Real-Time PCR

A qPCR Comparability Study to Demonstrate the Analytical Equivalenceof CFX96 Deep Well Dx and CFX Opus Deepwell Dx Systems

Srikanth Perike, Malekah Isa, Jacqueline Gonzalez Rubio, Angelica Olcott, Candice Cox, and Elizabeth Jordan Dreskin
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547

Abstract

A comparability study is intended to provide analytical evidence that transitioning from one platform
to a different one will not adversely affect an approved process. Prior bridging studies have shown
comparability between the Bio-Rad Laboratories, Inc. research use only (RUO) CFX96 Touch and
CFX Opus 96 Real-Time PCR Systems* (Woo et al. 2021). Here we designed studies to evaluate thenewer CFX Opus Deepwell Dx Real-Time PCR System and its equivalency to the CFX96 Deep Well Dx
Real-Time PCR System. Specific performance criteria were examined, including specificity, linearity,
range, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), robustness, and system
suitability for a quantitative PCR (qPCR) application. The purpose of this study is to guide users who plan
to transition their process from the CFX96 Deep Well Dx System to the CFX Opus Deepwell Dx System.
Introduction
The CFX Opus 96 Dx, CFX Opus 384 Dx, and CFX Opus Deepwell
Dx Real-Time PCR Systems from Bio-Rad provide a robust andreliable open platform for the development and performance of invitro diagnostic (IVD) assays. The family of CFX Opus Dx Systems
offers exceptional thermal uniformity, providing sensitive andprecise detection and quantification, and can perform multiplexed
detection of up to five targets per well for 96-well formats and fourtargets per well for 384-well formats. The format of the CFX Opus
Deepwell Dx System is designed for the detection of DNA and RNA
using either standard 20 µl volume or larger volume applications,
up to 125 μl. Unlike the previous CFX96 Dx and CFX96 Deep
Well Dx Systems, which use CFX Manager Dx Software, the CFX
Opus Deepwell Dx System uses the more recent CFX Maestro
Dx SE Software. This updated software provides easy setup,
run monitoring, data analysis, and report generation, including
traceable audit-ready reports to ensure the integrity of patientresults. The purpose of this comparability study is to demonstratethat the new CFX Opus Deepwell Dx System performs equivalently
to the previous CFX96 Deep Well Dx System.

To perform this qPCR comparability study, the guidelines set out
by the United States Pharmacopeia Convention (USP, 2017) andthe U.S. Food and Drug Administration (FDA, 2021) were followed.
In defining the acceptance criteria for transferring molecular assays
to the newer CFX Opus Deepwell Dx System, we referred to Gurtler
et al. (2018) and Bio-Rad's earlier bridging study (Woo et al. 2021).
Experiments were conducted using serial dilutions of SARS-CoV-2
in vitro transcribed (IVT) RNAs and detection by a SARS-CoV-2
triplex diagnostic Emergency Use Authorization (EUA) assay. Data
analysis was performed using the two different diagnostic software
packages and the performance of the CFX96 Deep Well Dx and
CFX Opus Deepwell Dx Systems were shown to be comparable.
This study is intended to guide diagnostic users who are interested
in transitioning from the CFX96 Deep Well Dx System to the new
CFX Opus Deepwell Dx System. Diagnostics users will need to use
a 510(k) cleared kit on an instrument platform that is cleared by
the FDA.

* Comparability studies also performed for CFX384 Touch and CFX Opus 384 Real-Time PCR Systems.

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A qPCR Comparability Study to Demonstrate the Analytical Equivalence of CFX96 Deep Well Dx and CFX Opus Deepwell Dx Systems

Experimental Design
The Bio-Rad Reliance SARS-CoV-2 RT-PCR Assay Kit, an EUA kit,has been validated for use on the Bio-Rad CFX96 Dx System.* This
reverse transcription quantitative PCR (RT-qPCR) comparability
study was designed (Figure 1), first, to show that the Reliance
SARS-CoV-2 Assay Kit provides equivalent performance on the
CFX96 Dx System and the CFX96 Deep Well Dx System. Second,
the analytical equivalency of the CFX96 Deep Well Dx Systemwith the newer CFX Opus Deepwell Dx System was compared.
As in the recent comparability study of the CFX Opus 96 and
CFX Opus 384 Systems (Woo et al. 2021), the key performance
characteristics measured here for a total of nine systems werelinearity (R2), range, accuracy, precision, LOD, LOQ, robustness,and system suitability. The detailed definitions for each of theseanalytical characteristics are provided in Appendix A and thecharacteristics must be tested by diagnostic labs intending to add
the CFX Opus Deepwell Dx System to existing SOPs approved foruse with other real-time PCR systems.

For the bridging study (Figure 1) between the CFX96 Deep Well
Dx and CFX Opus Deepwell Dx Systems, on each of three days,
standard curves were prepared and tested simultaneously on threeunits of each platform. Testing was carried out such that each day
a different CFX96 Deep Well Dx System was paired with a different
CFX Opus Deepwell Dx System (see Table 1).
CFX Manager Dx Software version 3.1 was used to run andanalyze assay performance experiments on the CFX96 Dx
and CFX96 Deep Well Dx units. For the CFX Opus Deepwell
Dx bridging study, CFX Maestro Dx SE Software was used tooperate the CFX Opus Deepwell Dx System, and to analyze both
the CFX96 Deep Well Dx and CFX Opus Deepwell Dx data files.
Regression analysis was also performed on the linear range ofquantification cycles (Cq) values obtained for the CFX96 Deep
Well Dx System in both software packages and a high correlation
coefficient (r = 1) was obtained (Supplementary Figure 1).
The acceptance criteria for the runs in this study had to meet thefollowing attributes and were aligned with those described in theprior comparability study between the CFX Touch and CFX Opus
Real-Time PCR platforms (Woo et al. 2021):■

CFX96 Dx

CFX96 Deep Well Dx

CFX Opus Deepwell Dx

Assay Performance■

Instrument Performance
Evaluation of Assay Performance■Detection capabilities for the N1 and N2 regions of the SARS-CoV-2 N gene
– Linearity– PCR efficiency– LOD and LOQ■
Equivalence (Bland-Altman plots)■
Deming regression and bias estimation■■

Evaluation of Instrument Precision andReproducibility■
Intraday comparisons■
Intrasystem comparisons fficiency between 90 and 110%, and linearity requirements
Eused an R2 greater than or equal to 0.99
 ll 4 replicates of the last dilution must cross the threshold withA
a standard deviation (SD) less than 0.6 to be included in thelinearity and efficiency calculations
 o calculate the LOD at 95%, the criteria specified that allT
4 replicates of the last dilution, or 100% of all replicates, mustcross the threshold q for no template controls (NTCs) must be at Cq greater thanC
40. However, if any NTCs are positive, the Cq must not be lessthan the Cq of the lowest standard concentration (Cq < 36)

Combined, these acceptance criteria also define system suitability.

Evaluation of Data Analysis■
System usability, data comparisons using
CFX Maestro Dx SE Software

Materials and Methods
Real-Time PCR Assay for the Comparability Study

The Reliance SARS-CoV-2 RT-PCR Assay Kit (Bio-Rad, catalog#12014115), a triplex EUA RT-qPCR assay, was used to 1)
demonstrate assay performance on the CFX96 Deep Well Dx
System, and 2) evaluate the analytical performance of the
CFX Opus Deepwell Dx System compared to the CFX96 Deep
Well Dx System. The SARS-CoV-2 RT-PCR Kit was designed forspecific detection of the SARS-CoV-2 nucleocapsid gene at two
regions, termed N1 and N2 using the same primer/probe sets
reported by the Centers for Disease Control and Prevention (CDC,#2019-nCoVEUA-01). An additional primer/probe set was used todetect the human RNase P (RP) gene.

Fig. 1. Schematic representation of experimental plan for determining assay
and instrument performance of CFX96 Dx, CFX96 Deep Well Dx, and CFX
Opus Deepwell Dx Systems. For the Reliance SARS-CoV-2 RT-PCR Assay Kit,
assay performance had already been validated for standard volume formats on
the CFX96 Dx System but not on the CFX96 Deep Well Dx System. Therefore, theprocess of transferring the assay to a deep well format (CFX96 Deep Well Dx System)
was evaluated first. Once assay performance had been demonstrated in the deep
well format, the assay was used to evaluate comparability between the CFX96 Deep
Well Dx and CFX Opus Deepwell Dx Systems by measuring instrument precisionand reproducibility. Finally, data were analyzed to demonstrate equivalence in thecalculations provided by CFX Manager Dx and CFX Maestro Dx SE Software. The
evaluations were performed across three days with three systems per platform.
LOD, limit of detection; LOQ, limit of quantitation.

Assay performance between the standard volume and deep well
assay format (Figure 1) was determined using three instruments
each of the CFX96 Dx and CFX96 Deep Well Dx Systems, withthree standard curves, each completed on three different days.
The evaluation included replicate plates prepared and run each day.
* Reliance SARS-CoV-2 RT-PCR Assay Kit also validated on CFX Opus 96, CFX96 Touch, CFX Opus 384, and CFX384 Touch Systems.
© 2023 Bio-Rad Laboratories, Inc.

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A qPCR Comparability Study to Demonstrate the Analytical Equivalence of CFX96 Deep Well Dx and CFX Opus Deepwell Dx Systems

Threefold serial dilutions of Exact Diagnostics SARS-CoV-2 IVT
RNA (Bio-Rad, custom product) were made in Tris EDTA buffer(TE) containing 5 ng/ml yeast tRNA (Thermo Fisher Scientific Inc.,#AM7119). A constant input of 5 ng human genomic DNA (gDNA)(Takara Bio USA, Inc., #636401) was added to each 20 μl reaction
as an assay control that detects the human RNase P gene. All
RT-qPCR reactions were performed in 20 µl, according to the
SARS-CoV-2 RT-PCR Assay Kit instructions with the following
exception: 10 μl of SARS-CoV-2 IVT RNA serial dilution, instead ofextracted RNA sample, was added per well. For each individual
experiment, reactions were run in quadruplicates on white wellHard-Shell™ 96-Well PCR Plates (Bio-Rad, #HSP9655) and sealedwith Microseal™ 'B' PCR Plate Sealing Film (Bio-Rad, #MSB1001).

set up according to the kit protocol. The Exact Diagnostics SARSCoV-2 Standard (Bio-Rad, #COV019) and SARS-CoV-2 Negative(Bio-Rad, #COV000) with human genomic DNA background wereused as controls for the assay. The following reverse transcription
PCR (RT-PCR) protocol was used to run all ddPCR experiments:
50ºC for 10 min, followed by enzyme activation at 95ºC for 10 min,followed by 40 cycles of 94ºC for 30 sec, 55ºC for 60 sec, enzyme
deactivation at 98ºC for 10 min, and droplet stabilization at 4ºC
for 30 min. The droplets were read on the QX200™ Droplet
Reader (Bio-Rad, #1864003). Copy number was determinedusing QuantaSoft™ Analysis Pro Software (Bio-Rad, download),
refer to the Bio-Rad SARS-CoV-2 ddPCR Kit Instructions for Use
12013769 for more information.

The following RT-qPCR protocol was run for all experiments:
50ºC for 10 min, followed by inactivation and activation at 95ºC for
10 min, followed by 45 cycles of 95ºC for 10 sec, 60ºC for 30 sec.
Instrument operation and data analysis were performed using
Bio-Rad CFX Manager Dx Software for the CFX96 Dx and CFX96
Deep Well Dx Systems, whereas CFX Maestro Dx SE Software
was used for CFX Opus Deepwell Dx Systems. The PCR efficiency
and Cq values were obtained by setting the threshold for eachfluorophore at 10% of the maximum relative fluorescence units(RFU). The data were exported to Excel 2022 and analyzed with
the Analyse-it Method Validation Edition version 6.15 (Analyse-it
Software, Ltd.).

Results
For this study, because the Reliance SARS-CoV-2 RT-PCR Assay
Kit was approved under EUA for the CFX96 Dx System, we first
had to demonstrate that the kit showed comparable performanceon the CFX96 Deep Well Dx System. Following that, we were able
to use that assay to compare the CFX96 Deep Well Dx with the
CFX Opus Deepwell Dx System.
To ensure the platforms were all performing as expected, theintrasystem repeatability of the CFX96 Dx, CFX96 Deep Well Dx,
and CFX Opus Deepwell Dx instruments was studied. Deming
regression analysis was performed on the Cq values obtained fromthe six-point dilutions curves for SARS-CoV-2 N1 (FAM channel)
and N2 targets (HEX channel). Intrasystem equivalence wasobserved based on the correlation coefficients of r = 0.99 for both
FAM and HEX channels in all CFX Opus Deepwell Dx, CFX96 Dx,
and CFX96 Deep Well Dx Systems (Table 1, Supplementary Figures
2 and 3).

Droplet Digital™ PCR (ddPCR™) Assay for Confirming LOD/LOQ

The SARS-Cov-2 ddPCR Kit (Bio-Rad, #12013743) containing
the 2019-nCoV CDC ddPCR Triplex Probe Assay (Bio-Rad,#12008202) was used to determine copy number for LOD, LOQ,
and linear range. The oligonucleotide primers and probes for
the SARS-CoV-2 ddPCR Assay were specific for detecting theN1 and N2 gene regions as reported by the CDC. An additional
primer/probe set was used to detect the human RNase P gene inexperimental and control samples. The droplets were preparedusing the Automated Droplet Generator (Bio-Rad, #1864101), andamplification was on a C1000 Touch Thermal Cycler with 96–DeepWell Reaction Module (Bio-Rad, #1851197). Reactions of 20 µl were

The Bland-Altman test was also used to demonstrate equivalence
between systems of the same platform and estimate the bias.
Representative results are shown with the predicted 95% limit ofagreement (LOA) between systems (Figure 2), and full datasets ofBland-Altman LOA plots for all intrasystem comparisons are also
provided (Supplementary Figures 4 and 5).

Table 1. Equivalence of three systems for each platform. Deming regression analysis of data from CFX96 Dx, CFX96 Deep Well Dx, and CFX Opus Deepwell Dx
Systems in the FAM and HEX detection channels for the two targets, N1 and N2.
Real-Time PCR System
Intrasystem Comparison
Fluorophore
Correlation, rSlope
Confidence IntervalIntercept
CFX96 Dx
A vs. B
FAM
0.997
1.016
0.968–1.064–0.584
CFX96 Dx
B vs. C
FAM
0.993
0.982
0.910–1.055
0.889
CFX96 Dx
A vs. C
FAM
0.987
0.997
0.897–1.096
0.382
CFX96 Deep Well Dx
A vs. B
FAM
0.999
0.999
0.969–1.029
0.023
CFX96 Deep Well Dx
B vs. C
FAM
0.999
1.012
0.985–1.039–0.289
CFX96 Deep Well Dx
A vs. C
FAM
0.999
1.012
0.984–1.039–0.265
CFX Opus Deepwell Dx
A vs. B
FAM
0.999
0.996
0.959–1.032
0.090
CFX Opus Deepwell Dx
B vs. C
FAM
1.000
1.007
0.990–1.023–0.241
CFX Opus Deepwell Dx
A vs. C
FAM
0.999
1.002
0.968–1.036–0.151
CFX96 Dx
A vs. B
HEX
0.995
1.019
0.966–1.071–0.718
CFX96 Dx
B vs. C
HEX
0.992
1.005
0.937–1.072
0.008
CFX96 Dx
A vs. C
HEX
0.995
0.986
0.917–1.053
0.805
CFX96 Deep Well Dx
A vs. B
HEX
0.998
1.014
0.973–1.054–0.342
CFX96 Deep Well Dx
B vs. C
HEX
0.998
0.998
0.966–1.028
0.179
CFX96 Deep Well Dx
A vs C
HEX
0.998
1.012
0.968–1.055–0.164
CFX Opus Deepwell Dx
A vs. B
HEX
0.998
1.025
0.981–1.069–0.642
CFX Opus Deepwell Dx
B vs. C
HEX
0.998
0.966
0.923–1.008
0.912
CFX Opus Deepwell Dx
A vs. C
HEX
0.999
0.990
0.965–1.015
0.301© 2023 Bio-Rad Laboratories, Inc.

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A qPCR Comparability Study to Demonstrate the Analytical Equivalence of CFX96 Deep Well Dx and CFX Opus Deepwell Dx Systems

B. CFX96 Deep Well Dx, FAM

26

28

30
32
Mean Cq FAM,
(CFX96 Dx A + B)/2

34

36

29

28

30
32
34
Mean Cq FAM,
(CFX96 Deep Well Dx A + B)/2

31
33
Mean Cq HEX,
(CFX96 Dx A + B)/2

35

37

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

29

Mean (–0.047)

26

28

30
32
34
Mean Cq FAM,
(CFX Opus Deepwell Dx A + B)/2

36

F. CFX Opus Deepwell Dx, HEX

Mean (0.102)

27

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

36

E. CFX96 Deep Well Dx, HEX

Mean (–0.114)

27

C. CFX Opus Deepwell Dx, FAM

Mean (0.009)

26

ΔCq HEX,
CFX96 Deep Well Dx B – A

ΔCq HEX,
CFX96 Dx B – A

D. CFX96 Dx, HEX
1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

ΔCq FAM,CFX Opus Deepwell Dx B – A

Mean (–0.087)

ΔCq HEX,CFX Opus Deepwell Dx B – A

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

Δ Cq FAM,
CFX96 Deep Well Dx B – A

ΔCq FAM,
CFX96 Dx B – A

A. CFX96 Dx, FAM

31
33
35
Mean Cq HEX,
(CFX96 Deep Well Dx A + B)/2

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

37

Mean (0.164)

27

29

31
33
35
Mean Cq HEX,
(CFX Opus Deepwell Dx A + B)/2

37

Fig. 2. Demonstration of equivalence among systems of the same platform type. Individual systems selected to represent each platform using Bland-Altman LOA plots
for pairwise comparisons between two CFX96 Dx Systems A, FAM and D, HEX channels; two CFX96 Deep Welll Dx Systems B, FAM and E, HEX channels; two CFX Opus
Deepwell Dx Systems C, FAM and F, HEX channels. Dashed blue lines represent the 95% LOA between units of each platform, and the solid line represents the mean. The
mean is an estimate of the average bias between instruments. Cq, quantification cycle; LOA, limit of agreement.

The acceptance criteria for specificity required that NTCs must notcross the threshold, or must have a Cq value greater than or equal
to 40; no NTCs crossed the threshold in any experiment within
the CFX96 DX, CFX96 Deep Well Dx, or CFX Opus Deepwell Dx
Systems tested.

Mean Cq FAM, CFX96 Deep Well Dx

AAssay performance was first determined on the CFX96 Dx and
CFX96 Deep Well Dx Systems using the Bio-Rad Reliance SARSCoV-2 RT-Kit under EUA. As described in the experimental design,
three units of each platform were tested over three days. Deming
regression analysis was used to calculate the repeatability correlation
coefficients between the two instrument platforms for all Cq
measurements in the FAM and HEX detection channels (Figure 3).
The correlation coefficients of r = 0.99 for the plots shown in Figure
4A and 4B suggest very similar assay performance on the CFX96
Dx and CFX96 Deep Well Dx Systems. Additional data are includedin Supplementary Table 1.

Deming fit(y = –0.001859 + 0.9963x)

36
34
32
30
28

r = 0.996

26
26

Mean Cq HEX, CFX96 Deep Well Dx

B

Next, the analytical characteristics between the CFX96 Deep
Well Dx and CFX Opus Deepwell Dx Systems were evaluated forequivalency using a six-point standard curve, which resulted in
Cq values of approximately 26–36 Cq on both platforms. Table 2
shows linearity, PCR efficiency, range, LOD, and LOQ for detectionof the SARS-CoV-2 N1 and N2 targets in assays run on the CFX96
Deep Well Dx and CFX Opus Deepwell Dx Systems. Linearities (R2)and PCR efficiencies (%) were observed within acceptable limits(R2 ≥ 0.99 and 90–110% respectively) and were almost identicalbetween platforms (Table 2).

© 2023 Bio-Rad Laboratories, Inc.

38

39

28
30
32
34
36
Mean Cq FAM, CFX96 Dx

38

Deming fit(y = 0.01247 + 0.9921x)

37
35
33
31
29

r = 0.997

27
27

29
31
33
35
37
Mean Cq HEX, CFX96 Dx

39

Fig. 3. Deming regression analysis between CFX96 Dx and CFX96 Deep Well
Dx Systems. Similar assay performance for CFX96 Dx and CFX96 Deep Well Dx
Systems based on correlation of mean Cq measurements. Triplicate measurementsof three independent runs over three days on three systems of each platform wereanalyzed and the results plotted as mean Cq results for CFX96 Dx System (x-axis)
versus CFX96 Deep Well Dx System (y-axis) for the FAM channel (A), the HEX
channel (B), and Deming fit plotted ( ) as shown. Cq, quantification cycle.
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Table 2. Summary of analytical characteristics for CFX96 Deep Well Dx and CFX Opus Deepwell Dx Systems. LOD, LOQ, linear range, linearity and efficiency on
CFX96 Deep Well Dx and CFX Opus Deepwell Dx Systems, average of three systems on three days. LOD, limit of detection; LOQ, limit of quantification.
Paramater
AnalyteFluorophore
CFX96 Deep Well Dx
CFX Opus Deepwell Dx
LOD, copies
SARS-CoV-2 N1
FAM
3.81
3.81
SARS-CoV-2 N2
HEX
3.21
3.21
LOQ, copies
SARS-CoV-2 N1
FAM
11.01
11.01
SARS-CoV-2 N2
HEX
6.11
6.11
Linear range, molecules
SARS-CoV-2 N1
FAM
8,026–11.01
8,026–11.01
SARS-CoV-2 N2HEX4,454–6.11
4,454–6.11
Linear range, Cq
SARS-CoV-2 N1
FAM
26.51–34.94
26.61–35.01
SARS-CoV-2 N2HEX
27.58–36.20
27.59–36.33
Linearity, R2
SARS-CoV-2 N1
FAM
0.996
0.997
SARS-CoV-2 N2
HEX
0.997
0.996
Efficiency, %SARS-CoV-2 N1
FAM
93.12
92.87
SARS-CoV-2 N2
HEX
90.66
90.35

To evaluate the analytical equivalence of the CFX96 Deep Well Dx
and CFX Opus Deepwell Dx Systems, six serial dilutions of SARSCoV-2 IVT RNA were prepared and run in replicate plates on threeof each instrument type on each of three days. Consistent detectionof six points was observed on the standard curve in both the FAM
channel targeting SARS-CoV-2 N1 and the HEX channel targeting
SARS-CoV-2 N2 on all systems tested in this study (Figure 5),demonstrating linearity. We conclude that the CFX96 Deep Well Dx
and CFX Opus Deepwell Dx Systems performed similarly to eachother in this study.

Range, LOQ, and LOD for N1 (FAM) and N2 (HEX) targets were also
determined to be equivalent between the CFX96 Deep Well Dx and
CFX Opus Deepwell Dx Systems (Table 2). These results support
comparable analytical characteristics between the CFX96 Dx and
CFX Opus Dx platforms.
Deming regression analysis was used to compare system
performance. As shown in Figure 4, the correlation coefficient
between mean Cq of CFX96 Deep Well Dx and CFX Opus
Deepwell Dx Systems had an excellent correlation value (r = 0.99),
demonstrating the equivalence of CFX96 Deep Well Dx and
CFX Opus Deepwell Dx Systems.

A. CFX96 Deep Well Dx
38
36

38

Deming fit(y = 0.02222 + 1.002x)

34

Mean Cq HEX, CFX Opus Deepwell Dx

32
30
28

32

26
24

30

3.0

3.5

4.0

28

39

6.0

6.5

6.0

6.5

38

28
30
32
34
36
38
Mean Cq FAM, CFX96 Deep Well Dx

36
34

Deming fit(y = –0.4504 + 1.015x)

37

4.5
5.0
5.5
log starting quantity

B. CFX Opus Deepwell Dx

r = 0.998

26
26

B

34
Cq

36

Cq

Mean Cq FAM, CFX Opus Deepwell Dx

A

32
30
28

35

26
24

33

3.0

31

r = 0.996
27

4.0

4.5
5.0
5.5
log starting quantity

Fig. 5. Demonstration of multiplex qPCR assay performance on A, CFX96
Deep Well Dx; B, CFX Opus Deepwell Dx Systems. Linearity of detection for the
SARS-CoV-2 nucleocapsid gene regions N1 [FAM ( )] and N2 [HEX ( )].
Cq, quantification cycle.

29
27

3.5

29
31
33
35
37
39
Mean Cq HEX, CFX96 Deep Well Dx

Fig. 4. Deming regression analysis between CFX96 Dx Deep Well and CFX
Opus Deepwell Dx Systems. Quadruplicate measurements of three independentruns over three days and with three instruments of each platform were analyzed.
Mean Cq for CFX96 Deep Well Dx Systems were plotted against CFX Opus Deepwell
Dx Systems for FAM channel (A), HEX channel (B), and Deming fit plotted ( ) as
shown. Cq, quantification cycle.

© 2023 Bio-Rad Laboratories, Inc.

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Table 3. Precision of CFX96 Deep Well Dx and CFX Opus Deepwell Dx Systems calculated from one input. Standard deviation results are within the 95% CI of theinter-run and interday measurements.
Inter-Run

InterdayIntermediate

Platform

Fluorophore

AnalyteSD

95% CISD

95% CISD

CFX96 Deep Well Dx

FAM

SARS-CoV-2 N1

0.300

0.240–0.398

0.302

0.251–0.436

0.312

HEX

SARS-CoV-2 N2

0.146

0.116–0.199

0.165

0.135–0.267

0.183

CFX Opus Deepwell Dx FAM

SARS-CoV-2 N1

0.053

0.041–0.073

0.061

0.049–0.110

0.129

HEX

SARS-CoV-2 N2

0.081

0.064–0.110

0.111

0.087–0.201

0.241

CI, confidence interval; SD, standard deviation.

Conclusions
Demonstrations of equivalent performance are critical to specificuser environments. This series of comparability studies was
conducted to show the analytical equivalence of the CFX Opus
Deepwell Dx System with the CFX96 Deep Well Dx System andused performance characteristics described in the USP (2017) andInternational Conference on Harmonisation (ICH, 2005) guidelines.
This study is intended to provide a guide to laboratories whowish to transfer assays from the CFX96 Deep Well Dx System to
the CFX Opus Deepwell Dx System or use these two platforms
together. If differences are seen when transferring protocols to new
platforms, additional system optimization may be required.

References
Gurtler C et al. (2018). Transferring a quantitative molecular diagnostic test to
multiple real-time quantitative PCR platforms. J Mol Diag 20, 398–414.
International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH) (2005). Validation of Analytical
Procedures: Text and Methodology Q2(R1). https://www.ich.org/page/qualityguidelines, accessed July 2, 2023.
United States Food and Drug Administration (2021). Document FDA-2017-D-6821;
Q2(R1) Validation of Analytical Procedures: Text and Methodology Guidance forIndustry. www.fda.gov/media/152208/download. Accessed July 2, 2023.
United States Food and Drug Administration Office of Regulatory Affairs (2020).
Document ORA-LAB.5.4.5: Methods, Method Verification and Validation,
Laboratory Manual Volume II. https://www.fda.gov/media/73920/download,
accessed July 2, 2023.
United States Pharmacopeial Convention (2017). Validation of Compendial Methods.
http://www.uspbpep.com/usp29/v29240/usp29nf24s0_c1225.html, accessed
July 2, 2023.

The study results demonstrate comparable performance
between the new CFX Opus Deepwell Dx System and the CFX96
Deep Well Dx System. An investigation of intrasystem variation
between instruments of the same platform type (CFX96 Dx,
CFX96 Deep Well Dx, and CFX Opus Deepwell Dx), conductedby both Deming regression analysis and Bland-Altman LOA plots,
demonstrated that all units performed equivalently. To evaluate
assay performance, a multiplexed RT-qPCR assay for the detectionof SARS-CoV-2 was shown to perform similarly on both the CFX96
Dx and CFX96 Deep Well Dx Systems. Comparisons of the CFX96
Deep Well Dx and CFX Opus Deepwell Dx Systems for analyticalcharacteristics determined that the PCR efficiency, linearity, LOD,
LOQ, and linear range of Cq values are all equivalent between thetwo platforms. The study also included repeatability and precision
analyses (Table 3) to investigate any variations occurring between
PCR runs performed over the three consecutive days of thiscomparability study, and no differences were detected. Finally,
the analysis of CFX96 Deep Well Dx data in CFX Maestro Dx SE
Software and CFX Manager Dx Software yielded identical results(Supplementary Figure 1), demonstrating the ability to provide
equivalent results.

Woo et al. (2021). A model qPCR system comparability study: CFX Touch and
CFX Opus Real-Time PCR Platforms. Bio-Rad Bulletin 7444.

Users of CFX96 Deep Well Dx Systems can be confident thatwhen performing their own comparability study, they can expect
to obtain similar results on the CFX Opus Deepwell Dx System.

© 2023 Bio-Rad Laboratories, Inc.

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Appendix A: Definition of Characteristics
The following definitions were compiled from ICH (2005),
USP (2017), and U.S. FDA (2020) guidelines.

Range — the interval between the upper and lower concentrationof analyte in a sample for which it has been demonstrated thatthe analytical procedure has an acceptable level of accuracy,
precision, and linearity.

Accuracy — the nearness of a result or the mean of a set ofmeasurements to the true value.

Robustness — an analytical procedure’s capacity to remain
unaffected by small but deliberate variations in method parameters.
It provides an indication of the procedure’s reliability during
normal usage.

Limit of detection (LOD) — the lowest concentration level thatcan be determined as statistically different from a blank at a
specified level of confidence. It is determined from the analysis
of sample blanks.

Specificity — the ability to assess unequivocally an analytein the presence of impurities, degradation products, or othercomponents that may be present.

Limit of quantification (LOQ) — the level above whichquantifiable results may be determined with acceptableaccuracy and precision.

System suitability — system suitability testing is an integral partof many analytical procedures. The tests are based on the conceptthat the equipment, electronics, analytical operations, and samples
to be analyzed constitute an integral system that can be evaluatedas such. System suitability test parameters to be established fora particular procedure depend on the type of procedure being
validated. See pharmacopeias for additional information.

Linearity — the ability of a method to elicit results that are
directly proportional to analyte concentration within a given range.
Precision — agreement between a set of replicate measurements.
Precision does not necessarily refer to the true value. The precisionof test results is described by statistical methods, such as astandard deviation or confidence limit. Repeatability expresses
precision under the same operating conditions over a shortperiod of time. Intermediate precision expresses precision withinlaboratory variations, such as different days, different analysts,
and different equipment. Reproducibility expresses the precision
between laboratories.
Appendix B: Supplementary Data
38
36

B
Deming fit(y = 0 + 1x)

34
32
30
28
r=1

26

26
28
30
32
34
36
38
Mean Cq FAM, CFX96 Deep Well Dx with
CFX Maestro Dx SE Software

Mean Cq HEX, CFX 96 Deepwell Dx
with CFX Manager Dx Software

Mean Cq FAM, CFX 96 Deepwell Dx
with CFX Manager Dx Software

A

38
36

Deming fit(y = 0 + 1x)

34
32
30
28
r=1

26

26
28
30
32
34
36
38
Mean Cq HEX, CFX96 Deep Well Dx with
CFX Maestro Dx SE Software

Suppl. Fig 1. System usability. Comparison of CFX96 Deep Well Dx System data using CFX Manager
Dx and CFX Maestro Dx SE Software. Cq, quantification cycle.

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Suppl. Table 1. Summary of RT-qPCR performance. LOD, LOQ, linear range, linearity, and efficiency on the CFX96 Dx System.
Paramater

AnalyteFluorophore

CFX96 Dx

LOD, copies

SARS-CoV-2 N1

FAM

3.81*

SARS-CoV-2 N2

HEX

3.21

SARS-CoV-2 N1

FAM

11.01

SARS-CoV-2 N2

HEX

6.11

SARS-CoV-2 N1

FAM

8,026–11.01

SARS-CoV-2 N2HEX4,454–6.11

SARS-CoV-2 N1

FAM

26.67–35.09

SARS-CoV-2 N2HEX

27.86–36.48

SARS-CoV-2 N1

FAM

0.995

SARS-CoV-2 N2

HEX

0.995

SARS-CoV-2 N1

FAM

93.4

SARS-CoV-2 N2

HEX

90.47

LOQ, copies
Linear range, molecules
Linear range, Cq
Linearity, R2
Efficiency, %* The LOD for FAM channel was detected between 3.81 and 9.02 copies/μl, within the acceptable threefold limit.
Cq, quantification cycle; LOD, limit of detection; LOQ, limit of quantification.
Suppl. Table 2. Precision of CFX96 Dx Systems calculated from one input.
Inter-Run

InterdayIntermediate

Platform

Fluorophore

AnalyteSD

95% CISD

95% CISD

CFX96 DxFAM

SARS-CoV-2 N1

0.317

0.251 to 0.431

0.405

0.324 to 0.715

0.405

HEX

SARS-CoV-2 N2

0.308

0.244 to 0.419

0.381

0.307 to 0.659

0.381

CI, confidence interval; SD, standard deviation.
A. CFX96 Dx, A vs. B instruments

36

33
32
31
30
29
28
27
26
26

28

30

32

34

33
32
31
30
29
28

36

26

Mean Cq FAM, CFX96 Dx A

33
32
31
30
29
28
r = 0.999

26
26

28

30

32

34

Mean Cq FAM, CFX96 Deep Well Dx C

Mean Cq FAM, CFX96 Deep Well Dx B

34

27

32

34

32
31
30
29
28
r = 0.987

26

36

26

34
33
32
31
30
29
28
27

r = 0.999

26

36

26

Mean Cq FAM, CFX96 Deep Well Dx A

28

30

32

30

32

34

36

F. CFX96 Deep Well Dx, A vs. C instruments
36

Deming fit(y = –0.2896 + 1.012x)

35

28

Mean Cq FAM, CFX96 Dx A

E. CFX96 Deep Well Dx, B vs. C instruments
36

Deming fit(y = –0.02351 + 0.9995x)

35

30

33

Mean Cq FAM, CFX96 Dx B

D. CFX96 Deep Well Dx, A vs. B instruments
36

28

34

27

r = 0.993

26

Deming fit(y = 0.3821 + 0.9966x)

35

34

27

r = 0.997

36
Mean Cq FAM, CFX96 Dx C

34

C. CFX96 Dx, A vs. C instruments

Deming fit(y = 0.8889 + 0.9829x)

35
Mean Cq FAM, CFX96 Dx C

35
Mean Cq FAM, CFX96 Dx B

B. CFX96 Dx, B vs. C instruments

Deming fit(y = –0.5839 + 1.016x)

34

Mean Cq FAM, CFX96 Deep Well Dx C

36

Deming fit(y = –0.2651 + 1.012x)

35
34
33
32
31
30
29
28
27

r = 0.999

26

36

26

Mean Cq FAM, CFX96 Deep Well Dx B

28

30

32

34

36

Mean Cq FAM, CFX96 Deep Well Dx A

Deming fit(y = 0.0904 + 0.9956x)

35
34
33
32
31
30
29
28
27

r = 0.999

26
26

28

30

32

34

Mean Cq FAM, CFX Opus Deepwell Dx A

36

36

Deming fit(y = –0.2408 + 1.007x)

35
34
33
32
31
30
29
28
27

r=1

26
26

28

30

32

34

36

Mean Cq FAM, CFX Opus Deepwell Dx B

Mean Cq FAM, CFX Opus Deepwell Dx C

36

Mean Cq FAM, CFX Opus Deepwell Dx C

Mean Cq FAM, CFX Opus Deepwell Dx B

G. CFX Opus Deepwell Dx, A vs. B instruments H. CFX Opus Deepwell Dx, B vs. C instruments I. CFX Opus Deepwell Dx, A vs. C instruments
36

Deming fit(y = –0.1508 + 1.002x)

35
34
33
32
31
30
29
28
27

r = 0.999

26
26

28

30

32

34

36

Mean Cq FAM, CFX Opus Deepwell Dx A

Suppl. Fig 2. Deming regression analysis of FAM channel measurements for the CFX96 Dx, CFX96 Deep Well Dx, and CFX Opus Deepwell Dx Systems. Threereplicates for each system in three independent runs over three days on three systems of each platform were analyzed. Results were plotted using mean Cq for the FAM
channel and pairwise comparisons were made within each platform. Cq, quantification cycle.
© 2023 Bio-Rad Laboratories, Inc.

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A. CFX96 Dx, A vs. B instruments

B. CFX96 Dx, B vs. C instruments
39

35
33
31
29
r = 0.995
27

29

31

33

37

37
35
33
31
29

r = 0.994

27

39

27

Mean Cq HEX, CFX96 Dx A

33
31
29
r = 0.998
27

29

31

33

37

Mean Cq HEX, CFX96 Deep Well Dx C

Mean Cq HEX, CFX96 Deep Well Dx B

35

27

37

33
31
29
r = 0.998
29

31

33

37

Mean Cq HEX, CFX Opus Deepwell Dx A

31
29
r = 0.992

27
27

29
r = 0.998
29

31

33

37

39

35
33
31
29
r = 0.998

27
27

29

31

33

37

39

35
33
31
29
r = 0.998

27
27

37

39

Mean Cq HEX, CFX Opus Deepwell Dx B

29

31

33

37

39

Mean Cq HEX, CFX96 Deep Well Dx A

Deming fit(y = 0.912 + 0.9659x)

37

33

Deming fit(y = –0.1639 + 1.012x)

37

39

H. CFX Opus Deepwell Dx, B vs. C instruments
39

31

F. CFX96 Deep Well Dx, A vs. C instruments
39

31

27

29

Mean Cq HEX, CFX96 Dx A

33

27

Mean Cq HEX, CFX Opus Deepwell Dx C

Mean Cq HEX, CFX Opus Deepwell Dx B

35

27

33

Mean Cq HEX, CFX96 Deep Well Dx B

Deming fit(y = –0.642 + 1.025x)

27

35

39

35

39

G. CFX Opus Deepwell Dx, A vs. B instruments
37

37

Deming fit(y = 0.1785 + 0.9979x)

Mean Cq HEX, CFX96 Deep Well Dx A
39

33

E. CFX96 Deep Well Dx, B vs. C instruments
39

Deming fit(y = –0.3419 + 1.014x)

37

31

Deming fit(y = 0.07751 + 1.005x)

37

Mean Cq HEX, CFX96 Dx B

D. CFX96 Deep Well Dx, A vs. B instruments
39

29

Mean Cq HEX, CFX96 Deep Well Dx C

27

39

I. CFX Opus Deepwell Dx, A vs. C instruments
Mean Cq HEX, CFX Opus Deepwell Dx C

37

C. CFX96 Dx, A vs. C instruments

Deming fit(y = 0.8046 + 0.9855x)
Mean Cq HEX, CFX96 Dx C

Deming fit(y = –0.7182 + 1.019x)
Mean Cq HEX, CFX96 Dx C

Mean Cq HEX, CFX96 Dx B

39

39

Deming fit(y = 0.3009 + 0.99x)

37
35
33
31
29

r = 0.999

27
27

29

31

33

37

39

Mean Cq HEX, CFX Opus Deepwell Dx A

Suppl. Fig 3. Deming regression analysis of HEX channel measurements for the CFX96 Dx, CFX96 Deep Well Dx, and CFX Opus Deepwell Dx Systems.
Three replicates for each system in three independent runs over three days on three systems of each platform were analyzed. Results were plotted using mean Cq
for the HEX channel and pairwise comparisons were made within each platform. Cq, quantification cycle.

© 2023 Bio-Rad Laboratories, Inc.

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B. CFX96 Dx, B vs. C instruments

28
30
32
34
Mean Cq FAM, (CFX96 Dx A + CFX96 Dx B)/2

36

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

28
30
32
34
Mean Cq FAM, (CFX96 Deep Well Dx A +CFX96 Deep Well Dx B)/2

28
30
32
34
Mean Cq FAM, (CFX Opus Deepwell Dx A +CFX Opus Deepwell Dx B)/2

ΔCq FAM, CFX Opus Deepwell Dx C –CFX Opus Deepwell Dx A

ΔCq FAM, CFX Opus Deepwell Dx B –CFX Opus Deepwell Dx A

28
30
32
34
Mean Cq FAM, (CFX96 Deep Well Dx B +CFX96 Deep Well Dx C)/2

36

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

26

28
30
32
34
Mean Cq FAM, (CFX Opus Deepwell Dx A + CFX Opus
Deepwell Dx C)/2

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

36

28
30
32
34
Mean Cq FAM, (CFX96 Dx A + CFX96 Dx C)/2

36

Mean (0.093)

26

28
30
32
34
Mean Cq FAM, (CFX96 Deep Well Dx A +CFX96 Deep Well Dx C)/2

36

I. CFX Opus Deepwell Dx, A vs. C instruments

Mean (–0.036)

26

Mean (0.277)

F. CFX96 Deep Well Dx, A vs. C instruments

Mean (0.084)

26

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

36

H. CFX Opus Deepwell Dx, B vs. C instruments

Mean (–0.047)

26

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

36

G. CFX Opus Deepwell Dx, A vs. B instruments
1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

28
30
32
34
Mean Cq FAM, (CFX96 Dx B + CFX96 Dx C)/2

E. CFX96 Deep Well Dx, B vs. C instruments

Mean (0.009)

26

26

ΔCq FAM, CFX96 Deep Well Dx C –CFX96 Deep Well Dx B

Δ Cq FAM, CFX96 Deep Well Dx B –CFX96 Deep Well Dx A

D. CFX96 Deep Well Dx, A vs. B instruments

Mean (0.365)

ΔCq FAM, CFX96 Deep Well Dx C –CFX96 Deep Well Dx A

26

C. CFX96 Dx, A vs. C instruments

ΔCq FAM, CFX96 Dx C –CFX96 Dx A

Mean (–0.087)

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

ΔCq FAM, CFX Opus Deepwell Dx C –CFX Opus Deepwell Dx A

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

ΔCq FAM, CFX96 Dx C –CFX96 Dx B

ΔCq FAM, CFX 96 Dx B –CFX96 Dx A

A. CFX96 Dx, A vs. B instruments

36

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

Mean (–0.083)

26

28
30
32
34
Mean Cq FAM, (CFX Opus Deepwell Dx A +CFX Opus Deepwell Dx C)/2

36

Suppl. Fig 4. The Bland-Altman test demonstrates equivalence between systems of the same platform in limits of agreement (LOA) plots. Representative results areshown using pairwise comparisons between three CFX96 Dx, three CFX96 Deep Well Dx, and three CFX Opus Deepwell Dx Systems in the FAM channel.

© 2023 Bio-Rad Laboratories, Inc.

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B. CFX96 Dx, B vs. C instruments

29
31
33
35
Mean Cq HEX, (CFX96 Dx A + CFX96 Dx B)/2

37

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

29
31
33
35
Mean Cq HEX, (CFX96 Deep Well Dx A +CFX96 Deep Well Dx B)/2

ΔCq HEX, CFX Opus Deepwell Dx B –CFX Opus Deepwell Dx A

Mean (0.165)

27

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

37

G. CFX Opus Deepwell Dx, A vs. B instruments
1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

29
31
33
35
Mean Cq HEX, (CFX96 Dx B + CFX96 Dx C)/2

29
31
33
35
37
Mean Cq HEX, (CFX Opus Deepwell Dx A + CFX Opus
Deepwell Dx B)/2

29
31
33
35
Mean Cq HEX, (CFX96 Deep Well Dx B +CFX96 Deep Well Dx C)/2

Mean (–0.181)

27

27

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

37

H. CFX Opus Deepwell Dx, B vs. C instruments
1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

Mean (0.226)

29
31
33
35
Mean Cq HEX, (CFX96 Dx A + CFX96 Dx C)/2

37

F. CFX96 Deep Well Dx, A vs. C instruments

Mean (0.111)

27

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

37

E. CFX96 Deep Well Dx, B vs. C instruments

Mean (0.102)

27

27

ΔCq HEX, CFX96 Deep Well Dx C –CFX96 Deep Well Dx B

ΔCq HEX, CFX96 Deep Well Dx B –CFX96 Deep Well Dx A

D. CFX96 Deep Well Dx, A vs. B instruments

C. CFX96 Dx, A vs. C instruments

Mean (0.339)

ΔCq HEX, CFX96 Deep Well Dx C –CFX96 Deep Well Dx A

27

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

ΔCq HEX, CFX96 Dx C –CFX96 Dx A

ΔCq HEX, CFX96 Dx C –CFX96 Dx B

Mean (–0.114)

29
31
33
35
37
Mean Cq HEX, (CFX Opus Deepwell Dx B + CFX Opus
Deepwell Dx C)/2

Mean (0.212)

27

29
31
33
35
Mean Cq HEX, (CFX96 Deep Well Dx A +CFX96 Deep Well Dx C)/2

37

I. CFX Opus Deepwell Dx, A vs. C instruments
ΔCq HEX, CFX Opus Deepwell Dx C –CFX Opus Deepwell Dx A

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

ΔCq HEX, CFX Opus Deepwell Dx C –CFX Opus Deepwell Dx B

ΔCq HEX, CFX96 Dx B –CFX96 Dx A

A. CFX96 Dx, A vs. B instruments

1.4
1.2
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1

Mean (–0.017)

27

29
31
33
35
Mean Cq HEX, (CFX Opus Deepwell Dx A +CFX Opus Deepwell Dx C)/2

37

Suppl. Fig 5. The Bland-Altman test demonstrates equivalence between systems of the same platform in limits of agreement (LOA) plots. Representative results areshown using pairwise comparisons between three CFX96 Dx, three CFX96 Deep Well Dx, and three CFX Opus Deepwell Dx Systems in the HEX channel.
Cq, quantification cycle.

© 2023 Bio-Rad Laboratories, Inc.

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U.S. and foreign patents and/or pending patent applications owned by or under license to Bio-Rad Laboratories, Inc. See https://www.bio-rad.
com/en-us/trademarks.

Bio-RadLaboratories, Inc.
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