Product ProfiledPCR Microbial DNA Detection Assays
Detect microbial targets – bacterial, fungal, parasitic, viral,
antibiotic resistance and virulence factor genes – using digital PCR
The dPCR Microbial DNA Detection Assays are hydrolysis
probe-based assays for precise and sensitive identificationof a wide range of microbial targets using digital PCR(dPCR). They consist of a primer pair and a hydrolysis
probe with a configurable dye (fluorophore) that allows
multiplex detection of up to five targets per reaction.
The assays are intended for use with the QIAcuity® Probe
PCR Kit (for DNA targets) or the QIAcuity OneStep
Advanced Probe Kit (for RNA or RNA+DNA targets) on
the QIAcuity Digital PCR System.
Step 1Reaction setup in a PCR pre-plate• Sample, Primer/Probe,Probe Master Mix• Up to ~27 µl of template for
26k Nanoplate and~8 µl for 8.5k Nanoplate•Identify more than 680 microbial targets•Detect up to five targets per reaction using fivefluorophores – FAM™, HEX ™, TAMRA, ROX ™, Cy5®
•Follow a simple and fast dPCR workflow on theQIAcuity Digital PCR System•Combine microbial DNA and viral RNA detection inone reaction

Step 2

Transfer to a single Nanoplateor multiple Nanoplates

Step 3

Load single (QIAcuity One) or multiple Nanoplates
simultaneously (QIAcuity Four and Eight) and start the run
QIAcuity Probe cycling:• 2 min at 95°C
• 15 sec at 95°C + 60 sec at 58°C (40 cycles)

Figure 1. Microbial dPCR in about two hours with minimal hands-on time.
This example shows a Nanoplate 8.5k 96-well. The recommended Nanoplate for higher sensitivity is Nanoplate 26k 24-well.

Explore the virtual workflow: www.qiagen.com/applications/digital-pcr/workflow/qiacuity-demo

Sample to InsightHighly sensitive detection of over 680 microbial targets
dPCR Microbial DNA Detection Assays can be used totest the presence of over 680 bacterial, fungal, parasitic,
viral, antibiotic resistance or virulence factor genes(Table 1). The high sensitivity of the QIAcuity Digital PCR
System allows detection of microorganisms or their genes

present at extremely low levels such as in metagenomic
samples, isolated colonies or other challenging sample
types. A streamlined workflow gets you to results in just
two hours with minimal hands-on time (Figure 1).

Table 1. Targets covered by the dPCR Microbial DNA Detection Assays

Bacterial targets

Fungal targets

Viral targets

Virulence and antibiotic
resistance targets

387 targets

35 targets

46 targets

202 targets

Examples:E. coli, Salmonella, Yersinia,
Legionella, Vibrio, Clostridia,
Streptococcus, Mycobacteria,
Mycoplasma species

Examples:Aspergillus, Candida, Trichophyton,
Fusarium, Microsporum species

Examples:SARS-CoV-2 (N1, N2),
Influenza A&B, RSV,Norovirus GI & II

Examples:Beta lactamases, MDR efflux pumps,
fluoroquinolone resistances, toxins,
components of microbial secretion
systems, surface proteins

For a detailed target list, see the Technical Information: www.qiagen.com/TI_dPCR-microbial-detection

Accurate quantification of microbial targets
To demonstrate accurate quantification of microbial
targets using the dPCR Microbial DNA Detection Assays
on the QIAcuity Digital PCR System, we quantified theNIST reference standard 8376 (https://www-s.nist.
gov/m-srmors/certificates/8376.pdf) using
genomic DNA (gDNA) from Shigella sonnei as input.

2246
2321Copies/µl reaction
10000
2Figure 2. Accurate quantification of a reference standard.
The NIST reference standard 8376 was quantified using gDNA from S. sonnei
as input. The dPCR assay that targets a single copy gene of S. sonnei revealed
accurate absolute quantification of the expected copy numbers in all dilutions of
the reference DNA template (1x – 10,000x). Dark blue bars represent the mean
measured copies/µl values with error bars from three replicates each run using
a 26k 24-well Nanoplate on the QIAcuity Digital PCR System.

232381000
23.8
523.2
1100
2.

122.
32

100.

1800.
232

10.1

NTC
Measured

The measured concentration (copies/µl) of the target
gene closely matches the expected concentration, asshown in Figure 2.
Figure 3 illustrates a four-log dynamic range of linear
quantification of two bacterial targets – Enterococcus
faecalis and Shigella sonnei – down to 0.4 copies/µl.

1x10x

100x

1000x10,000x

Expected2 	dPCR Microbial DNA Detection Assays 05/2022

Enterococcus faecalis (yellow channel, HEX)A
Fluorescence intensity (RFU)
A1A2A3200

B1B2B3C1C2C3D1D2D3E1E2E2G1G2G3000000000000000150
100500000

Analyzed partition

Shigella sonnei (red channel, ROX)
Fluorescence intensity (RFU)
A1A2A3B1

B2

B3

C1C2C3

D1

D2

D3E1E2E2

G1G2G3000000000000000140120100806040
200000Analyzed partition

Enterococcus faecalisBShigella sonnei

Copies/µl reaction
10,000Copies/µl reaction
10,0001000

1000

1001001010R2 = 1

10.10.1

R2 = 0.9999

10.1

110100Template dilution factor

1000

10,0000.1

110100Template dilution factor

1000

10,000Figure 3. Four-log dynamic range of linear quantification of target species.
A 1D scatter plots of replicates (three each) of five template dilutions and a no template control (NTC) for E. faecalis and S. sonnei using gDNA as input template.
B Respective plots of the mean measured copies/µl values against the dilution factor of the input template. R2 values indicate a strong positive linear relationship between
the measured copies/µl values and the dilution factor of the input template. dPCR was performed using 26k 24-well Nanoplates on the QIAcuity Digital PCR System.

dPCR Microbial DNA Detection Assays 05/2022	3

Precise and specific quantification in singleplex and multiplex

2242
21 .00
62.00

180.

0.
240.
30

272.

2.

122.
32

42
2.
42

23.7
22 1.7923.0
23 7.3423
525 .430.
60

23724 .30
0.
33

0.

0.

0.
200.
220.1

250.
35

2.

1

3.

04
2.
14

1009
3.
14

22.4
21 6.91100

31.5
33 4.2523
4
23 .47
4.
90

1000

32
931 .50
9.
40

1987
19 .7019.4
731
47
28 .8092.6
3Copies/µl reaction
10,0005-plex bundles are available; other combinations need
to be verified by the customer. The performance of thedPCR Microbial DNA Detection Assays are comparable
in singleplex and multiplex (Figure 4). Figure 5 shows thestrong signal to noise separation observed in multiplex
assays. Figure 6 illustrates multiplex detection of fivewater microbial pathogens in a single run.

2308
24 .23
26.63

The choice of multiple fluorescent dye combinations
enables quantification of up to five targets per reaction.
This saves time, money and precious sample material.
Experiments can be set up using fewer (than usual)wells in a Nanoplate, allowing higher sample throughput.
Furthermore, you can make your own multiplex assay byordering single assays with different dyes (FAM, HEX,TAMRA, ROX, Cy5). Several wet-lab tested and validated

1xTCNx100xx1000x00

1010,0TC
N

1xx
100x10x00x00

1010,0NTC

1xx1000x100xx00

1010,0TCN

1xx100xx1000

1010,000

x0.01

Pseudomonas aeruginosaEnterococcus faecalis

Shigella sonnei

Staphylococcus epidermidis

FAM

HEX

ROX

Cy5Multiplex

Singleplex

Figure 4. Singleplex versus multiplex setup quantifying four different bacterial targets.
Four assays were run in singleplex and 4-plex reactions using the same template gDNA material. In both setups, the same concentrations were observed for template
dilutions between 1x and 10,000x. dPCR, with three replicates per condition, was performed using 26k 24-well Nanoplates and the QIAcuity Probe PCR Kit on theQIAcuity Digital PCR System.

Fluorescence intensity (RFU) – sul1 (HEX)
80
7060504030201001020304050060
70
80
90Fluorescence intensity (RFU) – OXA-48 group (FAM)

100

Fluorescence intensity (RFU) – OXA-58 group (Cy5)8070605040302010001020304050
Fluorescence intensity (RFU) – QepA (TAMRA)

60

Fluorescence intensity (RFU) – QepA (TAMRA)
60
504030201000102030405060
Fluorescence intensity (RFU) – sul1 (HEX)70

4 	60Figure 5. Strong signal separation between channels in multiplex.
2D scatter plots of various dye combinations from 4-plex multiplex runs.
Four assays targeting four bacterial resistance genes were run in multiplex
reactions using 26k 24-well Nanoplates and the QIAcuity Probe PCR Kit on
the QIAcuity Digital PCR System.

dPCR Microbial DNA Detection Assays 05/2022

Mix 1

Mix 2

Fluorescence intensity (RFU)
B4C480

Mix 3
D3

Mix 4E4NTC
F460
Green

Clostridium
perfringens

4020000
Fluorescence intensity (RFU)
8000X

60
Yellow

0

Legionella
pneumophila

4020000000

Fluorescence intensity (RFU)
10080

X

Orange 60

Vibriocholerae

40200Red00

Fluorescence intensity (RFU)
70
60
50
40
30
20
10000000X

Fluorescence intensity (RFU)
706050Crimson 40302010000

Leptospiraalexanderi

000X
Yersinia
enterocolitica000

Analyzed partition
Figure 6. Water microbial pathogen detection in 5-plex.
1D scatter plot showing five reactions with four different template mixes and a no template control (NTC). Each gDNA mix contained different amounts of gDNAs fromfour out of five target organisms (red crosses mark the missing organism in a particular mix); mix 1 lacks Leptospira template; mix 2 lacks Legionella template; mix 3 lacks
Yersinia template; mix 4 lacks Vibrio template. Each target DNA was quantified in the 5-plex reaction. dPCR, with three replicates per condition, was run using 8.5k24-well Nanoplates and the QIAcuity Probe PCR Kit on the QIAcuity Digital PCR System.

dPCR Microbial DNA Detection Assays 05/2022	5Detect viral RNA and microbial DNA together in a multiplex reactiondPCR Microbial DNA Detection Assays, together with thenew QIAcuity OneStep Advanced Probe Kit, enables
detection of RNA targets or a combination of RNA andDNA targets in one multiplex reaction. This is particularly
useful for the simultaneous detection of DNA and RNA
Copies/µl reaction
1000511.010010518.2
53.7

561.4

530.2

4.5

542.4
56.9

52.3

50.9

44.7
4.4viruses. Figure 7 illustrates simultaneous detection of theSARS-CoV-2 (N1) RNA target and four bacterial targets
in multiplex. Viral target-only combinations are also
possible, for example, multiplex detection of SARS-CoV-2
and Influenza or Norovirus (data not shown).

5.1

4.3

5.7

10.1

500 50 5
SARS-CoV-2 N1

500 50 5
Clostridioides difficile

500 50 5Vibrio cholerae

500 50 5Yersinia enterocolitica

500 50 5Listeria monocytogenes

Figure 7. Multiplex detection of RNA and DNA targets.
A mixture of four bacterial gDNAs (Clostridioides difficile, Vibrio cholerae, Yersinia enterocolitica, Listeria monocytogenes) and SARS-CoV-2 RNA was used as input.
dPCR, with three replicates per condition, was run using 8.5k 96-well Nanoplates and the QIAcuity OneStep Advanced Probe Kit on the QIAcuity Digital PCR System.
Three template dilutions with 500, 50 and 5 copies/µl were used. Barchart shows the mean measured concentrations (copies/µl) of three replicates each for each of thefive targets.

Ordering InformationProductContents

Cat. no.

dPCR Microbial DNA Detection Assays

One tube Microbial DNA dPCR Assay, 200 x 40 µl reactions inNanoplate 26k, 666 x 12 µl reactions for Nanoplate 8.5k

250207(RespectiveAssay Cat. No.
to be specified in
GeneGlobe®)

QIAcuity Probe PCR Kit (1ml, 5 ml)

1 ml or 5 ml Master Mix for the QIAcuity dPCR instrument; water

250101, 250102

QIAcuity OneStep Advanced Probe Kit(1 ml, 5 ml)

1 ml or 5 ml OneStep Master Mix for the QIAcuity dPCR instrument,
RT Mix, Internal Control RNA, Enhancer GC, Water

250131, 250132For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook oruser 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.

For configuring and ordering the assays, please visit:
geneglobe.qiagen.com/products/analysis-type/analysis-type-dpcr

Trademarks: QIAGEN®, Sample to Insight®, QIAcuity®, GeneGlobe® (QIAGEN Group); Cy5® (GE Healthcare); FAM™, HEX ™ (Life Technologies Corporation); ROX ™ (Thermo Fisher Scientificor its subsidiaries). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.
© 2022 QIAGEN, all rights reserved. PROM-20505-002 1128145 05/2022

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