Assurance GDS

Why develop another PCR system?
Industry Needs
 Choices in PCR systems
 Improved Accuracy
 Reduction in “indeterminates”
 Fewer “False Positives”
 Greater confidence in results
 Faster Results
 Test and policies
 Control costs associated with holding inventory
 Larger return on investment

What is Assurance GDS?
Genetic Detection System
 Contains multiple levels of specificity
 Immuno-magnetic sample concentration
 Primers
 Probe
 Offers real-time results

 Utilizes an innovative, highly efficient rotary cycler
 Practical, user-friendly sample preparation
What is PCR? DNA
Polymerase Chain Reaction

 Nobel prize winning
discovery
 Rapidly amplifies a single
molecule or specific
fragments of DNA into many
millions copies
 Many applications, e.g.
 crime scene PCR Amplification: cycle N = 2N copies
investigation/forensics e.g. Cycle 25 = 34 million copies = 3.4 x 107
 human disease research
 industrial diagnostics /
pathogen detection
How does PCR work?
Required components:
Target -Double stranded DNA Primer -Single strand DNA
sequence specific to the A sequence complementary portions
organism you are trying to detect DNA found in the target
C
-DNA sequence that is
T -Responsible for initiating the
amplified in PCR process
G copying of the “target” DNA
A sequence
-Used in pairs ( forward and

reverse primers)
Taq polymerase -Thermal Nucleotides – A, T, C, G DNA

stable enzyme responsible for building blocks used to assemble
copying “target” DNA copies of the “target” DNA
-Isolated from the bacteria

Thermus aquaticus discovered
in Yellowstone hot springs
How does PCR work?
PCR Amplification – One reaction (cycle)
Step 1 - DNA denaturation Step 3 - Extension
A T Separation A A T Taq enzyme
C G of the 2
C C G manufactures new
T A strands
T A T A
strands of DNA
when heated (72°C)
G C (95°C) GC C
A T A T T
Step 2 - Primers anneal Step 4 - Repeat cycle

A T Primers bind to their
The entire process is repeated
by cycling the temperatures
C G complementary target
T A sequences when
C Step 5 - Detection
G cooled
T (55°C)
A End product of this process must be
detected to determine if target was
present
Advances in multiple areas
Sample Reagent
Preparation System
Instrument
Platform
Accuracy + Speed + Ease of Use

Traditional Approach to
Sample Preparation
Issues with PCR:

 Many food samples contain PCR inhibitors
 Existing DNA extraction methods are tedious and impractical
Traditional approach = Sample Dilution

 reduces inhibitors but compromise sensitivity & accuracy
 requires higher levels of organisms = longer enrichment
times
 Results in more hands-on time
Assurance GDS
Sample Preparation
Patent-pending IMS-based method

 1st of 3 levels of specificity
 Antibody coated magnetic particles
 captures & concentrates target organisms
 physically separates target from food matrix leaving behind PCR inhibitors
 Greater accuracy
 Dilution protocols provides 1.25 uL target DNA
 IMS concentration brings 800 - 1000 uL of target DNA to the PCR tube
Reagent System Evolution
1st generation PCR

 Primers to start amplification
 Results determined by reading of gels
 Open tube system, susceptible to cross
contamination
 Primers only tool to determine specificity
 Gels cumbersome and require interpretation
Reagent System Evolution
2nd generation PCR

 Amplification and detection occurs inside the same PCR tube
 Non specific fluorescent dyes ( e.g. SYBR Green ) bind to any
double stranded DNA, including
 target amplicons
 Internal control amplicons
 non-specific products
Reagent System (Test kit)
2nd generation PCR

 Non specific fluorescent signal reflects presence &
amplification of all above components
 Extra time - curve analysis needed before results are
available
 Potential for questionable results - interpreting melt curve
shape and location
Assurance GDS
Reagent System
Next generation PCR

 Greater Accuracy
 2 levels of specificity
 Primers – responsible for amplifying target DNA
 Probe – responsible for detection of target DNA
 True internal control validates results
 No melt curves
How the Probe Works
F = Fluorophore
hv Q= Quencher
Probe in solution
• Arranged in a random coil MGB = Minor Groove
• Fluorescent marker quenched Binder
B
MG
Probe bound to target
• Probe becomes linear
• Fluorescent marker exposed
MGB
• MGB molecule attaches to stabilize
MG
B
Direct correlation between the amount of fluorescence and the amount of target
Instrument Platform
1st generation instrumentation

 Amplification and detection on separate instruments
 Peltier block based heating / cooling
2nd generation instrumentation

 Amplification and detection on a single instrument
 PC & software package control cycling and interpretation of results
 Single channel = 1 signal
 Peltier block based heating / cooling
Peltier Block-Based
Instrument Platform
Limitations
 Inconsistent temperature cycling
 Solid block format produces variable temperature
gradients
 Accuracy of PCR results from cold wells?
 Standard practice to avoid use of outer wells
Peltier Block-Based
Instrument Platform
Limitations
 Transfer of heat through a solid
surface
 Increased dwell times at each temp
change
 All wells must equilibrate and hold
temp to allow PCR process to occur
 Slow heat transfer leads to slow PCR
process and prolonged cycle times
Assurance GDS
Instrument Platform
Assurance GDS Rotor-GeneTM

 Samples arranged in a rotary format for direct
heating / cooling
 Centrifugal motion with constant air exchange
 Ensures uniform temperature among all
reactions
 Eliminates lengthy dwell times required by
block based systems
Assurance GDS
Instrument Platform
Improved Accuracy
 Rotor-Gene controlled
PCR reactions are highly
consistent
 Produces accurate &
reliable test results
les
 Generates confidence
Test #
Single Channel
Instrument Platform
Limitations of Single Channel

 Can not read separate signals
 Dependent upon non-specific indicator dyes (Sybr Green) and melt curve analysis
 1 signal for target, internal control, and PCR artifacts
 Extra time – melt curve analysis requires > 1hr
 Ambiguous interpretation of results - melt curve shape and location
Assurance GDS
Instrument Platform
Assurance GDS Rotor-GeneTM

 Multi Channel system – 3 discrete channels
 Each channel has separate light source
 Separate target and IC signals
 Alleviates dependency on melt-curves and provides
real-time detection
 Provides true multiplexing capabilities
Assurance GDS
Instrument Platform
Faster Results
PCR Step Rotary Block Time savings
Starting denaturation time 3 min 10 min 7 min
Typical cycle time <2 min 4 min --
Total cycle time ( 42 cycles) 70 mins 170 min 100 min
Melt Curve 0 60 min 60 min
TOTAL 73 min 240 min >2.5 hours!
Assurance GDS
Instrument Platform
Definitive +/- results

 Real-Time results
1-button reports
Secure data files
Advantages of Assurance GDS
Sample Prep Reagent System Instrument Platform
Speed IMS concentrates target = Probe eliminates melt Thermal efficiency

shorter enrichment times curves decreases cycle times
Specificity IMS provides 1st level of Primer - only target is Multi Channel system
specificity amplified allows for use of highly
Probe - only target is specific probes
detected
Ease of use PickPen – simple 20 min Lyophilized reagents in Definitive results - no melt
sample prep amp tubes curve interpretation
Accuracy IMS separates target from Dual specificity of primers Consistent PCR reaction in
PCR inhibitors & probes each tube
Work Flow
Sample Prep
Add Concentration Reagent
Sample Block + 1 ml enriched sample
Add Resuspension Buffer

Resuspension (RSP) Plate
Transfer samples from

sample block to RSP plate.
Sample Block RSP Plate
Amplification & Detection
Add Polymerase to each

amp tube.
Transfer from RSP plate
to amp tube.
Place amp tubes into Assurance

Rotor-Gene™
Click Start.
A complete family of assays
 E. coli 0157:H7
(AOAC OM 2005.04)
 E. coli 0157:H7 Shiga toxins
(AOAC OM 2005.05)
 Listeria monocytogenes
 Listeria spp.
 Salmonella spp.

Assurance GDS

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Why develop another PCR system?

 Fewer “False Positives”

 Greater confidence in results

 Control costs associated with holding inventory

 Larger return on investment

 Offers real-time results

Polymerase Chain Reaction

-Used in pairs ( forward and

Taq polymerase -Thermal Nucleotides – A, T, C, G DNA

-Isolated from the bacteria

Step 2 - Primers anneal Step 4 - Repeat cycle

Accuracy + Speed + Ease of Use

Issues with PCR:

Traditional approach = Sample Dilution

Patent-pending IMS-based method

1st generation PCR

2nd generation PCR

 Internal control amplicons

2nd generation PCR

Next generation PCR

1st generation instrumentation

2nd generation instrumentation

Assurance GDS Rotor-GeneTM

Limitations of Single Channel

Assurance GDS Rotor-GeneTM

Definitive +/- results

Sample Prep Reagent System Instrument Platform

Speed IMS concentrates target = Probe eliminates melt Thermal efficiency

Add Resuspension Buffer

Transfer samples from

Amplification & Detection

Add Polymerase to each

Place amp tubes into Assurance

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