RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and Microbial...

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:

[email protected]

1

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

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

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

Sample to Insight

Title, Location, Date 5

Reaction Chamber

PMT Detector

AssemblyLED Light

Source Assembly

Tubes in Rotor Spin Past Optics

Lens

Detection Filters

Spindle/Motor Assembly


Unique design for thermal and optical precision

Cross-section view (front)

Sample to Insight


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)

Sample to Insight

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

Sample to Insight

Title, Location, Date8

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

Sample to Insight


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

Sample to Insight

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:

Sample to Insight

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)

Sample to Insight

4

Agenda

Pathogen detection with HRM and multiplex RT-qPCR 12




time PCR setup

Applications:



talk



Applications

1

2

3

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


Sample to Insight

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

Sample to Insight15

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)

Sample to Insight

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


Sample to Insight

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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


Sample to Insight

4

Agenda

18




time PCR setup

Applications:



talk



1

2

3

Sample to Insight

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

19

Sample to Insight

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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Sample to Insight

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

21

Sample to Insight

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

22

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)

23

Sample to Insight

4

Agenda

24




time PCR setup

Applications:



talk



1

2

3

Sample to Insight


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

…

Sample to Insight

Bring multiple colors to your qPCR – The new QuantiNova Multiplex PCR KitFor 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

Sample to Insight

The latest generation in real-time multiplex PCRBuilt-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

27

Sample to Insight

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

28

Sample to Insight

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

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Faster, most sensitive multiplex qPCR

Sample to Insight

QuantiNova MP PCR Kit – Improvement of performanceStability 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

30

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

31

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)

32

Sample to Insight

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

33

Sample to Insight

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

. F

luoro

.

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

. F

luoro

.

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

. F

luoro

.

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

. F

luoro

.

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

34

Sample to Insight

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

35

Sample to Insight

QuantiNova MP PCR Kit: Multiplex PCR of up to 6 targetsOutstanding 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

20n

g/rx

n

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

36

Sample to Insight


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(T a lle le)

Wild types(C a lle le)

Heterozygotes

Mutants(T a lle le)

Wild types(C a lle le)

HeterozygotesThe 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)

37

Sample to Insight

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

Norm

alised F

luorescence

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

Norm

alised m

inus 50%

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

Norm

alis

ed m

inus w

t

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

Norm

alis

ed F

luore

scence

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

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.

Sample to Insight

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 Runwild type

heterozygote

mutant

A/T Class IV SNP

minute differences between homozygote alleles (< 0.1°C)

40

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

41

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

Norm

alis

ed m

inus V

5

55

50

45

40

35

30

25

20

15

10

5

0

-5

-10

JE341/Pro805Normalized to V5:

V6 V5

V3

V7

ºC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

Norm

alis

ed m

inus J

E341/P

ro805

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

42

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

Norm

alis

ed F

luore

scence

100

80

60

40

20

13003

13009

13000

13026

1303113027

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

43

Sample to Insight

Clustering Analysis by ScreenClust on human references for potential health monitoring

44

Sample to Insight

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.

45

Sample to Insight

Multiplex PCR products in Applied Testing applications

46

y = -3,2871x + 40,0720

25

30

35

40

0 2 4 6

CT

Log target copy number

Aspergillus niger

y = -3,2356x + 41,0420

25

30

35

40

0 2 4 6

CT


Klebsiella pneumoniae

y = -3,1423x + 41,649

20

25

30

35

40

0 2 4 6

CT


Mucor/Rhizopus spp.

y = -3,1608x + 41,204

20

30

40

0 2 4 6

CT


PanAspergillus/Penicillium


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

Sample to Insight

T. Foetus (Fam) Internal Control (Yellow)

TH = 0.05

Tritrichomonas Foetus and InType IC – A clean multiplex assay

47

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

48

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

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-plexTH = 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

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)

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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:

[email protected]

[email protected]

[email protected]

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mailto:[email protected]

mailto:[email protected]

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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Sample to Insight

FAQs II


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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Sample to Insight

FAQs III


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).

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