Making

the invisible
visible
A versatile workflow for the detection
Placeholder Image
of low-abundance microbes
 Microbial detection workflow​
Instruments:

•TissueLyser II • QIAcube® Connect •QIAcuity® Digital • QIAxcel® Connect

•PowerLyzer® 24 • QIAcube HT PCR System
Homogenizer • EZ2® Connect •Rotor-Gene® Q
•Vortex Adapters • QIAsymphony® •QIAquant®

• QIAxcel® Connect •QIAamplifier® 96

NGS workflow

Sample Detection:
Sample Detection: Data PCR
stabilization PCR/qPCR
extraction* NGS analyses validation
& disruption /dPCR

Microbes are ubiquitous and touch all Reagents, consumables

and analysis tools:
PCR workflow

• QIAGEN CLC Genomics

aspects of our lives, from health to food

•PowerProtect • PowerFecal® Pro Kits • QIAcuity OneStep • QIAseq® xHYB Viral
Workbench Premium
DNA/RNA • QIAwave DNA Blood Advanced Probe Kit and Bacterial Panels
• GeneGlobe Analysis
•RNAprotect® & Tissue Kit • dPCR Microbial DNA • QIAseq DIRECT
Detection Assays SARS-CoV-2 Kit

production. They can trigger various harmful

•PowerBead Pro • AllPrep® Kits
Tubes & Plates • QIAamp® Kits •GT-Digital SARS-CoV-2 • QIAseq FX DNA
Wastewater Surveillance Library Kit
• DNeasy PowerSoil
® ®
Assay Kit for QIAcuity v1.0** • QIAseq 16S/ITS
Pro Kit

or beneficial effects on us. And this makes the

• Microbial DNA qPCR Screening Panels
• EZ1&2™ Kits Assays and Arrays
• QIAsymphony DSP • QuantiNova®
Virus/Pathogen Kit PCR/RT-PCR Kits

specific detection and monitoring of microbes • UCP Probe and SYBR®

Green qPCR Kits
• UCP Multiplex PCR Kit
*Our extraction kits cover wastewater, sludge, biofilm, stool, soil, swab samples and more
** Customers from North America: Order from GT Molecular. Customers from other
countries: Contact your QIAGEN sales representative.

important for understanding their biological

function, especially in infection or colonization
of our body. Stabilization Disruption​ Extraction

• PowerProtect DNA/RNA is • Robust sample disruption • Dedicated kits for the

Conventional molecular techniques for
sample enrichment and DNA-based
quantification coupled with advances in
sequencing technologies have been able
to push certain boundaries of microbial
and infectious disease research, public
health and epidemiology. However, the
low concentrations of microbes, complex
sample matrices, lack of simplified and
a bulk stabilization solution
fast end-to-end workflows and limited for stool samples that ensures
the integrity of your microbial
throughput capabilities of existing
community and functional
techniques have impeded the rapid profile before nucleic acid
profiling and identification of antibiotic extraction​
resistance and virulence genes from • Specially designed for use with
our extraction chemistry​
diverse samples, including wastewater • For samples other than stool,
samples, the human microbiome, multiple choose the RNAprotect portfolio
for the best results
drug resistance, sepsis, food production
or environmental samples.
instruments provide fast simultaneous extraction of DNA
and efficient lyses and and RNA from the same sample
homogenization for various to maximize yields
sample types and throughput​ • Our patented Inhibitor Removal
• PowerBead Pro Tubes and Technology® (IRT) included
Plates combined with optimized in the Power kits eliminates
chemistry provides efficient lysis, contaminants, so you get the
reducing the risk of sample bias best starting material for your
towards easy-to-lyse microbes downstream application​
• Achieve higher reproducibility
and reliability with automation​

2  Microbial detection workflow 06/2022 Microbial

Title  XX/XXXX 
detection workflow 06/2022  3
Sample-derived Reliable fecal sample
PCR/RT-PCR inhibitors stabilization using PowerProtect DNA/RNA
A RNA Yield — room temperature storage
RNA yield – room temperature storage (UV-Vis)
RNA (µg/50 mg stool) RNA (µg/50 mg stool)
Other components, such as amplification inhibitors, are also released when breaking 25 50
QIAGEN Supplier Z QIAGEN Supplier Z
open cells to release nucleic acids. These include humic/fulvic acids in soil, 20 40
polysaccharides/polyphenolics in plants and bile, bilirubin and heme in stool.1 15 30
Two stool samples were
collected, and four replicates
of each were processed using
10 20 the QIAGEN or the Supplier
Z workflow. The unstabilized
5 10 sample at t0 was used as a
reference. The other samples
0 0 were stored in PowerProtect
n=4

ed

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sample 2
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(A) Whether unstabilized or
RNA (µg/50 mg stool) RNA (µg/50 mg stool)
stabilized for up to 28 days,
25 50 the stool samples processed
QIAGEN Supplier Z QIAGEN Supplier Z
with the QIAGEN workflow
20 40 deliver greater RNA yields
than Supplier Z.
15 30
(B) The RIN value is a tool
10 20
designed to estimate the
(1) Examples of additional matrices: Rådström, P. et al. (2004) Pre-PCR processing: Strategies to generate
5 10 integrity of total RNA
PCR-compatible samples. Mol. Biotechnol. 26, 133–46
samples. The higher the
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ed

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workflow on samples
Stool sample 1 Stool sample 2 stabilized over time are
superior to Supplier Z.

The patented Inhibitor Removal RNA integrity

B RNA Integrity — average RIN value (stool samples 1 and 2)
Technology (IRT) assures
representative results. The
RIN value
amplification plot shows the 10
influence of the inhibitors on the QIAGEN Supplier Z
PCR/RT-PCR reaction. In the 8
absence of inhibitors, 6
amplification shows in the early
cycles, but when the DNA is 4
co-isolated with inhibitors, there 2
appears a significant delay in
0
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4  Microbial detection workflow

Title  06/2022
XX/XXXX Microbial detection workflow 06/2022  5
 This saves time, money and precious sample material. dPCR Microbial DNA Detection Assays are comparable
Experiments can be set up using fewer (than usual) in singleplex and multiplex (Figure 4). Figure 5 shows the
wells in a Nanoplate, allowing higher sample throughput. strong signal to noise separation observed in multiplex
Furthermore, you can make your own multiplex assay by assays. Figure 6 illustrates multiplex detection of five
ordering single assays with different dyes (FAM, HEX, water microbial pathogens in a single run.
C RNA Quality (C) QIAxcel ScreenGel
electropherograms Precise and specific quantification in singleplex and multiplex
confirm RNA quality TAMRA, ROX, Cy5). Several wet-lab tested and validated
in terms of the integrity
of total RNA samples.
Copies/µl reaction
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Pseudomonas aeruginosa Enterococcus faecalis Shigella sonnei Staphylococcus epidermidis
FAM HEX ROX Cy5

Multiplex Singleplex

Figure 4. Singleplex versus multiplex setup quantifying four different bacterial targets.
Singleplex
Four assays were versus multiplexand
run in singleplex setup quantifying
4-plex reactionsfour
usingdifferent
the samebacterial
template targets.
gDNA material. In both setups, the same concentrations were observed for template
Fourbetween
dilutions assays were run in10,000x.
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and 4-plex reactions
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condition, genomic
was DNA using
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26kIn24-well
both setups, the same and
Nanoplates concentrations wereProbe
the QIAcuity observed
PCR for
Kit concen-
on the
trations
QIAcuity between
Digital PCR 0.25 and 2500 copies/μl. dPCR, with 3 replicates for each condition, was performed using 26K 24-well Nanoplates and the QIAcuity Probe PCR Kit on
System.
the QIAcuity Digital PCR System.

Fluorescence intensity (RFU) – sul1 (HEX) Fluorescence intensity (RFU) – OXA-58 group (Cy5)
80 80
70 70
60 60

Detecting and quantifying what goes unseen 50

40
30
Detect viral RNA and microbial DNA together in a multiplex reaction​ 50
40
30
20 20
A
A 10
10
0 0
Copies/µl reaction
As often in nature, the targets with low and rare abundance give you the most interesting 0 10Copies/µl
20 reaction
30 40 50 60 70 80 90 100 0 10 20 30 40 50 60
1000 511.0
Fluorescence 518.2 530.2 561.4 542.4
1000 511.0 intensity (RFU) – OXA-48
518.2 group (FAM) 530.2 561.4 Fluorescence intensity542.4
(RFU) – QepA (TAMRA)
insights. However, finding those targets is often a needle in a haystack problem. That’s where
digital PCR comes in handy with its high precision and sensitivity. 100 (RFU) – QepA
Fluorescence intensity
100
53.7 (TAMRA)
53.7
44.7 50.9 52.3
52.3
56.9
56.9
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10
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Benefits using dPCR NEW! dPCR Microbial DNA 1
1 0.1
30 0.1
Detection Assays 20
0.1
• Absolute, precise and sensitive quantification of pathogens 0.1 Figure 5. Strong signal separation between channels in multiplex.
without the need for a standard curve – now on GeneGlobe 10 500 50
500 50 5 5 NTC
NTC 500 500 50 50 5 5 NTC
NTC 500
500 50
50 2D55scatter
NTCplots500
NTC 500 50 dye
50
of various
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from
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5 NTC runs.
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SARS-CoV-2
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N1 Clostridioides
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60
difficile 70
Vibrio cholerae
Four assays targeting Yersinia
60Vibrio cholerae
reactions using 26k
Yersinia
enterocolitica
24-well Nanoplates and
Listeria
four bacterial resistance genes were run in multiplex
Listeria
monocytogenes
the QIAcuity Probe PCR Kit on
tolerance to inhibitors found in complex sample matrices For use with the QIAcuity Probe PCR Fluorescence intensity (RFU) – sul1difficile
(HEX) enterocolitica
the QIAcuity Digital PCR System.
monocytogenes
• Enhanced analytical performance for rare target detection Kit (for DNA targets) or the QIAcuity
OneStep Advanced Probe Kit (for RNA B
B
or a mix of RNA and DNA targets) Multiplex detection of RNA and DNA targets. A mixture of four bacterial gDNAs (Clostridioides difficile, Vibrio cholerae, Yersinia enterocolitica, Listeria monocyto-
Green channel SARS-CoV-2 N1
Green
genes) and channel
SARS-CoV-2 RNASARS-CoV-2
was used as input. N1dPCR, with three replicates per condition, was run using 8.5k 96-well Nanoplates and the QIAcuity OneStep Advanced
Probe Kit on the B4 B5
QIAcuity Digital B6
PCR System. ThreeB7 B8 with 500,
template dilutions B9 50 andC4 C4 C5
5 copies/μl C6
were used. Bar chart C7 C8 measured
shows the mean C9concentrations (cop-
• Identify >680 targets including 4
ies/μl) of100
B4 B5 B6 B7
three replicates each for each of the five targets.
B8 B9 C5 C6 C7
dPCR Microbial C8 C9
DNA Detection Assays 05/2022
Benefits using nanoplate dPCR bacterial, fungal, parasitic, viral, 100
80
antibiotic resistance and virulence 80
60
• High-order multiplexing of up to 5 targets per well factor genes 60
40
• User-friendly workflow with rapid time-to-result (~2 h) • Detect up to 5 targets per reaction 40
20 For more details on the assay performance, see the Product Profile:
• Large sample volumes (up to 28 µl) are possible for enhanced • Combine microbial DNA and viral 20
0 www.qiagen.com/PP_dPCR-microbial-detection
sensitivity RNA detection in one reaction 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Yellow channel Clostridioides
Yellow channel Clostridioides difficile
difficile
B4 B5 B6 B7 B8 B9 C4 C5 C6 C7 C8 C9
70 B4
70
B5 B6 B7 B8 B9 C4 C5 C6 C7 C8 C9
60
60
6  Microbial detection workflow 06/2022 Microbial
50detection workflow 06/2022  7
50
40
 High-quality enrichment of respiratory viral isolates

Mapped reads/fold enrichment

1,000,000
100,000
10,000
1000
Not ready for dPCR? 100

Try our Microbial DNA 10

1
qPCR Assays and Arrays

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QIAseq xHYB gives an average of 2400x enrichment of viral targets vs. standard shotgun sequencing

Sequencing for deeper insights For more details on high-quality targeted viral and bacterial sequencing, visit:
www.qiagen.com/QIAseq-xHYB-Viral-and-Bacterial-Panels

NGS determines the nucleic acid sequence that makes up a genome or targeted genomic
regions of the pathogen without any previous knowledge of its existence. This allows the
identification of novel pathogens. The unbiased nature of NGS also enables co-infections to be Making sense of complex
identified that are otherwise ignored by other assays. Moreover, it can be used for pathogen
surveillance to detect genetic variants that may be evolving. microbial genomic data
Tackle some of the challenges involved in implementing NGS for routine pathogen detection with QIAseq kits
and panels (for example, pair the QIAseq xHYB Viral and Bacterial Panels with the QIAseq FX DNA Library Only well-structured and clear data help
UDI Kit): you understand what is going on and Strain typing
Antimicrobial
allow you to take action. However, the and outbreak
resistance
• Unique Dual Index (UDI) adapters volume and complexity of the data can analysis
available for multiplexing up to
be quite overwhelming.
384 samples
• High library complexity and QIAGEN CLC Genomics Workbench
uniform coverage maximizes Microbiome
Premium is just one of many
interpretable data profiling and
bioinformatics platforms that is easy to Viral analysis
• Eliminate the data bottleneck and functional
use by bioinformaticians and non- metagenomics
reduce overall turnaround time
bioinformaticians alike. It offers various
with rapid data analysis and
tools and customizable workflows for
variant interpretation
diverse application areas, including
microbial and metagenomics.

8  Microbial detection workflow 06/2022 Microbial detection workflow 06/2022  9

Ordering information Ordering information

Product Description Cat. no. Product Description Cat. no.

TissueLyser II For convenient and secure sample disruption for variable throughput, from tubes to 85300 dPCR Microbial For digital PCR detection of microbial targets, including bacterial, fungal, parasitic, viral, 250207
96-well plates DNA Detection antibiotic resistance or virulence factor genes
Assays
PowerLyzer 24 For the fast homogenization of any biological sample in 2 ml tubes 13155
Homogenizer Microbial DNA For real-time PCR detection of microbial species, virulence genes or antibiotic resistance 330025
qPCR Assays and genes 33026
PowerProtect Bulk reagent for ambient temperature stabilization of nucleic acids in stool samples over 14800
Arrays
DNA/RNA time 14810
QuantiNova PCR/ For highly sensitive, specific and ultrafast probe-based real-time PCR/RT-PCR and 208152
QIAcube Connect* For fully automated, spin-column-based nucleic acid extraction managed from outside the 9002864
RT-PCR Kits* multiplex PCR/RT-PCR, and unparalleled results using SYBR Green-based qPCR 208352
lab using QIAsphere
UCP PCR Kits For SYBR Green-based and probe-based, real-time quantitative PCR for 208012
QIAcube HT* For automated mid- to high-throughput nucleic acid purification in a 96-well format 9001896
microbiome or quality control applications using reagents from Ultra Clean 208212
EZ2 Connect For end-to-end automation of nucleic acid extraction, from reagent setup to elution 9003210 Production
QIAsymphony* For automated sample preparation of DNA, RNA, and bacterial and viral nucleic 9001751 UCP Multiplex PCR Ultra-Clean Production master mix for multiplex hot-start PCR and microbiome 206742
acids from a wide range of starting materials Kit applications 206744
QIAxcel Connect For effortless, cost-effective, high-resolution DNA or RNA gel electrophoresis – all in a 9003110 QIAseq xHYB Viral For reverse transcription, hybrid capture and downstream post-hybrid capture 333325
single instrument Respiratory Panel* amplification of viral respiratory targets; can be paired with QIAseq FX DNA 333322
Library Kit
RNeasy PowerFecal For the isolation of microbial RNA from stool and gut samples, sludge, or wastewater 78404
Pro Kit QIAseq FX DNA Buffers and reagents for DNA fragmentation (including end repair and A-addition), 180477
Library UDI Kit ligation and library amplification; for use with Illumina instruments 180479
QIAwave DNA For a more eco-friendly alternative to our standard kit for extracting total DNA from 69556
Blood & Tissue Kit animal blood and tissues, cells, yeast, or bacteria QIAseq DIRECT For fast, targeted whole genome library preparation of SARS-CoV-2 for genomic 333891
SARS-CoV-2 Kits* surveillance and variant detection
AllPrep PowerViral For isolating viral or bacterial total nucleic acids from waste water and stool samples 28000-50
DNA/RNA Kit QIAseq 16S/ITS For next-generation sequencing-based Sample to Insight profiling of bacterial and 333812
Screening Panels fungal communities 333815
QIAamp DNA Micro For purification of genomic and mitochondrial DNA from small samples 56304
Kit QIAGEN CLC For single cell analysis, microbial profiling, pathogen typing and outbreak analysis 832023
Genomics using state-of-the-art bioinformatics
DNeasy PowerSoil For the isolation of microbial genomic DNA from all soil types 47014
Workbench
Pro Kit 47016
Premium Desktop
QIAcuity Digital For absolute and accurate quantification of nucleic acid molecules in nanoplates 911035
PCR System* 911045
Rotor-Gene Q* For outstanding performance in real-time PCR in a rotary format 9001620
9001640
9001660
QIAquant* For fast and high performance real-time PCR in a 96- or 384-well format 9003010
9003011
QIAamplifier 96 For fast and high performance end-point PCR experiments in a 96-well format 9002991
9002990
QIAcuity OneStep For one-step RT-PCR on the QIAcuity digital PCR instruments 250131
Advanced Probe Kit 250132

* Additional instrument, kit, panel, assay and service bundles are available. For all systems, Installation and Training are included but are additionally available as separate
service offerings. For specific catalog numbers and additional information, visit www.qiagen.com or contact your local sales representative

10  Microbial detection workflow 06/2022 Microbial detection workflow 06/2022  11

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit instructions for use or user operator manual. QIAGEN kit instructions
for use and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services (or your local distributor).

Trademarks: QIAGEN®, Sample to Insight®, QIAamp®, QIAamplifier®, QIAcube®, QIAcuity®, QIAseq®, QIAsymphony®, QIAquant®, AllPrep®, DNeasy®, Inhibitor
Removal Technology®, PowerLyzer®, PowerFecal®, PowerSoil®, PowerViral®, QuantiNova® (QIAGEN Group); GT Molecular® (GT Molecular LLC). Registered names,
trademarks, etc. used in this document, even when not specifically marked as such, may still be protected.

PROM-20709-003 1127765 06/2022 © 2022 QIAGEN, all rights reserved.

Ordering www.qiagen.com/shop
Technical Support www.support.qiagen.com
Website www.qiagen.com