The document describes the Rotor-Gene Q real-time PCR instrument. It has a unique centrifugal design that allows for excellent thermal and optical precision compared to block-based instruments. Each reaction tube spins individually, providing very precise temperature uniformity between wells. It also uses LED technology for excitation and filters for detection, allowing for multiple detection channels without need for normalization or reference dyes. Applications mentioned include quantitative PCR, high resolution melt analysis, genotyping, pathogen detection, and multiplex PCR. QIAGEN products are highlighted such as kits, controls, and the QIAgility liquid handler for automated real-time PCR setup.

1. Sample to Insight
RotorGene Q – A Rapid, Automatable real-time PCR Instrument for Genotyping and
Microbial Identification w/ Excellent efficiency, Reproducibility, and Support
James Qin
Senior Scientist, MDx Applications,
Qiagen
Webinar-related questions:
QIAwebinars@QIAGEN.com
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2. Sample to Insight
Legal disclaimer
2
 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.
Pathogen detection with HRM and multiplex RT-qPCR

3. Sample to Insight
4
Agenda
3
Introduction to Rotor-Gene Q
Unique real-time PCR chemistries and kit portfolios
QIAglity - An efficient, sensitive liquid handler for real-
time PCR setup
Applications:
• Sensitive target measurement in quantitation
• Good multiplex-PCR with no inhibition and cross-
talk
• Quantitative high-resolution melting (HRM) in
genotyping and microbial ID
1
2
3

4. Sample to Insight
4August 2013
Thermal cyclers (block): Peltier Technology
Characters of a “Traditional” Real-time PCR Instrument
Thermal precision (uniformity) in a block-based cycler
 + 0.50 ºC variance across the 96 well block (1.0 oC difference)
 Corner and edge wells most affected (fluorescence  1/temperature)
 Can cause localized “hotspots” at Peltier device junctions
 Hot spots can reduce Taq polymerase activity
 Can adversely affect QPCR data

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Title, Location, Date 5
Reaction Chamber
PMT
Detector
Assembly
LED Light
Source
Assembly
Tubes in Rotor
Spin Past Optics
Lens
Detection
Filters
Spindle/Motor
Assembly
Introduction to Rotor-Gene Q
Unique design for thermal and optical precision
Cross-section view (front)

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Title, Location, Date 6
Cool air in
Centrifugal fan Drives
air into chamber
Centrifugal fan drives
air around chamber
Chamber vent opens
expelling hot air
Heater
elements
switch off
Note: holes in
the rotor allow
free airflow
Rapid air exchange for fast heating and cooling
Temperature uniformity from well-to-well is + 0.02oC
Cross-section view
(side)

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7
The Rotor-Gene Q: Optics
High power light-emitting diodes (LED):
• Highly robust LED technology (Mean time between service visits >50 months)
• Constant and consistent output from run to run (no decay).
• Uniform light-path to well (all wells excited at same intensity), so no optical
normalization and no reference dye (ROX) required.
• Different wavelengths to optimally excite fluorophors at abs max
• LEDs are highly stable with lifetime guarantee (no lasers or lamps to replace)
Sensitive PMT
(photomultiplier tube)
detector
Excitation
source: LED
Rotor spins tubes
at 400 rpm Filter set
(rotates for each channel)
Lens

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Title, Location, Date 8
Excitation (LEDs)
Detection (Filters)
 Supports all QPCR detection chemistries:
 Intercalating dyes (eg. SYBR Green)
 Hydrolysis probes (TaqMan)
 Hybridization probes (FRET)
 Up to 6 individual channels with
wavelengths spanning UV to infra-red.
 Ideal for multiplexing
Unmatched versatility in dyes and channels
Rotor-Gene Q Optical Range

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Title, Location, Date 9
Rotor-Gene Q accessories
 Easy to set-up!
 Rotors are interchangeable and snap-in like a DVD.
 Locking ring secures tubes in place during the run
 Option to use 0.2 mL tubes or 0.1 mL
microtubes (strips of 4) or Rotor-Disc (100-well)
 RGQ includes accessories pack with
multiple rotors, setup blocks, rotor-holder and
plasticware starter set.
 1 year warranty

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10
*OTV disc
 The OTV system* uses the chemical properties of three
different thermochromic liquid crystals (TLCs) as an
absolute
temperature reference.
 TLCs change from opaque to transparent at a very precise
temperature s (transition point) of 50, 75 and 90oC
 The reported value is compared to the known calibrated
value to verify the instrument is within specification. If not
within specification, automatic re-calibration of the Rotor-
Gene can be done at the press of a button.
*patent pending
Easy, routine verification of in-tube temperature in 30 minutes!
Temperature verification on the Rotor-Gene Q:

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11
Rotor-Gene Q Instrument Options
To meet your specific laboratory requirements:
Rotor-Gene Q 2plex
 2 channel intro offer with highest performance
Rotor-Gene Q 2plex HRM
 a cost effective entry point to QPCR & HRM
Rotor-Gene Q 5plex
 full flexibility for all qPCR applications
Rotor-Gene Q 5plex HRM
 maximum versatility in your research
Rotor-Gene Q 6plex
 unmatched multiplexing possibilities
 All packages include:
 Real time PCR System & laptop
 General accessories & plasticware starter set
 1 year warranty
 1.5 day installation & training (optional)

12. Sample to Insight
4
Agenda
Pathogen detection with HRM and multiplex RT-qPCR 12
Introduction to Rotor-Gene Q
Unique real-time PCR chemistries and kit portfolios
QIAglity - An efficient, sensitive liquid handler for real-
time PCR setup
Applications:
• Sensitive target measurement in quantitation
• Good multiplex-PCR with no inhibition and cross-
talk
• Quantitative high-resolution melting (HRM) in
genotyping and microbial ID
Applications
1
2
3

13. Sample to Insight
13
QIAGEN Sample to Insight solutions for pathogen and microbial extraction
Detection
and analysis
• QIAmp DNA Mini Kit
• QIAmp UCP Pathogen Mini Kit
• QIAmp DNA Stool Fast/ Mini Kit
• QIAamp DNA Microbiome Kit
• QIAamp 96 DNA QIAcube HT Kit
• QIAsymphony mericon Bacteria Kit
MO BIO Laboratories for NGS
• PowerSoil DNA Isolation Kit
• PowerFecal DNA Isolation Kit
• …
• QuantiNova Probe PCR kits
• QuantiNova RT-PCR kit
• QuantiFast Pathogen +IC kits
• QuantiTect Virus kits
• Type-it HRM PCR kit for
genotyping assays
• Microbial DNA qPCR Arrays
and Assay Kits
• Rotor-Gene Q
• QIAxcel DNA Kits
• Allprotect
• InhibitEx
• RNAlater
Pathogen detection workflow: From Sample to Insight
Sample
isolation
Sample
collection &
Stabilization
qPCR or
qRT-PCR
Pathogen detection with HRM and multiplex RT-qPCR

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 The enzymes:
▪ HotStarTaq Plus and antibody-mediated hot-start DNA
Polymerases
▪ Reactivation within 5 minutes
▪ Unmatched specificity and sensitivity
 The buffer:
▪ Unique combination of K+ and NH4
+ ions
▪ High specificity
▪ No optimization necessary
 Q-BondTM:
▪ Enables fast cycling by promoting annealing of Taq and
primer/probe to the template
 Synthetic Factor MP
▪ Supports macromolecular crowding
▪ Enables equal amplification of templates that differ in
abundance
Unique buffer composition – Solving problems with fast real-time PCR
High PCR specificity and macromolecular crowding
Pathogen detection with HRM and multiplex RT-qPCR 14

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QIAGEN Genotyping product
 Type-it HRM PCR Kit
▪ Fast and accurate detection of gene mutations and SNPs by High-Resolution Melting (HRM) analysis
Scan for mutation
Type mutation
Dye Saturation/Relocation w/
SYBRGreen II Dye (FAM)
Dye Saturation/Relocation w/
SYBRGreen III Dye (EvaGreen,
CYTO9)

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16
QIAGEN RT-qPCR and multiplex PCR products
 QuantiTect Multiplex PCR Kit and QuantiTect Probe PCR Kit
▪ Real-time PCR and two-step RT-PCR
Wide dynamic rangeHigh specificity
Fast: Faster results with time savings of up to 50%
Multiplex: Successful multiplex PCR without the need for
optimization
Sensitive: Detect up to 4 targets in 1 tube
Reliable: Quantify low- and high-abundance targets, no optimization
Pathogen detection with HRM and multiplex RT-qPCR

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QIAGEN RT-qPCR and multiplex PCR products (2)
 QuantiNova Probe RT-PCR Kit and QuantiNova SYBR® Green RT-PCR Kit
▪ Novel QuantiNova two-phase hot-start mechanism
 Reliable: Increased reliability of gene expression results using gDNA reduction
 Convenient: Room-temperature reaction setup without compromising
 Visual pipetting control: limit pipetting errors
 Internal controls: positive in-process verification of successful RT-PCR and monitoring RT-
PCR inhibition
 Sensitive: High sensitivity for low-copy RNA targets
Pathogen detection with HRM and multiplex RT-qPCR

18. Sample to Insight
4
Agenda
18
Introduction to Rotor-Gene Q
Unique real-time PCR chemistries and kit portfolios
QIAglity - An efficient, sensitive liquid handler for real-
time PCR setup
Applications:
• Sensitive target measurement in quantitation
• Good multiplex-PCR with no inhibition and cross-
talk
• Quantitative high-resolution melting (HRM) in
genotyping and microbial ID
1
2
3

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QIAgility Liquid Handler Overview & Comparison
QIAgility
QIAgility was specifically developed for PCR setup,
and other liquid handling actions.
Advantages
 Easy to use software
 Small footprint
 Programmed applications
 Plug & play
Large Liquid Handlers
Hamiltons, Beckmanns, Tecans and more….
These instruments are real liquid handlers not
specifically developed for PCR setup.
Disadvantages
 Complex to programm
 Huge instruments – lab space limited
 No programmed applications
 long installation
Examples: Starlet, Biomek, Freedom Evo 75
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No matter what real-time instrument is used, QIAgility
is compatible
 The system is pre-configured to work with all PCR
tube types and all plate formats, including varieties
of 96-well, 384-well and Rotor-Disc
 Up to 4 different setups can be performed in parallel
 Beside any PCR setup the system can perform
▪ Set-up of quantification standards (dilution
series)
▪ Normalization of sample concentration
▪ Sample pooling
▪ Transfer of samples from one tube format to
another
▪ Plate replication
▪ Restriction digest setup
▪ Cherry picking from archived sample banks
The Hallmark of QIAgility Is Its Versatility
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QIAgility means Time Savings
 You have more important things to do…
 QIAgility performs complex set-ups faster than can be
done manually
 Staff can be attending to other duties while the QIAgility is
running
 Complicated runs don’t get put off because they will take
too long to set up
 Simple Transfer Actions 1:1
 96 samples
▪ 17 minutes
▪ 6 minutes with tip reuse
 Transfer Actions with pre-MM
 96 samples PCR set-up
▪ ~25 minutes
 Transfer Actions with pre-MM
 192 samples PCR set-up
▪ ~46 minutes
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QIAgility Applications and Protocol Support
 Applications
 Pre-written QIAGEN protocols at launch available
▪ Rotor-Gene SYBR Green PCR kit
▪ Rotor-Gene Multiplex PCR Kit
▪ QIAGEN OneStep RT-PCR kit
▪ HotStar Taq DNA Polymerase
▪ Cador BVDV RT-PCR kit / Reagent
 Application lab supports customized QIAGEN protocols
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23. Sample to Insight
RotorGene: Tests with 100-Ring Disc (100 replicates)
PMT ND5A on DRE-Urine Sample 3932 (1: 10) Setup by QIAgility
81.0 ⁰C
Run-1
(100 wells)
Run
Mean
Run
Stdev
Run
%CV
TH0.10 18.69 0.0752 0.0752
Linear Curve
Threshold 0.10
Log Curve
Threshold 0.10
Melt Curve
Threshold 0.10
Run-2
(100 wells)
Run
Mean
Run
Stdev
Run
%CV
TH0.125 18.72 0.0802 0.0802
QIAgility PCR Setup in 100 Disc-ring reactions by RotorGene
(10-ul reaction volume)
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4
Agenda
24
Introduction to Rotor-Gene Q
Unique real-time PCR chemistries and kit portfolios
QIAglity - An efficient, sensitive liquid handler for real-
time PCR setup
Applications:
• Sensitive target measurement in quantitation
• Good multiplex-PCR with no inhibition and cross-
talk
• Quantitative high-resolution melting (HRM) in
genotyping and microbial ID
1
2
3

25. Sample to Insight
Title, Location, Date 25
Rotor-Gene Q: Applications
All QPCR applications supported:
 Absolute Quantification (standard curve)
 Relative Quantification
 High Resolution Melt Analysis
Applications:
 Gene expression
 Genotyping – allelic discrimination
 SNP analysis
 Mutation detection and screening
 Pathogen detection
 Methylation analysis
 STR and VNTR analysis
 Quantification of copy number variants
and mosaicism
…

26. Sample to Insight
Bring multiple colors to your qPCR – The new QuantiNova Multiplex PCR Kit
For fast real-time PCR and two-step RT-PCR with up to 6 targets
Stringent antibody-mediated hot-start activation with QuantiNova Guard
:
Improved specificity and room-temperature setup enabling automated handling
26

27. Sample to Insight
The latest generation in real-time multiplex PCR
Built-in pipetting control for QuantiNova Multiplex PCR Kit
From sample to multiplex data in 60 mins
Fast results in a high
multiplex format
20 min
2 min
5 s
30 s
RNA
template
gDNA
template
40
cycles
Eliminate errors during reaction setup
 QuantiNova MP Master Mix
contains an inert blue dye
 The sample dilution buffer
contains an inert yellow dye
 Dyes do not interfere with
the real-time PCR
Ensures accurate reaction setup without impacting performance
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Gene Expression Workflow
QuantiNova Reverse Transcription Kit for cDNA synthesis in 2-step qRT-PCR
QuantiNova Multiplex PCR Kit for multiplex DNA or cDNA quantification
RNA Stabilization
and Purification
cDNA Synthesis
(2-step RT-PCR)
q(RT)-PCR
Data Analysis
and Interpretation
RNA templates
Reverse transcription
cDNA template
Amplification
qPCR products
gDNA template
Amplification
qPCR products
cDNA analysis — two-step RT-PCR gDNA analysis — PCR
QuantiNova RT Kit
QuantiNova
Multiplex PCR Kit
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QuantiNova Multiplex PCR Kit -
Reliable multiplex PCR from gDNA and cDNA providing unique in-process safety measures
 High specificity due to novel hot-start mechanism
 Built-in pipetting control to minimize errors during reaction setup
 Highest sensitivity for robust detection of rare targets
 Increased master mix concentration for maximal template input
 Multiplex capability for up to 6 targets for more results per reaction
 Outstanding reaction stability — ideal for high-throughput assays
 Compatible with all real-time PCR cyclers for more flexibility
Reliable multiplex PCR offers:
 Higher accuracy by inclusion of Internal Control (IC RNA) or reference gene(s)
 More insight from limited sample material
 Increased workflow efficiency
29
Faster, most sensitive multiplex qPCR

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QuantiNova MP PCR Kit – Improvement of performance
Stability immediately after rxn set-up (1)
QIAGEN Roche* AB*
ACTB (500)
CDK1
LINE1
GAPD
* LC Multiplex DNA Kit * AB TaqMan MP Kit* QN MP PCR Kit
4plex PCR reactions
were set up at room
temperature and run
immediately after
reaction setup
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31. Sample to Insight
QuantiNova MP PCR Kit – Stability 24 hours after rxn set-up (2)
Better performance for all 4 assays with QN MP PCR Kit after 24 hours storage
Reaction is stable at room temperature for 1 day after rxn setup, allowing worry-free automation
QIAGEN Roche AB
ACTB (500)
CDK1
LINE1
GAPD
4plex PCR reactions were set
up at room temperature and
left at 22.5°C for 24 hours
before PCR
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32. Sample to Insight
QuantiNova Multiplex PCR Kit –
Wide Linear Range; Ideal for Single Copy applications
Precise quantification of low and high abundant targets in a multiplex format: high template amount
LINE1
CDK1
4 targets with varying abundance were coamplified from gDNA starting w/ 800 ng, down to 50
ng in 1:2 dilutions (Examplary of 2 targets)
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QuantiNova MP PCR Kit – Wide Linear Range (2)
Precise quantification of low and high abundant targets in a multiplex format: low template amount
4 targets with varying abundance were coamplified from gDNA, starting w/ 48 ng, down to 6 pg in 1:2 dilutions
Reliable quantification of changes in gene expression profiles and copy numbers over a
wide range of template amount
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QN MP PCR Kit: Compatibility w/ automated setup by QIAgility vs. Manual
ACTB CDK1
Cycle
5 10 15 20 25 30 35 40 45
Norm.Fluoro.
0,30
0,25
0,20
0,15
0,10
0,05
0,00
Threshold
Cycle
5 10 15 20 25 30 35 40 45
Norm.Fluoro.
0,35
0,30
0,25
0,20
0,15
0,10
0,05
0,00
Threshold
Cycle
5 10 15 20 25 30 35 40 45
Norm.Fluoro.
0,95
0,90
0,85
0,80
0,75
0,70
0,65
0,60
0,55
0,50
0,45
0,40
0,35
0,30
0,25
0,20
0,15
0,10
0,05
0,00
Threshold
Cycle
5 10 15 20 25 30 35 40 45
Norm.Fluoro.
0,35
0,30
0,25
0,20
0,15
0,10
0,05
0,00
Threshold
LINE1 GAPD
A 4-plex PCR, using human genomic DNA (30 pg - 30 ng) was set up either manually or
with QIAgility and run side by side on the same plate.
Full compatibility with automated rxn setup, even after prolonged storage
time at room temperature
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Unbiased Multiplex PCR
Quantification of MYC in 1/2/3/4 plex PCR
Unaltered quantification upon coamplification of various targets with varying abundance
Template: 10 ng HeLa cDNA
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36. Sample to Insight
QuantiNova MP PCR Kit: Multiplex PCR of up to 6 targets
Outstanding performance, multiplexing capacity and process safety
6-plex PCR on RGQ from 20 ng HeLa cDNA
18SrRNA Marina Blue
LINE1 Cy5
GAPD Texas RED
MYC HEX
HSP90AA1 Quasar 705
TNF FAM
20ng/rxn
Note: Different fluorescence intensities are caused by
the use of different fluorophores in different channels
 Unique in-process safety measures differentiate us from
competition
 Inclusion of IC RNA and reference gene(s)
possible
 Visual pipetting control
 Excellent specificity and room temperature
stability by novel hot start mechanism
 Efficiency and performance increase through superior
multiplex format
 Up to 6-plex – outstanding capability to
streamline experimental design
– More insight from limited samples
 Highly concentrated enables sensitive assays
design
 Very fast, saving time
 Easy to use
Superior performance over existing chemistries ensuring reliable results for users
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37. Sample to Insight
Title, Location, Date 37
HRM characterizes double-stranded PCR products based on their detailed strand dissociation
(melt) behavior:
 Distinguishes samples with different sequence, length, GC content
 Sample characterization by melt curve shape and melt temperature
 Many applications such as:
▪ SNP Genotyping / allelic discrimination
▪ Quantitative DNA methylation analysis
▪ Gene scanning
▪ Sequence matching
 A high-intensity optical HRM channel
 An outstanding thermal resolution of 0.02°C
 High data acquisition rates
 A comprehensive HRM software package
 Use with special saturating intercalating dye (EvaGreen)
Mutants
(Tallele)
Wild types
(C allele)
Heterozygotes
Mutants
(Tallele)
Wild types
(C allele)
Heterozygotes
The Rotor-Gene Q HRM option features:
The Rotor-Gene Q can detect even the most difficult class IV SNPs by HRM
(with Tm difference of 0.1° C)
High Resolution Melt Anlaysis (HRM)

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deg.
75,0 75,5 76,0 76,5 77,0 77,5 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
100% methylated
70%
90% .
30%.
50%.
10%.
0% methylated
deg.
75,0 75,5 76,0 76,5 77,0 77,5 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0
Normalisedminus50%m50%u
35
30
25
20
15
10
5
0
-5
-10
-15
-20
-25
100% methylated
70%
90% .
30%.
50%.
10%.
0% methylated
Various ratios of methylated and unmethylated DNA-APC
Rotor-Gene HRM
+ EpiTect HRM PCR
Kit
Standard
normalized melt
curve
Difference plot
normalized to a
50% methylated sample
deg.
78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 84,5 85,0 85,5 86,0 86,5 87,0
Normalisedminuswt
15
10
5
0
Accurate SNP genotyping by HRM
Rotor-Gene HRM 5plex
+ Type-it HRM PCR Kit
+ 10 ng genomic DNA
Standard normalized melt curve
Difference plot
normalized to a
wild type sample
deg.
78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 84,5 85,0 85,5 86,0 86,5 87,0
NormalisedFluorescence
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
Homozygote
mutant
Wild Type
Heterozygote
Homozygote
mutant
Heterozygote
Wild Type
Genotyping with HRM on the Rotor-Gene Q is an fast and
cost-effective technique with high specificity
38
Applicaation example: Epigenetic research and Genotyping w/ HRM

39. Sample to Insight
For many datasets, most of the eigenvalues “lambda” are negligible and can be
discarded.
The eigenvalue
measures the variation
In the direction e:
Example:
ScreenClust S.W. for HRM and Principal Component Analysis (PCA)
Finding relationship from a large multi-layer sample set is always a challenge. One way to avoid the dimensionality is
by projecting the data onto a lower-dimensional space. It rotates multivariate dataset into a new configuration which
is easier to interpret.
Techniques for dimension reduction:
Principal Component Analysis (PCA) – most effective!
Fisher’s Linear Discriminant
Multi-dimensional Scaling.
Independent Component Analysis.

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Applications of PCA in Genomics
Purposes…
➢ Simplify data – PCA is the most commonly used dimension reduction technique.
➢ Look at relationships between variables – PCA is useful for finding new, more informative,
uncorrelated features.
➢ PCR reduces dimensionality by rejecting low variance features.
➢ Look at patterns of units – i.e. new mutations, targets
➢ Analysis of expression data
➢ Analysis of metabolomics data (Ward et al., 2003)
Example Run
wild type
heterozygote
mutant
A/T Class IV SNP
 minute differences between homozygote alleles (< 0.1°C)
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41. Sample to Insight
ºC
71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
dF/dT
3.75
3.50
3.25
3.00
2.75
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
JE341/Pro805
V6
V5
V3
V7
RGQ real-time PCR
Melt Curves
HRM: RotorGene Melt Curve Analysis on five assays for primer selection
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42. Sample to Insight
ºC
71.5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5
55
50
45
40
35
30
25
20
15
10
5
0
-5
-10
JE341/Pro805
Normalized to V5:
V6 V5
V3
V7
ºC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
0
-10
-20
-30
-40
-50
-60
Normalized to JE341/
Pro805:
JE341/Pro805
V6
V3
V5 V7
Example: RotorGene HRM Analysis on designed assays for primer selection (2)
HRM PCR Genotyping Kit/ RGQ – Offers Distinct Melt Curves
JE341/Pr
o805
V3
V5
V7
V6
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43. Sample to Insight
ºC
75 80 85 90
dF/dT
2.0
1.5
1.0
0.5
0.0
ºC
77 78 79 80 81 82 83 84 85 86 87 88 89
100
80
60
40
20
13003
13009
13000
13026
13031 13027
13003DS 13003DP13004
1300013029
HRM is able to pick up
individual differences
High Resolution Melt (V3 assay)
Distinct melt
curve
patterns
between
individual
subjects
HRM and melt curve analysis by ScreenClust on human references
in Microbiome research
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Clustering Analysis by ScreenClust on human references
for potential health monitoring
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Blood Duffy antigens (FY) Genotyping
HRM w. ScreenClust S.W. can be applied in blood genotyping application
Kidd antigens (FY) Genotyping:
PCA Analysis in
ScreenClust S.W.
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Multiplex PCR products in Applied Testing applications
46
y = -3,2871x + 40,07
20
25
30
35
40
0 2 4 6
CT
Log target copy number
Aspergillus niger
y = -3,2356x + 41,04
20
25
30
35
40
0 2 4 6
CT
Log target copy number
Klebsiella pneumoniae
y = -3,1423x + 41,649
20
25
30
35
40
0 2 4 6
CT
Log target copy number
Mucor/Rhizopus spp.
y = -3,1608x + 41,204
20
30
40
0 2 4 6
CT
Log target copy number
Pan
Aspergillus/Penicillium
Pathogen detection with HRM and multiplex RT-qPCR
Development of real-time
pathogen detection assays for
food and agricultural products
such as marijuana, organic drinks
Extraction chemistry: QIAamp DNA kit
Detection Kit: QuantiFast Pathogen+
PCR Kit
Sensitivity
▪ Standard curves from 0 to
1,000,000 copies were prepared
▪ 1000 copy Ct<31 or Ct<34 (low
end assays if NTC Ct=40)
▪ Primer efficiency> 80%, R>0.995,
LLOQ determined

47. Sample to Insight
T. Foetus (Fam) Internal Control (Yellow)
TH = 0.05
Tritrichomonas Foetus and InType IC – A clean multiplex assay
47

48. Sample to Insight
A clean Crypto assay in
4-plex reaction with
good dynamic range
Cryptosporidium in 4-plexes assay:
Good consistency and sensitivity of Crypto+ in the 4-plex
TH = 0.20
Calf scours panel: Ecoli K99/ Sal./Crypto/ InType 4-Plex assay
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49. Sample to Insight
Comparable target Cts.
A good specificity on POS vs Negs; and a
consistency of M. Haem in singleplex vs. 5-plex
assays.
Mannheimia Haemolytica in 5-plexes : Good consistency, specificity, and tighter range of M. Haem+
in the 4-plex
TH = 0.05
Good signal and tight range in IC controls.
InType ICM. Haem+
Bovine BRD Respiratory Panel: M. Haemolytica in 5-plexes
(M. haem/H. Somni/ M.bovis/ P.mult / InType IC)
49

50. Sample to Insight
Comparable target Cts.
Better specificity and great improvement on negative
samples in 5-plexes vs. H. Somni singleplex
Histophilus Somni in 5-plexes: Good consistency, specificity, and less cross-reactivity of M.
Haem+ in the 4-plex
TH = 0.05
A good signal and tight range in IC controls
in 5-plexes.
Bovine BRD Respiratory Panel: H. Somni in 5-plexes
(M. haem/H. Somni/ M.bovis/ P.mult / InType 5-Plex assay)
50

51. Sample to Insight
TH = 0.05
Better specificity and great improvement on negative
samples in 5-plexes vs. H. Somni singleplex after
optimization
Consistent, good signal and tight range in IC
controls.
Pasteurella multocida in 5-plexes: Good consistency, specificity, and tighter range of P. mult + in the 5-
plex
Bovine BRD Respiratory Panel: P. mult + Optimization
(M. haem/H. Somni/ M.bovis/ P.mult / InType 5-Plex assay)
51

52. Sample to Insight
Questions?
Thank you for attending
Contact QIAGEN Technical Service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
Email:
techservice-na@QIAGEN.com
techservice-eu@QIAGEN.com
QIAwebinars@QIAGEN.com
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53. Sample to Insight
FAQs I
QuantiNova Multiplex PCR Kit
 Is it possible to add the ROX passive reference to the master mix for long-term storage?
 If only Applied Biosystems cyclers will be used, QN ROX Reference Dye Solution can be added to the 4x
Master Mix for long-term storage, if desired. Corresponding instructions are given in the HB.
 Is the QuantiNova MP PCR mix compatible with partially purified / crude samples?
 The kit has been developed for efficient amplification of DNA/cDNA. Typical contaminations from crude
nucleic acid preparations (such as SDS or hemoglobin) will impair the results and will reduce the
sensitivity and specificity of detection.
 What is the stability of the QuantiNova MP PCR mix at -20°C, 4°C, and room temperature, respectively?
 Our (preliminary) data indicate that the stability will be at least 24 months (-20°C), 12 months (4°C) and
30 days (room temperature).
 Is the QuantiNova MP PCR Kit compatible with degenerated primers and probes?
 The mix has been developed to provide highest annealing specificity to allow selective target
amplification even if there are many similar target sequences. Therefore, degenerated primers and
probes will not work optimally.
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54. Sample to Insight
FAQs II
QuantiNova Multiplex PCR Kit
 Which reaction volumes can the QuantiNova MP PCR Kits be used with?
 Recommended rxn volume: 20 µl (96 well plate), 10 µl (384 well plate)
 Successfully tested rxn volume range: 8-25 µl (96 wells), 5-10 µl (384 wells)
 What is the maximal number of targets which can be coamplified?
 Our „official“ recommendation, as given in the QSP and the HB, are 5 targets. This is the phyisical limit of
most real-time cyclers on the market. However, we have shown, that up to 6 targets can be coamplified
successfully and in an unbiased manner.
 Which fluorophor combinations are recommended?
 Based on the number of different instruments on the market and fluorophore/quencher combinations offered
by the oligo suppliers, there is no „gold standard“ for the optimal probe combination. Actually each instrument
provider as well as any oligo supplier will provide corresponding information.
 On which cyclers has the QuantiNova MP PCR Kit successfully been tested?
 Basically almost every instrument from the major real-time PCR instrument suppliers. We did not have
access to AB‘s QuantStudio instruments; however, the technology is comparable to the ViiA7 instrument, on
which the QN MP PCR Kit works very efficiently.
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55. Sample to Insight
FAQs III
QuantiNova Multiplex PCR Kit
 What is the minimal/maximal template input?
 Template amounts (gDNA as well as cDNA) up to 800 ng can be used without compromising
quantification.
 Undiluted RT reactions can be added to the PCR reaction untill they make up 25% of the total reaction
volume without impairing the quantification results. This has been tested with RT kits from QIAGEN
(QuantiTect, QuantiNova) as well as with RT kits from all major suppliers (Thermo-Fisher/Invitrogen,
Bio-Rad, Roche).
 Can the QN MP PCR Kit be combined with contamination removal using UNG (uracil-N-glycosylase)?
 Please note that the QN MP PCR Kit neither contains dUTP as one of the nucleotides, nor the enzyme
UNG, both of which are prerequisites for an UNG treatment. However, we have very successfully
tested a modified version of the kit where dTTP has been replaced by dUTP and where UNG has been
added to the master mix. Amplification results were comparable to the „standard version“ and
contamination removal was highly efficient (clearly above 10E6).
If appropriate (revenue dependent), such a customized version can be made.
 Various types of probes?
 We have exclusively been testing TaqMan probes (both, standard TaqMan probes and MGB-containing
TaqMan probes). We do not expect any problems with other probe technologies such as Hybridization
probes and Molecular Beacons. Minor modifications of the protocol might be necessary (i.e. a 3-step
cycling protocol with separate annealing and extensions steps).
55