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 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
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 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
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
--
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)
E. coli 0157:H7 Shiga toxins (AOAC OM 2005.05)
Listeria monocytogenes Listeria spp. Salmonella spp.