Assurance Gds

Why develop another PCR system? Industry Needs  

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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 

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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 investigation/forensics  human disease research  industrial diagnostics / pathogen detection

PCR Amplification: cycle N = 2N copies e.g. Cycle 25 = 34 million copies = 3.4 x 107

How does PCR work? Required components: Primer -Single strand DNA

Target -Double stranded DNA sequence specific to the organism you are trying to detect

-DNA sequence that is amplified in PCR process

A C T G A

sequence complementary portions DNA found in the target -Responsible for initiating the copying of the “target” DNA sequence -Used in pairs ( forward and reverse primers)

Taq polymerase -Thermal

Nucleotides – A, T, C, G DNA

stable enzyme responsible for copying “target” DNA

building blocks used to assemble 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

A C T G A

T G A C T

Separation of the 2 strands when heated (95°C)

Step 2 - Primers anneal

A C T G A

T G A C T

Primers bind to their complementary target sequences when cooled (55°C)

Step 3 - Extension

A C T A GC A T

A T C G T A C T

Taq enzyme manufactures new strands of DNA (72°C)

Step 4 - Repeat cycle The entire process is repeated by cycling the temperatures

Step 5 - Detection End product of this process must be detected to determine if target was present

Advances in multiple areas Sample Preparation

Reagent 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  

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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  

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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 

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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 Probe in solution

Q= Quencher MGB = Minor Groove Binder

• Arranged in a random coil • Fluorescent marker quenched

Probe bound to target

• Probe becomes linear • Fluorescent marker exposed • MGB molecule attaches to stabilize

B MG

MGB

B MG

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  

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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

 Produces accurate & reliable test results  Generates confidence

les

 Rotor-Gene controlled PCR reactions are highly consistent

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

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70 mins

170 min

100 min

0

60 min

60 min

73 min

240 min

>2.5 hours!

Total cycle time ( 42 cycles) Melt Curve TOTAL

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 = shorter enrichment times

Probe eliminates melt curves

Thermal efficiency decreases cycle times

Specificity

IMS provides 1st level of specificity

Primer - only target is amplified Probe - only target is detected

Multi Channel system allows for use of highly specific probes

Ease of use

PickPen – simple 20 min sample prep

Lyophilized reagents in amp tubes

Definitive results - no melt curve interpretation

Accuracy

IMS separates target from PCR inhibitors

Dual specificity of primers Consistent PCR reaction in & probes each tube

Work Flow Sample Prep Add Concentration Reagent + 1 ml enriched sample

Sample Block

Add Resuspension Buffer Resuspension (RSP) Plate

Sample Block

RSP Plate

Transfer samples from sample block to 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)

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E. coli 0157:H7 Shiga toxins (AOAC OM 2005.05)

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Listeria monocytogenes Listeria spp. Salmonella spp.