Sample to Insight

Nanoplate Digital PCR 

for Wastewater Surveillance

DHS/NIST Workshop: Standards to Support an Enduring 

Capability in Wastewater Surveillance for Public Health

June 2021

Michael Bussmann, Ph.D.

Associate Director, Global Product Management Digital PCR
Sample to Insight

Legal disclaimer

QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or 

treatment of a disease.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit 

handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
Sample to Insight

Sources of variability in wastewater testing

Collected 

sample

Sample 

disruption

Sample 

extraction

Detection: 

PCR / digital 

PCR / NGS

Data 

analysis

Detection: 

digital PCR
Sample to Insight

Sources of variability in wastewater testing

Collected 

sample

Sample 

disruption

Sample 

extraction

Detection: 

PCR / digital 

PCR / NGS

Data 

analysis

Detection: 

digital PCR

Variability in detection

• Process complexity • Staff skill set • Partition volume and count
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Detection and absolute quantitation of pathogens in wastewater

• A standard curve is not required

• Better inter-laboratory comparability

• Higher precision due to absolute 

quantification

• Higher robustness for viral detection from 

complex samples

Why digital PCR for wastewater testing?

• Fully integrated workflow with time-to-result ~2 h

• Easy operating and workflow like qPCR

• Flexible throughput and resolution options

• High multiplexing capability (up to 5plex)

Why Nanoplate digital PCR?

Less expertise and experience needed to run 

digital PCR experiments
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Accuracy without complexity

Get accurate dPCR results with an easy-to-use and fast system

NIST Reference Material 2372a (human DNA quantification standard)

o Probe based detection (genomic targets are: CTNNB1, EGFR, ERBB2)

o NIST Reference Material was used to generate 10 and 100 copies/uL templates
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Scalable and comparable

Reliable and reproducible results over Different Systems

o Three runs per instrument and plate types

o Input (expected concentration): 500 copies/μl 

o Assay: QIAGEN dPCR Demo Assay (FAM)

Instrument type

/Nanoplate 8.5K 96-

well

Mean 

cp/μl SD CV%

QIAcuity One 523 23 4.3%

QIAcuity Four 530 27 4.9%

QIAcuity Eight 521 27 5.1%

Mean 523

SD 8.86

CV% 1.69%

Instrument type

/Nanoplate 26K 24-

well

Mean 

cp/μl SD CV%

QIAcuity One 480 16 3.3%

QIAcuity Four 484 15 3.1%

QIAcuity Eight 480 16 3.4%

Mean 481

SD 3.83

CV% 0.80%
Sample to Insight

Scalable and comparable

Reliable and reproducible results over Different Systems

o Three runs per instrument and plate types

o Input (expected concentration): 500 copies/μl 

o Assay: QIAGEN dPCR Demo Assay (FAM)

Instrument type – total Mean cp/μl

QIAcuity One 499

QIAcuity Four 507

QIAcuity Eight 501

Mean 502

SD 22.23

CV% 4.43%
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Predictable partition count

Robust quantification with high number of valid partitions

o Number of valid partitions per well for Nanoplate 26K 24-well and Nanoplate 8.5K 96-well 

23,000

23,500

24,000

24,500

25,000

25,500

26,000

Average number of valid partitions per well

0 2 4 6 8 10 12 14 16

Run (Nanoplate)

Valid partitions per run (Nanoplate 26K)

5000

5500

6000

6500

7000

7500

8000

8500

9000

0 2 4 6 8 10 12 14 16

Average number of valid partitions per well

Run (Nanoplate)

Valid partitions per run (Nanoplate 8.5K)
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Accuracy in all measurements without being a dPCR expert

The VPF (Volume Precision Factor)

• Nanoplates and the VPF enable control of the partition volume

• Ensure precision of the quantitation

• Nanoplate batch & reaction/well specific 

calibration of the partition volume

• Automatically applied 
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Example of results: SARS CoV-2 quantification

Sample: 10-fold serial dilution of positive control ATCC

• Concentration range between 104 to 1 copy/μl

• Test all samples in triplicates, 4 μl template for both

• Panel 1_3-plex SARS CoV2 N1/N2/E (HEX/Texas Red/FAM)

• “Low/high 10” à 10 copies of SARS-CoV-2 target in the presence of 105

copies each of the fecal indicator targets MS2, HF183 and crAssphage

N1 (HEX)

Reference: 

Taniuchi, Mami; Associate Professor, Medicine: Infectious Diseases and International Health; 

University of Virginia (Field tester of the QIAcuity One-Step Viral RT-PCR Kit, cus)
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Example of results: Norovirus G1 quantification

Experimental approach

• Samples – water from swimming pools

• Sample prep – QIAamp Viral RNA Mini kit

Consistent fluorescence intensity in 

reference channel is a good indicator of 

uniform partitioning across a nanoplate

Positive control

Cq value above 40

Negative with qPCR

Cq value above 40

Negative with qPCR
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Thank you for your attention.

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HorizonTM (Horizon Discovery Limited); ROXTM (Thermo Fisher Scientific or its subsidiaries); SYBR® (Life Technologies Corporation). Registered names, trademarks, etc. used in this 

document, even when not specifically marked as such, are not to be considered unprotected by law. PROM-18244-001 © 2021 QIAGEN, all rights reserved.