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

PrimePCR SARS-CoV-2 Single Mutation RT-qPCR Assays* can be validated by researchers totarget specific individual mutations identified in the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) genome believed to confer the evolutionary advantages of increased transmissibility
and the ability to escape immune response. These assays have been expertly designed against theoriginal SARS-CoV-2 genome derived from the strain first identified in Wuhan, China, using proprietarycomputational algorithms. They are intended to be validated by researchers for use in an allelicdiscrimination assay, an endpoint assay that detects down to a single nucleic acid sequence variation.
Assays include a primer set plus two probes: one probe (labeled with HEX) is a perfect match to thewild type, and the other probe (labeled with FAM) is a perfect match to the mutation. In addition, allassays contain primers and a probe to RPP40 (Cy5) as an RNA integrity and amplification control.
Each assay contains 18 µM primers and 5 µM of each probe in a ready-to-use single-tube format
and is optimal for use with the Reliance One-Step Multiplex RT-qPCR Supermix. Because it is anallelic discrimination assay, the actual quantity of the target sequence is not determined. Instead, thepresence of wild-type or variant mutation is determined by the researcher in comparing the endpoint
fluorescence values.
* For research use only (RUO). Not for use in diagnostic procedures.

Storage and Stability
The assays are stable for 12 months when stored at 4°C,
protected from light. The 20x assay mix can be kept at –20°C for
long-term storage.

■– SARS-CoV-2 S Gene Alpha Variant Control (B.1.1.7) (#COVA)– SARS-CoV-2 S Gene Beta Variant Control (B.1.351) (#COVB)

Product Contents
This product is a 20x concentrated, ready-to-use primer/probe mix
optimal for use with the Reliance One-Step Multiplex RT-qPCR
Supermix. It is provided as 200, 1,000, or 2,500 µl volumes,
sufficient for 200, 1,000, or 2,500 x 20 µl reactions.
Optimal Reagents and Equipment*■ 
Reliance One-Step Multiplex RT-qPCR Supermix(catalog #12010220, 12010221)■

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

Negative control: SARS-CoV-2 Negative (#COV000)* Bio-Rad reagents and equipment, unless otherwise noted. Reference, variant,and negative controls are from Exact Diagnostics, a Bio-Rad company.

10000143205

 ARS-CoV-2 Variant Controls for assays targeting spikeS
gene mutations:– 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)■ Blocks Gene Fragments can be validated for use as positivegcontrols for assays that target genes other than the spike gene.
The optimal gBlocks sequences can be found in the Assay
Control tab of each PrimePCR SARS-CoV-2 Single Mutation
Assay product, and can be ordered directly from Integrated DNA
Technologies, Inc.

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

 FX Opus 96, CFX Opus 384, CFX 96 Touch, or CFX 384
C
Touch Real-Time PCR System (#12011319, 12011452, 1855195,
or 1855485, respectively) or equivalent

which are research instruments equipped with five fluorescencechannels. For more detailed information, refer to the respective
instrument manual.

 ard-Shell 96-Well PCR Plates (#HSP9655, HSP9955),
H
Hard-Shell 384-Well PCR Plates (#HSP3805), or equivalent

Cycling Protocol Setup

1.

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

2.

Change Sample Volume to 20 μl.

The PrimePCR SARS-CoV-2 Single Mutation Assays amplify anddetect specific variants in purified genomic RNA samples.

3.

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

Reference Control

Table 1. Example of thermal cycling protocol.*■Microseal 'B' PCR Plate Sealing Film (#MSB1001)

Sample Preparation
Sample TypeThe 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.

Step Description

Time

Numberof Cycles

1

Reverse transcription

50

10 min1

2

DNA polymerase activation andtemplate denaturation95

10 min1

Template
denaturation95

10 secAnnealing/extension andplate read60

30 sec3
4

Positive Variant Controls

Temperature,°C

Amplification45* Set the lid temperature to 105°C.

The SARS-CoV-2 S Gene Variant Controls from Exact Diagnostics
are individually manufactured with synthetic RNA transcripts of the
S gene from current 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.

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.

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
Single Mutation Assay product, or the PrimePCR SARS-CoV-2
Single 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.

7.

Click OK to exit the Protocol Editor.

Negative Control

Plate Setup

1.

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

2.

Select Settings > Plate Size > choose 96 or 384 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.

No Template Control (NTC)

6.

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.

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

7.

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

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.

 ote: A minimum of two wells per mutation detection assay
Nmust be identified as NTC. These wells contain water insteadof sample. Reference, variant positive, and negative controls,
should also be identified as such. Samples should be
identified as Unknown.

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,© 2022 Bio-Rad Laboratories, Inc.

2

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

8.

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 as follows:
FAM — Mutant
HEX — WT
Cy5 — RP

9.

2.

Thaw all other components to room temperature. Mix
thoroughly by vortexing the tube to ensure homogeneity.
Centrifuge briefly to ensure that all components are at thebottom of the reaction tube. Store protected from light.

3.

Prepare samples and SARS-CoV-2 negative, reference, andvariant positive controls before setting up the reaction mix.

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. Scale volumes appropriately if using a 384-well plate
and a final volume of 10 μl.

Group the wells into separate well groups if more than one
variant assay is included on the plate.

10. Save the file by clicking File > Save As.
11. Name the plate file and click Save.

Table 2. Example of preparation of master mix.
Volume per Volume for
Component
Reaction, µl n Samples, µl

12. Click OK to exit the Plate Editor (Figure 1).
Suggested Reaction Setup
1.

Reliance One-Step Multiplex RT-qPCR Supermix

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.

5 µl

= (5 x n) x 1.1

20x PrimePCR SARS-CoV-2 Single Mutation Assay 1 μl

= (1 x n) x 1.1

Nuclease-free water*= (volume x n) x 1.1

4 µl* Adjust volume to accommodate larger sample volume. The volume of sample plus
water should equal 14 µl.

 ote: Reliance One-Step Multiplex RT-qPCR Supermix is aN
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.

5.

Pipet 10 µl of master mix into each well of a reaction plate.
Adjust the volume if accommodating a larger sample volume
or using a 384-well 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.

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

© 2022 Bio-Rad Laboratories, Inc.

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

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.

7.

Set the Cy5 threshold in the amplification plot by clicking anddragging the threshold line until it lies in the exponential phaseof the fluorescence curves and above any background signal.

8.

Load the PCR plate into the CFX System, select the plate andprotocol files defined in earlier steps, and start the run.

8.

Confirm that all wells identified as unknowns cross the threshold
before cycle 40 in the Cy5 channel. If an amplification curveof an unknown fails to cross the threshold before cycle 40,
the integrity or the amount of RNA in the sample may beinsufficient for that sample.

Suggested Data Analysis
Analysis should be performed in a two-step process. First, confirm
all samples contain amplifiable RNA and that the reactions were
set up correctly. Second, determine which allele is present byusing the allelic discrimination feature in CFX Maestro Software.

Determine Genotype Using Allelic Discrimination

The allelic discrimination function in CFX Maestro Software canbe used by researchers to determine which of the two variants
tested are present in the sample. The HEX fluorescent dye detector
is associated with the probe that is a perfect match to the wildtype allele. The FAM fluorescent dye detector is associated withthe probe that is a perfect match to the mutant allele. The allelicdiscrimination function plots the endpoint fluorescence signal ineach of the two channels for each well. Samples that contain themutation should have a high FAM signal relative to the HEX signal.
A dominant HEX signal in a well likely indicates a wild-type allele.
If multiple variant assays are present on a plate, assess each assay
individually by selecting specific well groups.

Confirm Expression of RNase P Control

RPP40 (Cy5) is an internal sample control. Failure to amplify thistarget suggests incorrect reaction setup, inadequate reagent
quality, or insufficient intact RNA in the sample by the researcher's
method. Use the Quantification tab to confirm the amplification of
the RPP40 target in each of the samples as follows.
1.

Select Settings > Cycles to Analyze. Enter 5 in the first cell
to replace the default setting of 1, and change the second cellto 45 as shown. Click OK to apply changes and exit.

2.

Select Settings > Baseline Setting > Apply Fluorescence
Drift Correction.

3.

Deselect FAM and HEX fluorophores by unchecking thecorresponding boxes under the amplification plot. Only theCy5 box should be selected.

4.

Select Log Scale by checking the box in the lower rightcorner of the amplification plot.

5.

Visually inspect the traces. Any well with amplification in the
Cy5 channel should show an exponential increase in relative
fluorescence units (RFU) values until the reaction plateaus.

6.

Manual baseline adjustment may be necessary if amplification
traces are not exponential. To manually define the baseline,
select Settings > Baseline Threshold. Highlight the well tobe adjusted, enter 2 in the Baseline Begin cell, and enter acycle number that is two cycles before the amplification trace
starts to rise in the Baseline End cell. Click OK to apply.

© 2022 Bio-Rad Laboratories, Inc.

4

1.

Click the Allelic Discrimination tab.

2.

Define axes using the dropdown menu in the SelectedFluorophores box located in the lower right quadrant of theallelic discrimination screen. Set X to FAM and Y to HEX toplace the mutant allele on the x-axis and the wild-type allele
on the y-axis. The selected cycle should be the last cycle ofthe thermal cycling protocol (endpoint).

3.

Confirm the NTC wells are correctly identified with black
diamonds in the lower left corner of the plot. These wells
should have only background fluorescence in both channels.
Significant RFU in either channel may indicate reagent or
sample contamination.

4.

Identify wells that have a high FAM and low HEX signal (orange
circles, Figure 2). These wells can be identified in the chart as
having an allele 1 call and contain the mutant allele.

5.

Identify wells that have a high HEX and low FAM signal (bluesquares, Figure 2). These wells can be identified in the chart
as having an allele 2 call and contain the wild-type allele.

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

6.

If a sample has a signal above background signal in both FAM
and HEX channels (NTC wells), then the well may be plotted asa green triangle and can be identified in the chart as having aheterozygote call. If a manual call is necessary, use the mouseto hover over the sample in the allelic discrimination plot, rightclick to access a pop-up menu, mouse over the well number,and select the desired call.

7.

Click View call map in the Selected Fluorophores box fora color-coded view of the plate layout.

Optimization Recommendations
The PrimePCR SARS-CoV-2 Single Mutation Assays andReliance One-Step Multiplex RT-qPCR Supermix cycling
protocols are optimal for assays with a predicted primer melting
temperature of 60°C. However, the protocol can be validated foruse by researchers in their laboratories. For example, if the RFU
differentiation between wild-type and mutant allele is suboptimal,
adjusting the annealing/extension temperature is suggested.
1.

Prepare a temperature gradient reaction plate using two
samples: one positive for the mutant allele and the othersample with a wild-type allele. Prepare a reaction mix for
at least 16 wells (two columns) according to Table 2.
Dispense the reaction mix into two columns. Add the wild-typeallele sample to one column and the mutant allele sample
to the other column.

2.

Change the thermal cycling protocol defined in Table 1 byreplacing the annealing/extension step with a thermal gradient.
Set the thermal gradient temperature to span 55–65°C.

3.

Run the plate set up in step 1.

4.

Identify the optimal annealing temperature based on signal
polarization: wells containing a sample with a mutant allele
should have a strong FAM signal relative to HEX and wells
containing wild-type samples should have a strong HEX signal
relative to FAM.

14

RFU for allele 2 (HEX)

12
10
8
6
4
2
0
0

1,000

2,000
3,000
4,000
5,000
RFU for allele 1 (FAM 103)

6,000

Fig. 2. Allelic discrimination data plot. Mutant allele ( ); wild-type allele ( );NTC ( ). RFU, relative fluorescence units.

Generate a Printable Report

1.

Select Tools > Reports.

2.

Select the output report in the tree window located in theupper left quadrant of the report screen. The optimal output
is shown.

3.

Customize the report details by assigning a Title, Sub-Header
Title, and alignment in the Header box located in the bottom
left quadrant.

	
Note: If this section is grayed out, select the Notes option in
the tree window under Header.
4.

Click Update Report.

5.

Select File > Print or Save Report.

© 2022 Bio-Rad Laboratories, Inc.

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

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

MSB1001

Microseal 'B' PCR Plate Sealing Film, adhesive, optical,
pkg of 100

HSP9655

Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted,
white shell/white well

HSP9955

Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted,
white shell/white well, barcoded

HSP3805

Hard-Shell 384-Well PCR Plates, low profile, thin wall, skirted,
clear shell/white well12010220

Reliance One-Step Multiplex RT-qPCR Supermix,
1,000 x 20 µl reactions

12010221

Reliance One-Step Multiplex RT-qPCR Supermix,
2,000 x 20 µl reactions

Instruments
12011319

CFX Opus 96 Real-Time PCR System

12011452

CFX Opus 384 Real-Time PCR System

1855195

CFX96 Touch Real-Time PCR System

1855485

CFX384 Touch Real-Time PCR System

Exact Diagnostics SARS-CoV-2 Reference Control (RUO)
COV019SARS-CoV-2 Standard

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

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.
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.
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 link at
bio-rad.com.

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)

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.

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)

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

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

SARS-CoV-2 Negative

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

Bio-RadLaboratories, Inc.
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10000143205 Ver C

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