SARS-CoV-2 Variant SurveillancePrimePCR SARS-CoV-2 Multiple Mutation RT-qPCR Assay
Using Reliance One-Step Multiplex RT-qPCR Supermix
ProtocolDescription

The PrimePCR SARS-CoV-2 Multiple Mutation RT-qPCR Assay* is a single-well multiplexed panel
which can be validated by researchers to detect and differentiate four key mutations identified in thesevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. The identification of mutations
targeted by the assay can be evaluated by researchers to assess the likelihood of a specific variant in thesample. The assay’s high sensitivity can be validated by researchers to assess low-level samples with
SARS-CoV-2 quantification cycle (Cq) values up to 40. The single tube assay is 20x concentrated andcontains a primer set plus up to six probes, including one probe that is the multiplexed control (MPC).
This probe has homology to a conserved region between the primers and serves as an internal RNA
integrity and experimental setup control. The other probes are perfect matches to the four mutationsof interest in the spike protein gene. The assay format is optimal for use with the Reliance One-Step
Multiplex RT-qPCR Supermix and should be run on a real-time PCR detection system equipped with fivefluorescence channels, such as the CFX96 Touch or CFX Opus 96 Real-Time PCR System. Additional
assays will become available as the virus continues to evolve and the need to identify novel variants
arises. Visit bio-rad.com/SC2Variants for a complete list of assays available.
*  For research use only (RUO). Not for use in diagnostic procedures.
** The multiplexed control (MPC) is designed in a conserved region of the S gene. MPC functionality may be compromised if the virus acquires mutations in this region.

Storage and Stability
The assays are stable for 12 months when stored at 4°C, protectedfrom light. The 20x assay mix can be kept at –20°C for long-term
storage.
Product Contents
This product is a 20x concentrated, ready-to-use primer/probe
mix, which can be validated for use with the Reliance One-Step
Multiplex RT-qPCR Supermix for detection of four mutations ina single well. It is provided as 200, 1,000, or 2,500 μl volumes,
sufficient for 200, 1,000, or 2,500 x 20 μl reactions.
Suggested Reagents and Equipment*■ 
Reliance One-Step Multiplex RT-qPCR Supermix (catalog#12010220, 12010221)■

Reference control: SARS-CoV-2 Standard (#COV019)■

SARS-CoV-2 Variant Controls:– SARS-CoV-2 S Gene Alpha Variant Control (B.1.1.7) (#COVA)

10000147102– SARS-CoV-2 S Gene Beta Variant Control (B.1.351) (#COVB)– SARS-CoV-2 S Gene Epsilon Variant Control (B.1.427 andB.1.429) (#COVE)– SARS-CoV-2 S Gene Gamma Variant Control (P.1) (#COVG)– SARS-CoV-2 S Gene Delta-Kappa Variants Control ​(Delta
B.1.617.2 and AY sublineages, Kappa B.1.617.1 and B.1.617.3)(#COVDK)
■

■

■

■

Negative control: SARS-CoV-2 Negative (#COV000)
 FX Opus 96 or CFX96 Touch Real-Time PCR System (#12011319
Cor 1855195, respectively) or equivalent 5-color instrument
 ard-Shell 96-Well PCR Plates (#HSP9655, HSP9955) orHequivalent
Microseal 'B' PCR Plate Sealing Film (#MSB1001)*Bio-Rad reagents and equipment, unless otherwise noted. Reference, variant,and negative controls are from Exact Diagnostics, a Bio-Rad company.

PrimePCR SARS-CoV-2 Multiple Mutation RT-qPCR Assay Using Reliance One-Step Multiplex RT-qPCR Supermix

Bio-Rad Instrument Setup Example
The following instructions are suggested for researchers to develop
a validation protocol in their laboratory. The example shown here isfor the CFX Opus 96 and CFX96 Touch Real-Time PCR Systems,
which are research instruments equipped with five fluorescencechannels. For more detailed information, refer to the respective
instrument manual.

Sample Preparation
Sample TypeThe optimal sample type is total RNA extracted from humanspecimen samples. Alternative sample types may be compatible
but should be optimized and validated before use by the
researcher. Assay performance depends on the amount andquality of template RNA validated. The PrimePCR SARS-CoV-2
Multiple Mutation Assay is for research use only and is notintended for patient diagnosis from clinical samples.

Cycling Protocol Setup

Reference Control

The SARS-CoV-2 Standard (#COV019) from Exact Diagnostics
is manufactured with synthetic RNA transcripts containing fivegene targets (the E, N, ORF1ab, RdRp, and S genes of SARSCoV-2). The product is optimal to validate reverse transcription,
amplification, and detection of SARS-CoV-2 RNA. This productalso contains human genomic DNA, which can serve as anamplification control (RPP40). Add 10 µl per reference control
reaction; extraction or dilution should not be required.

1.

Click File > New > Protocol in the menu bar to open the
Protocol Editor.

2.

Change Sample Volume to 20 μl.

3.

Modify the thermal cycling protocol according to theguidelines in Table 1.

Table 1. Example of thermal cycling protocol.*Temperature,
Step Description°C

Positive Variant Controls

The SARS-CoV-2 S Gene Variant Controls from Exact Diagnostics
are individually manufactured with synthetic RNA transcripts of
the S gene from SARS-CoV-2 variants of interest or concern.
These controls contain human genomic DNA, which serves as anamplification control. These variant controls can be validated for use
to monitor the reverse transcription, amplification, and detection
steps in assays that target S gene mutations. Add 10 µl per variant
control reaction; extraction or dilution should not be required.

Numberof Cycles

1

Reverse transcription

50

10 min1

2

DNA polymerase activation andtemplate denaturation95

10 min1

Template
denaturation95

10 secAnnealing/extension andplate read58

30 sec3
4

Amplification45* Set the lid temperature to 105°C.

gBlocks Gene Fragments from Integrated DNA Technologies, Inc.
are DNA oligos, which are optimal as positive controls in assays thattarget mutations other than the S gene. Ordering information canbe found in the Assay Control tab of each PrimePCR SARS-CoV-2
Multiple Mutation Assay product, or the PrimePCR SARS-CoV-2
Multiple Mutation Assay product page. Dilute gBlocks Controls in
1x Tris-EDTA (TE) buffer to 105–106 copies/µl (50–500 fM). Use 5 µl
per variant control reaction.

4.

Confirm the annealing/extension step includes a plate read,as indicated by a camera symbol in the step.

5.

To add a plate read, click on the step to highlight, then click
Add Plate Read to Step.

6.

Save the protocol by clicking File > Save As, assign a name,and click Save.

7.

Click OK to exit the Protocol Editor.

Plate Setup

Negative Control

The SARS-CoV-2 Negative (#COV000) from Exact Diagnostics
is formulated in a synthetic matrix and contains genomic DNA. It
should be used as negative reference material when testing for
SARS-CoV-2. Add 10 μl per negative control reaction; extraction or
dilution should not be required.

1.

Click File > New > Plate in the menu bar to open the Plate
Editor.

2.

Select Settings > Plate Size > choose 96 Wells.

3.

Select Settings > Plate Type > choose BR White.

4.

Expand the dropdown menu to the right of Scan Mode andselect All Channels.

5.

Highlight the wells where samples and controls will be on theplate. To highlight all the wells, click the upper left corner ofthe plate graphic.

6.

Click Select Fluorophores and choose FAM, HEX, Texas Red,
Cy5,* and Cy5.5 by checking the box to the right of the selected
fluorophore (uncheck SYBR). Click OK to apply changes.

No Template Control (NTC)

This control can be validated by researchers to monitor forreagent and environmental contamination of the reactions. Use
10 µl of nuclease-free water per NTC reaction. A minimum of two
NTC wells per plate is required to analyze data using the allelicdiscrimination module in CFX Maestro Software.

© 2022 Bio-Rad Laboratories, Inc.

Time

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PrimePCR SARS-CoV-2 Multiple Mutation RT-qPCR Assay Using Reliance One-Step Multiplex RT-qPCR Supermix

7.

Suggested Reaction Setup

Define the sample type for each well by highlighting the wells,
then choosing the appropriate identifier from the Sample Type
dropdown menu.

1.

Note: If samples contain very low amounts of viral RNA,
consider adding a second set of control samples that contain
SARS-CoV-2 Variant Controls and the SARS-CoV-2 Standard
diluted in TE buffer at 1:100, and undiluted.
8.

Note: Reliance One-Step Multiplex RT-qPCR Supermix is
a 4x ready-to-use solution. The viscosity of this solution isgreater than a 2x mix. If using an automated liquid handlingsystem, allow the supermix to equilibrate at room temperaturefor 10 min to reduce its viscosity.

Apply target names and fluorophores to all wells byhighlighting the wells, then checking the Load box to the leftof each of the fluorophores listed in the Target Name section.
To include the target names, replace <none> in the open text
box to the right of each fluorophore. For example, if using
PrimePCR SARS-CoV-2 Multiple Mutation Assay 1:
FAM — 484K/Q
HEX — MPC
Texas Red — 501Y
Cy5 — 417N
Cy5.5 — 452R

9.

Thaw the Reliance One-Step Multiplex RT-qPCR Supermix
on ice. Mix thoroughly by vortexing the tube to ensure
homogeneity and resuspend any precipitates that may haveformed during storage. Centrifuge briefly to collect contents atthe bottom of the tube. Store on ice protected from light until
ready to use.

Save the file by clicking File > Save As.

10. Name the plate file and click Save.
11. Click OK to exit the Plate Editor (Figure 1).

2.

Thaw the PrimePCR SARS-CoV-2 Multiple Mutation Assayat room temperature. Mix thoroughly by vortexing the tube
to ensure homogeneity. Centrifuge briefly to ensure that allcomponents are at the bottom of the reaction tube. Store
protected from light.

3.

Thaw samples on ice. Place the SARS-CoV-2 Standard,
Negative, and Variant Controls (stored at 4°C) on ice. Mix
thoroughly by vortexing each tube to ensure homogeneity.
Centrifuge briefly to ensure that all components are at thebottom of each reaction tube.

* Assays utilize an ATTO 647 fluorophore, which has a similar excitation and emission
spectra to Cy5.

Fig. 1. Plate layout in CFX Maestro Software Plate Editor.

© 2022 Bio-Rad Laboratories, Inc.

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PrimePCR SARS-CoV-2 Multiple Mutation RT-qPCR Assay Using Reliance One-Step Multiplex RT-qPCR Supermix

Note: The controls are supplied at 200,000 copies/ml andcan be validated for use in these assays without dilution andwithout extraction. However, if samples contain very low
amounts of viral RNA (<300 copies/ml or have Cq values ≥30),
it is recommended to prepare diluted SARS-CoV-2 Variant
Controls (1:100) so that the control wells cross the threshold at
approximately the same cycle as the samples.
4.

Prepare a master mix volume sufficient for the number ofsamples and controls to be tested, plus 10% more to accountfor pipetting variability (Table 2). The volumes in Table 2 arebased on 10 μl of sample being added to a final volume of 20 μl.

3.

Change lower cycles to 5 and upper cycles to 45.

4.

Click OK to apply changes and exit.

5.

Select Settings > Baseline Setting > Apply Fluorescence
Drift Correction.

6.

Deselect the check boxes for HEX, Texas Red, Cy5, andCy5.5 below the amplification plot (only FAM should beselected).

7.

Select Log Scale by checking the box in the lower rightcorner of the amplification plot. Change the log scale to aminimum value, such as 0.5, so that the baseline signals arevisible.

8.

Click and drag the bold horizontal threshold line until it lies inthe exponential phase of the fluorescence curve and above
any baseline signal.

9.

Repeat steps 4–8, for each of the remaining fluorophores(HEX, Texas Red, Cy5, and Cy5.5).

Table 2. Example of preparation of master mix.
Volume per Volume for
Component
Reaction, µl n Samples, µl
Reliance One-Step Multiplex RT-qPCR Supermix 5 µl

= (5 x n) x 1.1

20x PrimePCR SARS-CoV-2 Multiple
Mutation Assay

1 μl= (1 x n) x 1.1

Nuclease-free water*

4 μl= (volume x n) x 1.1* Adjust volume to accommodate larger sample volume. We recommend using
10 μl of sample. In each 20 μl reaction, the volume of sample plus water should
equal 14 μl.

5.

To obtain a final volume of 20 μl, pipet 10 μl of master mix into
each well of a Hard-Shell 96-Well PCR Plate.

6.

Add 10 μl of RNA sample, reference control, variant control,
negative control, or nuclease-free water (no template control)to the appropriate well of the reaction plate for a final volumeof 20 μl. Adjust volume, if necessary.

7.

Seal the plate with Microseal 'B' PCR Plate Sealing Film andvortex to mix reagents. Centrifuge the plate briefly to ensurethat all components are at the bottom of the reaction tube.

8.

Load the PCR plate into the CFX96 Touch Real-Time PCR
System, select the plate and protocol files defined in earlier
steps, and start the run.

Confirm Expression for Multiplexed Control

MPC (HEX) can be an internal sample control. The probe hashomology to a highly conserved region between the primers.
Failure to amplify MPC may suggest incorrect reaction setup,
inadequate reagent quality, or insufficient intact RNA by the
researcher’s method. Alternatively, the sample may contain a new
mutation that impacts the ability of the probe to bind.

Suggested Data Analysis
Analysis should be performed in a three-step process. First, adjust
the baseline and threshold setting for each fluorophore. Second,
confirm that reactions were set up properly and that all samples
contain amplifiable RNA by assessing the MPC amplification curvein the HEX channel for each sample. Third, assess the presence orabsence of each mutation by determining whether the amplification
plot indicates exponential growth and crosses the threshold foreach fluorophore.
The baseline and threshold settings should be confirmed andmanually defined, if necessary, in the Quantification tab of CFX
Maestro Software.
All detection channels should be selected by default during
plate setup; confirm before proceeding to step 2.

2.

Select Settings > Cycles to Analyze.

© 2022 Bio-Rad Laboratories, Inc.

Select HEX by unchecking all other fluorophores in the boxes
under the amplification plot.

2.

Confirm that all wells identified by researchers as unknowns
cross the threshold before cycle 45 in the HEX channel.

3.

Exclude from analysis any well containing an unknown thatfails to cross the threshold before cycle 45 in the HEX channel.

Assess Presence or Absence of Mutations

Baseline-Threshold Setting

1.

1.

4

1.

Select FAM by unchecking all other fluorophores in the boxes
under the amplification plot.

2.

Identify unknowns that may contain the mutation associatedwith the FAM fluorophore by determining whether theamplification curves in the FAM channel display exponential
growth and cross the threshold before cycle 40.

3.

Repeat steps 1 and 2 for fluorophores Texas Red, Cy5 (ATTO
647), and Cy5.5 sequentially to identify unknowns that containmutations that encode the other targeted mutations.

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PrimePCR SARS-CoV-2 Multiple Mutation RT-qPCR Assay Using Reliance One-Step Multiplex RT-qPCR Supermix

Data Interpretation
Researchers can determine the likelihood that a sample contains
a variant of interest by considering which of the mutations areassessed as positive in the sample. The currently reported mostcommon SARS-CoV-2 variants of interest or concern that canbe identified using the PrimePCR SARS-CoV-2 Multiple Mutation
Assay are listed in Table 3. To determine the likelihood that asample contains a specific variant, researchers should identify
which fluorophore(s) cross the threshold before cycle 45 in asample, and then confirm the sample is positive for MPC. Data canbe compared to the information about currently reported variantsof interest or concern in Table 3. For example, using PrimePCR
SARS-CoV-2 Multiple Mutation Assay 1, if a sample is determined
to be positive for FAM (E484K/Q), Texas Red (N501Y), ATTO 647(K417N), and HEX (MPC), then the sample mutation profile may beconsistent with the Beta variant (B.1.351).
Visit bio-rad.com/SC2Variants for a complete list of multiple
mutation assays and data interpretation tables.

Table 3. Example of mutation fingerprint of SARS-CoV-2 variants of interest or concern for assay 1.
WHO Label

Pango Lineage

E484K/Q (FAM)

N501Y (Texas Red)

K417N (ATTO 647)

L452R (Cy5.5)

Alpha*

B.1.1.7

—

•

—

—

MPC (HEX)•

Beta

B.1.351

•

•

•

—

•

Gamma

P.1

•

—

—

—

•

Delta**

B.1.617.2

—

—

—

•

•

Epsilon**B.1.427/B.1.429

—

—

—

•

•

Kappa

B.1.617.1/B.1.617.3

•

—

—

•

•

OmicronB.1.1.529

—

•***

•

—

•

WHO, World Health Organization.
*	Some Alpha strains contain the E484K mutation, but the majority do not. Those that do contain the E484K mutation will have a mutation pattern similar to the Beta variant.
In such cases, the PrimePCR SARS-CoV-2 Single Mutation Assay targeting the D80A or A701V assay for Beta or the HV69-70 deletion assay for Alpha can be used byresearchers to further distinguish the viral strain present in the sample.
**	In this panel, it is not possible to distinguish the Delta variant from the Epsilon variant. To differentiate between these strains, the singleplex T478K assay can be used byresearchers.
*** Omicron has the N501Y mutation but it may not be detected due to other mutations in the region.
Additional PrimePCR SARS-CoV-2 Multiple Mutation Assays will become available as the virus undergoes additional mutations and a need to survey for novel mutations
arises. Visit bio-rad.com/SC2Variants for a complete list of assays available and evaluation of results.

Bio-Rad PCR Plastics, Reagents, and Equipment
Catalog #Description

MSB1001

Microseal 'B' PCR Plate Sealing Film, adhesive, optical,
pkg of 100
Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted,
white shell/white wellHard-Shell 96-Well PCR Plates, low profile, thin wall, skirted,
white shell/white well, barcoded
Reliance One-Step Multiplex RT-qPCR Supermix,
1,000 x 20 μl reactions
Reliance One-Step Multiplex RT-qPCR Supermix,
2,000 x 20 μl reactions

HSP9655
HSP9955
12010220
12010221

Visit bio-rad.com/SC2Variants for a complete list of PrimePCR
SARS-CoV-2 Mutation Assays and ordering information.
Visit bio-rad.com/PCRPlastics for a full range of PCR plastics
and seals.
Visit bio-rad.com/RelianceOneStep for more information.
Visit ExactDiagnostics.com for more information about thesecontrols. Q-1678 COVID-19 Quality Control Solutions contains acomplete list of SARS-CoV-2 controls.

Instruments
12011319
CFX Opus 96 Real-Time PCR System
1855195
CFX96 Touch Real-Time PCR System
Exact Diagnostics SARS-CoV-2 Reference Control (RUO)
COV019

Bio-Rad Technical Support
The Bio-Rad Technical Support department in the U.S. is open
Monday through Friday, 5:00 AM to 5:00 PM, Pacific time.

SARS-CoV-2 Standard

Exact Diagnostics SARS-CoV-2 Variant Mutation Controls (RUO)
COVA

SARS-CoV-2 S Gene Alpha Variant Control (B.1.1.7)

COVB

SARS-CoV-2 S Gene Beta Variant Control (B.1.351)

COVE

SARS-CoV-2 S Gene Epsilon Variant Control (B.1.427
and B.1.429)

COVG

SARS-CoV-2 S Gene Gamma Variant Control (P.1)

COVDK

SARS-CoV-2 S Gene Delta-Kappa Variants Control ​(Delta
B.1.617.2 and AY sublineages, Kappa B.1.617.1 and B.1.617.3)

Phone: 1-800-424-6723, option 2
Email: Support@Bio-Rad.com (U.S./Canada only)
For technical assistance outside the U.S. and Canada, contact
your local technical support office or click the Contact us linkat bio-rad.com.

Exact Diagnostics SARS-CoV-2 Negative Control (RUO)
COV000

BIO-RAD, HARD-SHELL, and MICROSEAL are trademarks of Bio-Rad Laboratories, Inc.
in certain jurisdictions. SYBR is a trademark of Thermo Fisher Scientific Inc. All trademarks
used herein are the property of their respective owner.

SARS-CoV-2 Negative

PrimePCR SARS-CoV-2 Multiple Mutation Assays
Refer to bio-rad.com/SC2Variants for assays and ordering information.

For research use only. Not for use in diagnostic procedures.

© 2022 Bio-Rad Laboratories, Inc.

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10000147102

PrimePCR SARS-CoV-2 Multiple Mutation RT-qPCR Assay Using Reliance One-Step Multiplex RT-qPCR Supermix

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