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

SARS-COV-2 N501Y MUTATION SCREENING

As of February 3, 2021, all COVID-19 PCR-positive specimens with Ct value ≤35* in Ontario will be screened for the N501Y mutation.

This mutation is present in the following Variants of Concern:

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

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

P.1 (originally identified in Brazil)

This will be arranged with the individual testing laboratories and the results will be reported back to the submitter.

All screen positive results (and subsequent sequencing results) will be reported to the ordering provider and to public health.

Ongoing universal VOC screening of all PCR-positive specimens will be revisited in early March.

* Specimen cycle threshold (Ct) value must be ≤35 to ensure adequate viral load, which is required for successful N501Y mutation screening and subsequent gene sequencing. A subset of samples with a CT ≤ 35 may not be successfully screened for VOCs due to a low level of virus present or RNA degradation.

*Uncontrolled print copy. Valid only on day of print: 27 Feb 2021.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
SARS-COV-2 VARIANT SCREENING	Coronavirus Disease 2019 (COVID-19) Test Requisition	SARS-CoV-2 PCR-positive specimen	1 ml preferred (min 500 µl)	N/A

Submission and Collection Notes

As of February 3, 2021, all COVID-19 PCR-positive specimens with Ct value ≤35 in Ontario will be screened for the N501Y mutation. A submission form is not required until further notice.
The following submission notes only pertain to requests for VOC screening of a PCR-positive specimen that was tested prior to the implementation of universal screening.

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

If submitting a SARS-CoV-2-positive specimen that was tested at a non-PHO laboratory, submit each of the following to your nearest PHO Laboratory:

1mL (minimum volume 500 µL) of specimen maintained at refrigeration temperature (freeze if anticipated transport time to laboratory is >72 hours); and
a fully completed COVID-19 PCR Test Requisition. Order “SARS-CoV-2 Variant Screening” on the requisition; AND,
Inform PHO Laboratory by contacting the PHO Laboratory Customer Service Centre at 1-877- 604-4567 or 416-235-6556.

Limitations

This laboratory-developed investigational assay is being used for surveillance purposes and public health management.
VOC screening relies on successful amplification of a partial SARS-CoV-2 spike (S) gene, followed by sequencing.

Preparation Prior to Transport

For SARS-CoV-2-positive specimens being sent to PHO Laboratory for screening, 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.

Results will be available within 3 days.

Reporting

All SARS-CoV-2 VOC surveillance 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 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.

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

Sequencing involves PCR amplification, followed by sequencing, including a 700 base pair (bp) fragment of the SARS-CoV-2 spike (S) gene (extending from amino acid P330 to N540). This fragment 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, E484K, K417N
P.1 (originally identified in Brazil)	N501Y¹, E484K, K417T

¹Identified by the VOC SNP real-time PCR screening assay

Final classification is based on screening and additional sequencing data.

Algorithm

See “Surveillance Indications” above.

*Uncontrolled print copy. Valid only on day of print: 27 Feb 2021.

Updated 24 Feb 2021