC testing cannot be performed prior to laboratory detection of SARS-CoV-2.
SARS-CoV-2 VOC testing may be requested on SARS-CoV-2-positive specimens meeting acceptance criteria (see above).

Limitations

This laboratory-developed investigational assay is being used for surveillance purposes and public health management. It is not a clinical test.

Preparation Prior to Transport

For SARS-CoV-2-positive specimens being sent to PHO Laboratory for VOC mutation testing, 1 mL (minimum volume 500 µL) of specimen should be provided and maintained at refrigeration temperature during transportation (freeze if anticipated transport time to the laboratory is >72 hours).

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Surveillance testing will be performed at regular intervals.

SARS-CoV-2 VOC Multiplex real-time PCR results will be available within 3 days.

If performed, SARS-CoV-2 genome sequencing results will be available within 2-4 weeks.

Reporting

All SARS-CoV-2 VOC testing results are reported back to the ordering health-care provider as indicated on the requisition.

As a disease of public health significance, detection of SARS-CoV-2 VOC-related mutations and VOCs will be reported to the local public health unit.

Test Methods

This laboratory-developed investigational assay is being used for surveillance purposes and public health management.

Testing involves a multiplexed single nucleotide polymorphism (SNP) real-time PCR assay developed at PHO Laboratory. The assay screens for the N501Y and E484K mutations in the spike (S) gene, which are associated with VOCs (see table below). Specimens with the SARS-CoV-2 E484K S gene mutation detected with Ct ≤30 in the VOC real-time PCR assay undergo genome sequencing for VOC identification and classification.

Sequencing involves PCR amplification, followed by sequencing, including a 700 base pair (bp) region of the SARS-CoV-2 spike (S) gene (extending from amino acid P330 to N540). This region includes the receptor binding domain (RBD), where key VOC mutations listed in the table below are located.

VOC

Key RBD (S gene) Mutations

B.1.1.7 (202012/01) (originally identified in the UK)

N501Y1

B.1.351 (501Y.V2) (originally identified in South Africa)

N501Y1, E484K1, K417N

P.1 (originally identified in Brazil)

N501Y1, E484K1, K417T

1Identified by the VOC SNP multiplex real-time PCR assay.

Final classification is based on mutation detection and additional sequencing data, when performed.

Algorithm

  1. SARS-COV-2 positive specimen with Ct≤35 tested by multiplex real-time PCR assay for N501Y and E484K
  2. Based on results of multiplex real-time PCR assay:
    1. If positive for E484K mutation and VOC real-time PCR Ct is ≤30, reflex to whole genome sequencing for VOC identification and classification
    2. If negative for E484K mutation or Ct is >30, no whole genome sequencing performed, final report sent

Data and Analysis

Related Testing Resources

PHO has developed the following testing resources:

Frequently Asked Questions: SARS-COV-2 (COVID-19 virus) Variant of Concern (VOC) Testing Update, March 26, 2021

Updated 26 March 2021