technologies for multiple pathogen detection
TRANSCRIPT
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Technologies for Multiple
Pathogen Detection
Patrick Tang, MD, PhD, FRCPC
Medical Microbiologist,BCCDC Public Health Microbiology & Reference Laboratory
Clinical Assistant Professor, UBC
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Technologies for Multiple
Pathogen Detection
• Molecular diagnostic
technologies
• Challenges of multiplex
pathogen detection
• Future of molecular
microbiology
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BCCDC Public Health
Microbiology & Reference
Laboratory
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BCCDC Public Health
Microbiology & Reference
Laboratory
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Limitations of Traditional Methods
Speed
Sensitivity
Taxonomy-based
Spectrum of targets
Specialized training
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Ideal Microbiology Test
• Rapid
• Sensitive and specific
• Syndrome-based multiplexing
• Shared technology platform
• Common laboratory training
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Detection of Host Response
Serology(Host
Immune
Response)
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Microscopy
AntigensAntigens Nucleic AcidsNucleic Acids
Culture
Detection of Pathogen
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Nucleic Acid Detection
• Non-amplified Nucleic Acid Probe
– labeled DNA or RNA probe (enzyme, fluorescence, etc.)
• Signal Amplification
– increase concentration of labeled molecules attached to
target
• Target Amplification
– enzyme-mediated synthesis of copies of the target nucleic
acid
• Probe Amplification
– amplification products generated only from probes
• Metagenomics (sequencing)
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Non-Amplified Nucleic Acid Probe
• Liquid-phase hybridization protection assay
(Gen-Probe)
• Single-stranded DNA probe labeled with
acridinium ester is added to sample
• If the probe binds to its complementary target
sequence, the acridinium ester is protected
from alkaline hydrolysis
– otherwise, acridinium ester will be hydrolyzed
• Acridinium ester emits light upon addition of
peroxides
target sequence
DNA probe
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Quantum Dots
• Semiconductor nanocrystals
• Quantum confinement of electrons
• Shape and size determine fluorescence
spectrum
• 100 times brighter than regular fluorescent
report dyes
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• Electrochemical detection
• Capture probe is bound to sensor surface
• HRP-linked detection probe binds to second site on target DNA
• HRP reaction generates current proportional to target concentration
HRP
e-
GeneFluidics
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Signal Amplification
• Branched DNA (Siemens)– sandwich hybridization
assay with multiple sets of probes
– bDNA has 15 identical branches, each can bind 3 labeled probes
capture probes
target sequence target probes
bDNA
enzyme-labeled probes
microwell with capture probes
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Nanosphere
• Microarray-based platform
• Gold nanoparticle detection
• Signal amplification through deposition of elemental silver onto gold nanoparticles
• Measure light scatter from nanoparticles
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Target Amplification
• Polymerase Chain Reaction (PCR)
• Transcription-mediated amplification (TMA) /
Nucleic acid sequence-based amplification
(NASBA)
• Strand displacement amplification (SDA)
• plus many other strategies…
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Multiplexing
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectroscopy detection
• Sequencing
• Spatial multiplexing
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectroscopy detection
• Sequencing
• Spatial multiplexingR Q
TaqMan 5’ Exonuclease Probe
Molecular Beacon
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectroscopy detection
• Sequencing
• Spatial multiplexingLine Probe Assays
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectroscopy detection
• Sequencing
• Spatial multiplexing
AutoGenomics
INFINITI
Akonni
TruArray
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectroscopy detection
• Sequencing
• Spatial multiplexingLuminex
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectrometry
• Sequencing
• Spatial multiplexing Abbott PLEX-ID
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectroscopy detection
• Sequencing
• Spatial multiplexing
Roche GS Junior
Illumina MiSeq
Life Technologies
Ion Torrent
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Multiplex PCR
• Electrophoresis
• Fluorescent probes (FRET)
• Hybridization
– Microarrays
– Bead-based arrays
• Mass spectroscopy detection
• Sequencing
• Spatial multiplexing
Life Technologies
OpenArray
Idaho Technology
FilmArray
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LAMP
• Loop mediated isothermal amplification
• Precipitation of pyrophosphates released during DNA amplification
• Visible turbidity
– Can modify to produce
color or fluorescence
• Inexpensive
– No thermal cycler
– Visible to naked eye
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Nucleic Acid Lateral Flow
• Simple, fast, cheap– potential for developing world POC diagnostics
• May incorporate target amplification or signal amplification to increase sensitivity– detection using enzymes, quantum dots, colloidal gold, etc.
• Future devices may incorporate integrated solid phase isothermal nucleic acid amplification
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Which is the best one for my lab?
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Technical Challenges of Multiplexing
• Nucleic acid extraction
• Loss of sensitivity
• Mispriming
• Cross-hybridization
• Competition
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Expensive
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** Not for Diagnostic Testing **
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Centralized Testing
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Batch Testing
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New Pathogens
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Budgets
Perpetuating the Old Microbiology
Education
Laboratory infrastructure
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Where are we headed?
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BCCDC Public Health
Microbiology & Reference
Laboratory
Integration of new point-of-care diagnostics onto wards
Improved rapid serologic tests (antigen or antibody)
Simple, rapid chip-based or lab-in-a-box molecular tests
• Real-time PCR, microarrays, single-molecular detection
Target the most common pathogens• Syndromic-based testing
Expansion of clinical teamPhysicians, nurses, pharmacists
plus laboratory technologists• Operation and troubleshooting of advanced diagnostic
equipment
The Future…
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BCCDC Public Health
Microbiology & Reference
Laboratory
Hospital laboratoriesFocus on rapid tests targeting common pathogens (multiplexing based on syndrome)
More molecular tests, fewer culture-based tests
Reference laboratoriesTraditional culture and phenotypic tests
•Maintenance of sequence databases to ensure molecular assays reflect the circulating strains of various organisms
High volume tests•Tests which do not require rapid turn-around times
Advanced molecular tests•Microarrays
•Metagenomics
Genotyping and characterization of organisms•Whole genome sequencing
The Future…
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Point-of-Care Molecular Tests
• Integrated sample preparation
• Sealed lab-in-a-box systems
• Fast turn-around time
• High sensitivity
Cepheid GeneXpert
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Point-of-Care Molecular Tests
Idaho Technologies
FilmArrayIQuum Liat Analyzer
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Advancing the Molecular Revolution
• Revolution requires re-thinking of the entire
system of delivering laboratory services
• Many new technologies emerging
– What is hype and what is the real thing?
• New technology inherently costs more
– How do we pay for it all?
• POC molecular tests are here
– How will we manage POC testing?
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BCCDC Public Health
Microbiology & Reference
Laboratory
Questions