THE MAGIC BOXES. And The Molecular Biology Behind the Marvels. April 28, 2016
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1 THE MAGIC BOXES And The Molecular Biology Behind the Marvels April 28, 2016 Raymond P. Podzorski, ph.d., D(ABMM) Ph.d. Microbiologist St. Mary s Hospital Laboratory Wisconsin Region SSMHC Madison, WI raymond_podzorski@ssmhc.com
2 Disclosure Raymond P. Podzorski, Ph.D., D(ABMM) April 28, 2016 No relevant financial relationships do disclose.
3 Objectives Illustrate how the number of nucleic acid amplification methods has grown over the years Review the technology behind some of the popular nucleic amplification test systems Discuss some of the mechanisms used to detect amplified product in these systems Show the hardware associated with this technology
4 Nucleic Acid Amplification Systems Polymerase Chain Reaction (PCR) Nucleic Acid Sequence Based Amplification (NASBA) Strand Displacement Amplification (SDA) Transcription Mediated Amplification (TMA) Ligase Chain Reaction (LCR) Replicase Branched DNA (bdna)
5 Nucleic Acid Amplification Systems Polymerase Chain Reaction (PCR) Nucleic Acid Sequence Based Amplification (NASBA) Strand Displacement Amplification (SDA) Multiple Displacement Amplification (MDA) Transcription Mediated Amplification (TMA) Ligase Chain Reaction (LCR) Replicase Cycling Probe Reaction (CPR) Hybrid Capture (HCII) Cleavase-based Amplification (INVADER) Branched DNA (bdna) Nicking Enzyme Amplification Reaction (NEAR) Loop-Mediated Isothermal Amplification (LAMP) Helicase Dependent Amplification (HDA)
6 isothermal Amplification Systems Helicase Dependent Amplification (HDA) Loop-Mediated Isothermal Amplification (LAMP) Multiple Displacement Amplification (MDA) Nucleic Acid Sequence Based Amplification (NASBA) Nicking Enzyme Amplification Reaction (NEAR) Ramification Amplification Method (RAM) Rolling Circle Amplification (RCA) Stand Displacement Amplification (SDA) Signal Mediated Amplification of RNA Technology (SMART) Single Primer Isothermal Amplification (SPIA) Transcription Mediated Amplification (TMA)
7 isothermal Amplification Systems Helicase Dependent Amplification (HDA) Loop-Mediated Isothermal Amplification (LAMP) Multiple Displacement Amplification (MDA) Nucleic Acid Sequence Based Amplification (NASBA) Nicking Enzyme Amplification Reaction (NEAR) Ramification Amplification Method (RAM) Rolling Circle Amplification (RCA) Stand Displacement Amplification (SDA) Signal Mediated Amplification of RNA Technology (SMART) Single Primer Isothermal Amplification (SPIA) Transcription Mediated Amplification (TMA)
8 Target Amplification System - PCR
9 FRET CONCEPT
10 FRET Probes Scorpion Adjacent Hybridization Probes TaqMan (hydrolysis) Molecular Beacon
11 FRET Probes
12 Illumigene Loop Mediated Isothermal Amplification
13 LAMP I. Production of Starting Material - All 4 primers used at the start
14 LAMP I. Production of Starting Material - All 4 primers used at the start Starting material For LAMP cycling
15 LAMP III. Elongation and Recycling Step II. Cycling Amplification 0 4- P 2 O P 2 O 7 P 2 O
16 Illumigene P 2 O 7 + Mg 2 = Loop Mediated Isothermal Amplification
17 AmpliVue Ampli-fied DNA/RNA + Vue-able Results Helicase Dependent Amplification
18 Primers 3X FITC-Probe
19 Primers 3X FITC-Probe
20 Primers 3X FITC-Probe
21 Primers 3X FITC-Probe
22 Primers 3X FITC-Probe
23 Primers 3X FITC-Probe
24 Primers 3X FITC-Probe
25 Primers 3X FITC-Probe
26 Step 4 Primers 3X FITC-Probe
27 AmpliVue Ampli-fied DNA/RNA + Vue-able Results Strepavidin coated latex beads imbedded in strip colored red Anti-DIG Ab at C-line Anti-FITC Ab at T-line B. pertussis C. difficile toxin A S. pyogenes S. agalactiae HSV 1+2 T. vaginalis Probe target Probe control
28
29 Manual Detection
30 Solana Helicase Dependent Amplification
31 Primers RNaseH2 RNA probe F 3X
32 Primers RNaseH2 RNA probe F 3X
33 Primers RNaseH2 RNA probe F 3X
34 F F Primers RNaseH2 RNA probe F 3X
35 F F F F F F Primers RNaseH2 RNA probe F 3X
36 F F F F F F F Primers RNaseH2 RNA probe F 3X
37 Solana Helicase Dependent Amplification
38 Panther Transcription Mediated Amplification
39
40
41 Transcription Mediated Amplification 1: Primer 1 incl. T7-promotor sequence binds to target 2: Rev. Transcriptase (RT) creates first strand of cdna RT 3: RNA:DNA duplex is completed 4: RNAse H activity of RT degrades the target RNA RT 5: Primer 2 binds to cdna and creates the second strand cdna 6: Double stranded DNA template including T7- promotor sequence generated RT
42 Transcription Mediated Amplification 7. RNA polymerase initiates transcription of RNA from DNA template 6. Double stranded DNA template including T7-promotor 12: Primer 1 incl. the T7-promotor binds and RT creates a new double stranded cdna RNA Pol RT 8. Production of copies of RNA transcript RT 9. Primer 2 binds to RNA amplicon and creates first strand of new cdna 10: RNA:DNA duplex is completed 11: RNAse H activity of RT degrades the target RNA
43
44
45
46 Panther - Fusion TMA RT-PCR
47 Panther - Fusion TMA RT-PCR
48 Alere i Nicking Enzyme Amplification Reaction
49
50
51
52
53 One template is in a higher concentration to drive asymmetric product formation.
54 One template is in a higher concentration to drive asymmetric product formation.
55 One template is in a higher concentration to drive asymmetric product formation.
56 One template is in a higher concentrati on to drive asymmetric product formation.
57 Asymmetric Generation of Product F F F F F F F F F
58 Alere i Nicking Enzyme Amplification Reaction
59 The END HDA NASBA SDA LCR LAMP
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