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Current and Future Developments in Nucleic Acid-

Based Diagnostics

Gerrit J. Viljoen1, Marco Romito2 & Pravesh D. Kara2

FAO/IAEA Joint Division1, Austria,

Onderstepoort Veterinary Institute2, South Africa

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4. Nucleic acid amplification 5. Signal amplification and detection

3. Sample processing6. Real-time

1. Introduction

2. Bioinformatics7. Probes

Current Developments in Nucleic Acid-based Diagnostics

8. Fingerprinting

12. Integrated systems 9. Sequencing

11. Microarrays 10. Biosensors

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INTRODUCTION• Direct pathogen detection

• Advantages: Stable, sensitive, potentially highly informative

• Where serology is uninformative; allows for improved molecular characterisation - epidemiology, theranostics

• Contributing developments:

• R.E.s, plasmid cloning

• Hybridization: dot, Southern & northern blotting

• Sequencing

• NA amplification

• Future - improvements with:

• Target & signal amplification

• Direct detection without amplification

• Increased throughput

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BIOINFORMATICS

• Genomic data storage and analysis

• Probe and primer design

• Identification of diagnostic molecular targets

• Microarray data

• Pattern recognition tools

• Molecular typing & phylogenetics

- diagnostics, epidemiology, theranostics

• Improved computational methods, data banks, integration of datasystems

• Data bases; biomedical literature; data and text mining tools

• Communications technologies - internet and wireless systems

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SAMPLE COLLECTION AND PROCESSING

• Ensure stability of DNA / RNA with minimal interference

e.g. DNA/RNA ProtectTM (Sierra Diagnostics), BD VacutainerTM , CPTTM, PPTTM (Beckton Dickinson); swab systems (Medical Packaging Corporation)

• More rapid and efficient release of NAs -

commercial kits, cycled pressure devices, MagNALyser

• Immunomagnetic separation; paramagnetic beads

• DNA binding silica-coated beads; capture probes; poly dT

• Robotic devices e.g. MagNAPure (Roche)

• Integrated systems - processing, amplification, analysis

using microfluidics, microfabrication, nanotechnology

6Picard and Bergeron, 2002, DDT 7: 1092-1100

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NUCLEIC ACID-amplificationTarget amplification

• PCR

• RT-PCR, Hot Start, assymetric PCR, internal controls, multiplexing, contamination control, quantification, improved instrumentation, enzymes etc.

• Advantages:

• detection of difficult to isolate pathogens

• rapid, sensitive

• detection of latent carriers

• where serology is ambiguous

• potential to distinguish different strains, vaccine vs field type etc.

8Versalovic and Lupski, J.R., 2002, Trends Microbiology 10: S15-S21.

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Transcription mediated assays:

TMA, NASBA

Weusten J.J.A.M. et al., 2002, Nucleic Acids Research 30, E26.

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Transcription mediated assays:

Signal Mediated Amplification of RNA Technology ( SMART)

Wharam, S.D. et al ., 2001, Nucleic Acids Research 29, E54.

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Strand Displacement Amplification (SDA)

Spargo, C.A. et al., 1996, Molecular and Cellular Probes 10: 247-256.

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Loop-mediated Isothermal Amplification (LAMP)

Notomi, T. et al., 2000, Nucleic Acids Research. 28, E63.

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Probe amplification

• OLA, LDR → LCR

Lee, H.L., 1996, Biologicals, 24: 197–199

• PCR-LDR

• Gap-LCR

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Rolling Circle Amplification

Schweitzer, B. and Kingsmore, S. , 2001, Current Opinion in Biotechnology 12, 21-27.

Ramification Amplification Assay

Zhang et al., 2001, Molecular Diagnosis 6: 141-150.

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Signal amplification

• Branched DNA

Nygren, M., 2000, Royal Institute of Technology, KTH, Stockholm, Sweden.

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Invader assay (Third Wave Technologies, USA)

Arruda, M. et al., 2002, Expert Rev. Mol. Diagn. 2: 487-496.

17Picard and Bergeron, 2002, DDT 7: 1092-1100

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PROBES

• Hydrolysis probes: TaqMan

• Molecular beaconshairpin loop

Uhl, J.R. et al., 2002, Mayo Clin Proc. 77: 673-680

• Scorpion primer-probes: 5’ -hairpin loop

Primer extension followedby probe binding

Thelwell, N. et al., 2000, Nucleic Acids Research 28, 3752-3761.

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• Smart probes: hairpin loop, oxazine dye, guanosine quenching

• Hybridization probes: singly labelled dual probes; donor and acceptor fluorophore

Uhl J.R., et al. , 2002, Mayo Clin Proc. 77: 673-680

LightCycler (Roche)

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Hybridization probes - other:

• Acceptor-labelled primer with donor-labelled probe

• Labelled probe hybridizes adjacent to G (quencher)

Lyon, E. , 2001, Expert Rev. Mol. Diagn. 1: 92-101.

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•Padlock probes: ligation and circularisation;

• RCA and / or probe detection

• Chimeric probes - RNA-DNA hybrids -Cycling Probe Technology; RNase H cleavage

• Other -

• Catalytic probes - hybridization & ribozyme domain + beacon substrate

• Hybrid capture (Digene)RNA probes; enzyme-labelled Ab detectDNA-RNA hybrids

Baner et al. ,2001, Current Opinion in Biotechnology 2001, 12:11–15

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NUCLEIC ACID ANALOGUES• Peptide nucleic acids - rapid, stronger binding nuclease resistant, no amplicons; more robust detector probes • Stemless PNA probes -

e.g. LightSpeed PNA probes

• LightUp PNA probes

• Q-PNA PCR: probe masks labelled primer, unmasked during extension

• PD loop technology - dsDNA binding

• Locked nucleic acidsO2'- to C4' methylene-links; strong hybridization affinities & efficient mismatch discrimination - DNA & RNA

Demidov, V.V. (2001) Expert Rev. Mol. Diagn. 1, 343-351.

Stender et al., 2002, J Micro Meth 48: 1-17.

Kurreck, J. et al., 2002, Nucleic Acids Research. 30, 1911-1918.

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REAL-TIME ASSAYS

• Fluorescence intercalating dyes (SYBR Green) and FRET probes e.g. Taqman and dual hybridization probes

• Tube-based or microtitre plate-based platforms: ABI7700 (Applied Biosystems), MX4000 (Stratagene), iCycler (Bio-Rad) SmartCycler (Cepheid) and Robocycler (MJ Research)

• Air heating/ cooling: LightCycler (Roche), glass capillaries; melting curve analyses

• Quantification - exponential curve & cycle threshhold (Ct) determination

• Lower turn-around times

• Closed system

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PORTABLE DEVICES

• R.A.P.I.D.® (Idaho Technology, Salt Lake City, UT); RAZOR

• Advanced nucleic acid analyzer (ANAA)

• SmartCycler (Cepheid; Sunnyvale, CA)

• Hand-held advanced nucleic acid analyzer (HANAA) (Lawrence Livermore National Laboratory)

Hand Held Advanced Nucleic Acid Analyser

Lawrence Livermore National Laboratory, CA

PolyANAA a 24 sample PCR instrument for fly-away lab use

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Labelling and detection systems• Radioactivity

• DIG-dUTP; Biotin-dUTP

• Intercalating dyes (SYBR Green) and fluorescent (FRET) probes

• Incorporation by nick translations, PCR

• Chemical modification: Psoralen, alkylating agents and ULS® (Kreatech).

• PCR-Immunochromatography System

• Hybridization protection assay (chemiluminescence)

• Tessera Array Technology (TAT; Applied Gene Technologies; San Diego)

• Microspheres

• Nanoparticles - gold; quantum dots

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GENOTYPING AND FINGERPRINTING

• RFLP, ribotyping

• PCR:

• Real- time PCR, FRET probes ,

• PCR-RFLP, RAPD, rep-PCR, spoligotyping, AFLP

• DNA sequencing Post-amplification sequencing, dideoxy fingerprinting

• Reverse hybridization - linear probe arrays, microarray

• RNase A mismatch cleavage

• Separation techniques: PFGE, SSCP, DSCA, CDGE, DGGE, TGGE

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Amplification Fragment Length Polymorphism

Savelkoul et al. 1999, JOURNAL OF CLINICAL MICROBIOLOGY. Oct. 1999, p. 3083–3091

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SEQUENCING AND ALTERNATIVES

• ssDNA generation: solid phase sequencing; assymetric PCR;exonuclease generated ssDNA; transcript sequencing

• Cycle sequencing: Taq polymerase + single primer, thermocycling

• Mutated Taq DNA polymerase F667Y

• Capillary array electrophoresis

• Automated sequencing

Nygren, M.,(2000, Royal Institute of Technology, KTH, Stockholm, Sweden.

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Novel sequencing methods

• Sequencing by hybridization e.g. Microchips,

• Future: microfluidics, integration

• MALDI-TOF Mass Spectrometry

• Pyrosequencing

• Atomic Force Microscopy

• Minisequencing

Berg, L.M. et al (2002) Expert Rev. Mol. Diagn. 2, 361-369.

Nygren, M. ,2000, Royal Institute of Technology, KTH, Stockholm, Sweden.

•Nygren, M. ,2000, Royal Institute of Technology, KTH, Stockholm, Sweden.

Berg, L.M. et al. 2002, Expert Rev. Mol. Diagn. 2, 361-369.

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MICROARRAY TECHNOLOGIES• Addressable probes, immobilised on solid support

• > 500 features/cm2 (cf: < on macroarrays)

• Spotted microarrays

• High density microarrays

• Bead arrays - flow cytometry

• Glass or silicon chips

• Semiconductor chips - microelectronics, microfabrication technologies

• Detection: Fluorophores; enhancer molecules; gold nanoparticles, microfabricated cantilevers

• Allow multiple hybridization tests simultaneously

• Current applications: Species identification, strain typing, drug resistance, pathogenicity determinants

• Future: Microfluidics and integration

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BIOSENSORS• Biological receptors in DNA sensors : DNA, ion channels

• Transducers : electrodes, FET, optical, optoelectronical, thermistors, piezoelectric crystals, cantilevers

• Detection: Enzyme labelling; magentic beads; nanopatricles; redox mediators and intercalators; surface mass changes

• End goal: point-of-care testing devices

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Optical fibresSPR

Magnetic sensorsFET

Edelstein, R.L. et al. 2000, Biosensors and Bioelectronics. 14, 805-813.

Microfabricated cantilevers

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INTEGRATED MICROFABRICATED/MICROFLUIDIC SYSTEMS

• Integration of processing, amplification and detection

• Microfabrication

• Microfluidics: microchannels, electroosmosis, pneumatics

• “Lab-on-chip” technologies

• Point-of-care devices

• Nanotechnology Miniature thermal cycler

Shimadzu Developments in Biotechnology

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Conclusion

• Rapidly growing sequence data bases• Improved NA extraction from biological samples• Real-time and portable devices• Microchips, miniaturisation• Future goals: - Enhanced sensitivity, specificity

rapidity, versatility, direct target detection• Multiplexing and multiple pathogen detection • Point-of-care use• Integrated systems• Communications technologies

Picard and Bergeron, 2002, DDT Vol. 7, No. 21: 1092-1100