current and future developments in nucleic acid- based...
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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