Sensitive Codetection of Viral-Pathogen RNA
and DNA Using One-Step Multiplex RT-qPCR
Jason Ma, Michelle Kong, and Steve Okino
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547

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Abstract

RT-qPCR is a well-established method for the detection, quantification, and typing of different
microbial agents in the areas of clinical and veterinary diagnostics, as well as food safety.
There are multiple benefits of this technology: unparalleled sensitivity, quick time to results,
simultaneous detection of multiple targets, scalable throughput, and low cost. However, when
dealing with the challenges of pathogen detection, not all RT-qPCR reagents are createdequal. In this application note, we demonstrate a sensitive codetection of viral RNA and DNA
targets in a multiplex setting using a novel one-step multiplex RT-qPCR supermix.

Materials and Methods
We designed an experiment testing samples with threeviral nucleic acids, two in their RNA form — Zika (ZIKV) andchikungunya (CHIKV) — and the DNA version of dengue virus(DENV), with an internal positive control (IPC) RNA and humangenomic DNA (gDNA) control. The viral targets were tested at fivedifferent dilutions while the controls were kept constant. The copy
numbers for the viral RNA targets were determined by real-time
Droplet Digital PCR and were diluted to a single copy or less per
reaction in their lowest dilution series (Table 1). We made two
identical plates, each containing ten technical replicates of eachsample; one plate was analyzed immediately (time ( T ) = 0 hr),while the second plate was stored at room temperature (25°C)
and analyzed the next day ( T = 24 hr).

Table 1. Targets and templates used for a 5-plex reaction.
TargetReporter Dye

Template

AmountRNA Copies

ZIKV

FAM

Viral RNA

5 dilution series

1.17 to 11,668

CHIKV

HEX

Viral RNA

5 dilution series

0.15 to 1,498

DENV

Texas Red

Synthetic DNA

5 dilution series

N/A

IPC

Cy5

Synthetic RNA

Constant

N/A

RNaseP

Cy5.5

Human gDNA

Constant

N/A

T=040

ZIKV
CHIKV
DENV
IC
RNaseP

35

Quantitation cycle, Cq

Introduction
Bio-Rad’s Reliance One-Step Multiplex Supermix is a reagent
that enables researchers to sensitively and accurately detect
up to five targets in a single reaction. This mix is based on anovel enzyme, Reliance Reverse Transcriptase — a chimeric
enzyme derived from FLV (feline leukemia virus) and MMLV
(Moloney murine leukemia virus) reverse transcriptases.
Reliance Reverse Transcriptase, in combination witha powerful blend of DNA polymerases and an expert
formulation, gives the mix unparalleled robustness andstability. As a consequence of this, reaction plates preparedwith the Reliance One-Step Multiplex Supermix can be held atroom temperature for an extended period of time with no loss
in target detection sensitivity.

T = 24+++++●●●●●

302520156543Log dilution

210Fig. 1. Codetection of various nucleic acid targets in 5-plex reaction.

Sensitive Codetection of Viral-Pathogen RNA and DNA Using One-Step Multiplex RT-qPCR

Results and Discussion
Room Temperature Stability of Reliance One-Step Multiplex Supermix

Comparison to Other Reagents

As shown in Figure 1, we observe remarkable consistency
between the data produced at T = 0 hr (solid circles) andT = 24 hr (crosses). The T = 0 hr and T = 24 hr points generally
overlap, indicating that the amount of target quantified by
Reliance One-Step Multiplex Supermix is stable after holding
the preassembled reactions at room temperature for one day.
The detected amount of viral nucleic acid over the dilution serieschanges as expected with excellent efficiency and R2 values(Table 2). All viruses were detected in all five sample dilutions
at both time points (Table 2).

We also analyzed one-step multiplex reagents offered by othercompanies. At T = 0 hr, both companies’ products detect
the viruses (Tables 2 and 3). However, Company A had poor
efficiency amplifying CHIKV and only detected 3 of the 5
CHIKV dilutions and 4 of the 5 ZIKV dilutions. Company B
performed better than Company A, yet still missed detecting
CHIKV is sample dilution 5 (Table 3). At T = 24 hr, however,the performance of both companies’ reagents degradedsignificantly (Tables 2 and 3). This is a clear demonstrationof the uniqueness of Reliance One-Step Multiplex Supermix
for its ability to retain sensitivity and efficiency even after
24 hours at room temperature. This feature is critical whenscreening large numbers of samples, since high-throughput
workflows involve the testing of multiple plates. Adopting a
reagent which is stable at room temperature would circumventthe need for refrigerating plates, and therefore enable much
simpler automated solutions.

When working with fewer copies of RNA (one or less), not alltechnical replicates of a sample are expected to be positive.
For example, based on a Poisson distribution, a sample
that has, on average, one RNA copy per reaction should
lead to 6–7 positive reactions out of 10 tested. This type ofdistribution is observed in ZIKV dilution 5 where 1.17 RNA
copies are expected in each technical replicate (Table 3). Here,seven out of ten technical replicates are positive at T = 0 hr
and T = 24 hr, aligning perfectly with expectations. Similarly,
CHIKV is detected at both time points in a technical replicate
series where an average of only 0.15 RNA copies is expected(Table 3). These results imply that Reliance One-Step Multiplex
Supermix enables detection of single RNA copies at close to100% efficiency, even in reactions that have been prepared
24 hours earlier.
Table 2. Comparison of 5-plex detection at two time points.
T = 0 hr
R2

Efficiency

R2Dilutions Detected(Out of 5)

88%

0.999

95%

0.996

586%

0.996

55102%

0.996

DENV

92%

50.999

592%

0.999

ZIKV

582%

0.991

4

N/A

N/A

1

CHIKV

68%

0.990

3

N/A

N/A

0DENV

107%

0.999

579%

0.987

3

ZIKV

81%

0.998

582%

1.000

2

CHIKV

84%

0.996

4

N/A

N/A

1

DENV

96%

0.999

572%

0.959

4

Reagent

TargetEfficiency

Reliance One-Step
Multiplex Supermix

ZIKV
CHIKVCompany ACompany B

T = 24 hr
Dilutions Detected(Out of 5)

Table 3. Comparison of a number of ZIKV and CHIKV replicates detected at two time points over 24 hours.
Reliance One-Step
Multiplex Supermix
Viral Target

Company A

Company B

Log dilution

Viral RNA Copies

T = 0 hr

T = 24 hr

T = 0 hr

T = 24 hr

T = 0 hr

T = 24 hr

ZIKV

411.7

1010100100

ZIKV

51.17

770050CHIKV

315101090100

CHIKV

41.5890070

CHIKV

50.15240000© 2019 Bio-Rad Laboratories, Inc.

2

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Sensitive Codetection of Viral-Pathogen RNA and DNA Using One-Step Multiplex RT-qPCRConclusion
Reliance One-Step Multiplex Supermix can detect and quantify
five targets simultaneously with outstanding efficiency andsingle-copy detection sensitivity. Additionally, the supermix
can codetect RNA and DNA targets effectively, allowing for thedevelopment of panels detecting various types of pathogens(e.g., RNA viruses, DNA viruses, bacteria, yeast). Finally, thisreagent’s performance is not affected by extended storageof assembled reactions at room temperature thereby enabling
simpler and more reproducible automated workflows.
These results highlight the fact that Reliance One-Step
Multiplex Supermix can be used easily in high-throughput
screening workflows and applications that require highsensitivity detection, in particular for pathogen detection.
Combining Reliance One-Step Multiplex Supermix with
Bio-Rad’s CFX Automation System II (a robotic plate handler)
and two CFX Real-Time PCR Systems would allow for large
numbers of plates (up to 40, depending on the cyclingprotocol) to be screened for up to five RNA or DNA targets —with no operator assistance.

© 2019 Bio-Rad Laboratories, Inc.

3Bulletin 7196

Sensitive Codetection of Viral-Pathogen RNA and DNA Using One-Step Multiplex RT-qPCR

Visit bio-rad.com/RelianceOneStep for more information.
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