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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.