Mobile nucleic acid amplification and detection systems. Animal Production and Health Sub-programme

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1 Mobile nucleic acid amplification and detection systems Animal Production and Health Sub-programme Hermann Unger OIE, Berlin 2013

2 Our mandate To improve livestock production through the support of problem-orientated research that identifies the constraints on production and develops cost-effective and sustainable solutions uses nuclear and nuclear related technologies Transfer of technologies

3 Mobile molecular diagnostics Early systems were based on qpcr technology requiring bulky equipment, sample extraction and sophisticated data analysis.

4 The last remaining DXNA (2005); qpcr; complete from extraction to result, 2-3 pathogens

5 Pseudo-Isothermal Amplification Convection PCR or Insulated Isothermal PCR Primer design simple; < 100 bp Probe: design easy but short One step RT possible Extraction required

6 Isothermal amplification Early attempts before the development of PCR Since ~ 2000 a number of techniques evolved As no cycling is required these solutions claim to be Mobile Simple Low cost Robust Quick

7 Isothermal Amplification Basic need: Separate DNA strands without heating Helicase Endonuclease Recombinase Nicking Enzyme By a strand displacing polymerase (BST, BSM, Phi29 )

8 Helicase Depended Amplification (HDA) Process Helicase binding and DNA unwinding Primer binding DNA polymerisation DNA amplification Reporting: Fluorescent, LUX primer Requirements Thermostable Helicase (~65C) ATP dependent DNA extraction required Denaturation of sample required

9 Recombinase Polymerase Amplification Process Primers covered with Recombinase ssdna binding Prot. supports displacement Polymerase amplification & displacement Probe or intercalating dye Requirements Thermostable Recombinase (~42C) Complex mixture of enhancers Sample prep required Extremely quick Simple primer design RNA and DNA ELISA reader possible!

Loop mediated isothermal AMPlification Process Outer Primers attach to dsdna => displacement Inner primers attach and elongate Flipping of terminal loops (Loop primer attach) Next elongation

10 Loop mediated isothermal AMPlification Process Outer Primers attach to dsdna => displacement Inner primers attach and elongate Flipping of terminal loops (Loop primer attach) Next elongation duplicates the length Detection by ID, probe,..pyrophosphate Requirements Sample prep by simple dilution Temperature 55-65C Runs minutes Primer design not trivial RNA and DNA Mobile device available Highly specific

Rolling Cycle Process Target & primer bind to probe DNA polymerisation & amplification DNA displacement for next binding Rolling cycle amplification Reporting: Fluorescent probes

11 Rolling Cycle Process Target & primer bind to probe DNA polymerisation & amplification DNA displacement for next binding Rolling cycle amplification Reporting: Fluorescent probes Requirements Sample prep needed Pre treatment of DNA with Padlock P Temperature low (37C) RNA amplification complex Runs 1-4 hours Primer design not trivial Only DNA Highly specific

Strand Displacement Amplification Process Target generation by primer on ssdna Complete restriction site at each end Enzyme nicks the dsdna Polymerase attaches, extends and

12 Strand Displacement Amplification Process Target generation by primer on ssdna Complete restriction site at each end Enzyme nicks the dsdna Polymerase attaches, extends and displaces Exponential amplification of products Requirements Sample prep needed Temperature 30 55C DNA only Runs 1-4 hours Nuclease selection not trivial Only DNA Highly specific due to Φ29

polymerase Reporting: molecular beacon Requirements For RNA or ssdna!

13 NASBA Process Primer 1 with T7 promoter attaches Reverse transcription RNaseH splice & Primer 2 attachment dsdna template for T7 RNA polymerase Reporting: molecular beacon Requirements For RNA or ssdna! Sample extraction needed 3 enzymes!! Temperature first 65C then ~41C Runs 1-4 hours

14 Isothermal Amplification Advantages Only few operation steps Some work without sample extraction Disadvantages Quantification of template DNA/RNA not possible Quality control missing Only few tests available How to validate with crude samples??

15 Isothermal Detection Systems Chemical (LAMP) Pyrophosphate Hydroxy naphthol blue dye Calcein Intercalating dyes Probes and beacons Lateral flow systems Electrochemistry

Chemical detection (LAMP) Pyrophosphate Due to

Calcein Manganous ions attached to Calcein are

16 Chemical detection (LAMP) Pyrophosphate Due to excess of Mg+ complex build up => white salt Hydroxy naphthol blue dye The colour change is induced by chelation of Mg2+ ions by dntps Calcein Manganous ions attached to Calcein are absorbed by Pyrophosphate leading to fluorescence.

17 Intercalating dye detection Picogreen Sybr green EVA green

18 Probes and beacons Quencher probes: (LAMP) exploited by DIASORIN (IT) cleavage of a fluorophore quencher Multiplexing possible Quencher probes: (RPA) exploited by TwistDx LUX-primers (Light Upon extension) for RPA Molecular beacon: NASBA

19 Lateral Flow Systems Digoxin labelled primer Biotin labelled probe

20 Electrochemistry Several techniques Sensitive Basically cheap Problems with detergents Difficult interpretation

21 Sample Preparation Alkaline Lysis: NaOH + SDS, neutralization, ethanol Phenol and chloroform extraction (TRIZOL) Silica-based NA absorption in the presence of chaotropic salt and affinity chromatography (columns) Labelled antibodies in tube for pathogen extraction Boiling of samples Dilution of samples

22 Inherent Problems of Isothermal Assays Highly specific: => Too specific!! (LAMP) High amplification efficiency: Risk of false priming Risk of contamination Low temperature: design of primers Visual reading: difficult task Lateral Flow and contamination (open tube) Which QC system to employ??

23 Interpretation of Results Fluorometric systems use qpcr like thresholds Problem when samples are not extracted! Background Optical systems use best guess Turbidometry based on threshold Lateral Flow: easy but false pos. Probes and beacon systems Threshold?? Quality control??

24 Future for Result Interpretation? Increase / t for fluorescence => efficiency as a QC parameter Tm analysis: is an amplification product correct? Absolute or relative measurement of fluorescence? Influence of contaminants in crude samples.

25 And what means mobile??? Sample prep must be quick and easy LAMP: crude samples diluted in buffer or short boiling Pipetting steps must be few Reagents must be thermo-stable Must be cheap, as many Vets will carry tests around Must have quantifiable data for QC and GSM transfer Low tech for un-critical results Malaria, High tech for critical results: FMD, HP Influenza, ASF

26 Our Experience with LAMP A lyophilised Mastermix is stable for >1 year BSM polymerase is great for low Temperature primers Trehalose can be exchanged for Betain EVAgreen is not inhibiting reaction One step approaches works without problem Together with a reader one can perform QC in the field Samples with low enzyme activity can be used directly Milk and Urine need only 1 centrifugation step

27 Future Perspectives The high number of interfering patents and start up companies will lead to a concentration on the market Resources for enzymes are limited and thus costly Multiplex approaches are under development, but are they useful?? Tests without sample extraction will play a major role, but the result evaluation will need careful design A very easy, cheap and robust test is not yet on the market

28 Improving animal productivity and health through nuclear and molecular technolo LAMP in the field Thanks a lot!

29 Jasper Bouman:

30 Some good reading Diagnostic Devices for Isothermal Nucleic Acid Amplification Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices Miniaturized isothermal nucleic acid amplification, a review a

Technical Review. Real time PCR

Technical Review. Real time PCR Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously