Molecular Approaches to Diagnosis and Characterization of Bacterial Infections

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Molecular Approaches to Diagnosis and
Characterization of Bacterial Infections

• Juan Pablo Olano M.D.
• Associate Professor, Department of Pathology
• Director, Residency Training Program
• Member, Center for Biodefense and Emerging
  Infectious Diseases. UTMB
• Pathogenic Bacteriology, 2007

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General Principles

• Which organisms to test for? Is it better than
  traditional methods?
• Which clinical specimens should be tested
• Sensitivity, specificity, speed, simplicity and
  clinical relevance.

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Techniques

• Non-amplified nucleic acid probes
• Liquid phase
• Solid phase
• In situ hybridization.
• Specimens Pure cultures for final ID, body
  fluids, tissues.

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Techniques

• Liquid phase
• Fastest
• Detection Hybridization protection assay
  (alkaline hydrolysis) and acridinium ester.
• PNA probes (Peptide nucleic acid probes)
• Solid phase
• Nucleic acid bound to membranes and probed with
  nucleic acid in solution.
• Detection Fluorescent, luminescent, radioactive,
  enzymatic.
• Variants Southern and Northern blot.

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Techniques

• In situ hybridization
• Whole cells or tissue section affixed to glass
  slides.
• Size of the probe critical for success of the
  test.
• Clinical applications in formalin-fixed paraffin
  embedded tissues.

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Techniques

• Nucleic acid amplification techniques
• Signal amplification techniques
• Low risk of contamination (greater specificity).
• Sensitivity still lower than target nucleic acid
  amplification techniques.
• High background noise due to non-specific binding
  of reporter probes.
• Signal directly proportional to amount of target.
  
• Less demanding DNA extraction techniques.
• Direct measurements of RNA without cDNA synthesis

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Techniques

• Signal amplification techniques
• Hybrid capture assays. Labeled antibodies and CL
  detection
• Branched DNA (bDNA).
• Highly reproducible, less sensitive than enzyme
  amplification systems.
• Background noise has been reduced with use of
  isoC and isoG in labeled probes.

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Techniques

• Target amplification techniques
• PCR
• RT-PCR
• Nested PCR
• Multiplex PCR
• Arbitrarily primed PCR
• Broad range PCR
• Real time PCR

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Techniques

• PCR
• RT-PCR
• RT technology
• Single enzyme technology
• More specificity More stringency and less
  secondary structures from target mRNA
• Nested PCR
• Contamination as a risk
• Single tube technology or oil/wax separating both
  mixtures

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Techniques

• Multiplex PCR
• Two or more targets amplified in the same
  reaction.
• No complementarity between different primers
• Similar annealing temperatures
• Less efficient than single PCR reactions
• Multiplex and luminex technology (spectral
  adressess for microspheres).

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Techniques

• Arbitrarily primed PCR (ramdomly amplified
  polymorphic DNA assay).
• Single short primer under low stringency
• Detection of species, serotypes and subtypes.
• Falling into disuse
• Broad range PCR
• Primers designed for conserved sequences within
  genomes of infectious agents. In bacteria 16S
  rRNA subunit gene widely used.
• Strand displacement amplification (SDA)
• Target generation and exponential target
  amplification
• Isothermal after denaturation
• Not very specific in the presence of low target
  sequence (complex DNA mixtures.

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Techniques

• Target amplification techniques
• Transcription based amplification
• NASBA (Nucleic acid sequence-based amplification)
  and TMA (transcription-mediated amplification).
• Isothermal RNA amplification.
• Synthesis of cDNA
• In vitro transcription with cDNA as template
• Isothermal and three enzymes RT, T7 RNA
  polymerase and RNase H.

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Techniques

• Probe amplification techniques
• Ligase chain reaction (LCR litigation chain
  reaction).
• Increases specificity No blunt end ligation at
  low annealing temperatures.
• Inactivation of postamplification products is
  difficult.

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Techniques

• Probe amplification
• Cleavase invading technology
• Isothermal
• Isotyping, point mutations, viral load testing.
• Cycling probe technology
• DNA-RNA-DNA hybrid probe
• RNase H cuts RNA from probe.
• Detection system is based on antibodies to detect
  uncut probe.

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Techniques

• Postamplification detection and analysis
• Gel electrophoresis
• Colorimetric microtiter plate systems
• Chemiluminescence/Electrochemiluminescence
• Direct amplification product sequencing.
• Matrix hybridization
• Real time PCR
• SYBR green
• Fluorescent and quencher dyes (Taqman).
• FRET
• Molecular beacons
• Scorpion probes
• Dark quenchers

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Techniques

• Gel electrophoresis
• Ethidium bromide staining and other DNA
  intercalators (more sensitive).
• Southern blotting More specificity and
  sensitivity
• Single strand conformational polymorphism (SSCP)
• Differential migration of strands with minimal
  sequence differences. Useful in analysis of
  resistance-related mutations.

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Techniques

• Gel electrophoresis (cont.)
• Postamplification RFLP
• Enzymatic digestion of amplified DNA.
• Latter two techniques will probably be placed by
  sequencing technology.

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Techniques

• Colorimetric microtiter plate systems
• Amplified DNA captured in microwells coated with
  complementary oligonucleotides. Detection based
  on colorimetric methods (enzyme-based,
  biotinylated primers).
• More detection sensitivity than gels (10-100
  times).
• More specificity (capture probe provides
  specificity).
• Speed.

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Techniques

• Chemiluminescence and electrochemiluminescence
• Attomole levels.
• Acridinium esters
• Ruthenium bipyridyl esters
• Quantitative potential. Linear responses over 3-4
  logs.

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Electrochemiluminescence
hv(622 nm)
Ru(bpy)32
Ru(bpy)33
Microbead
TPA
TPA
Ru(bpy)32
Ru(bpy)32
e
e
Anode
Magnet
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Tyramide based amplification
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ELF 97 based amplification
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ELF based amplification
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Techniques

• Direct amplification product sequencing
• PCR plus dideoxynucleotide chain termination.
• Sequencing
• Electrophoretic
• Slab gels
• Glass capillaries
• Solid phase
• Matrix hybridization

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Techniques

• Electrophoretic sequencing
• Fluorescent based
• Capillary Less contamination and less labor
  intensive.
• Matrix hybridization
• Thousands of oligonucleotides attached to solid
  support.
• Labeled amplicon
• Hybridization pattern.
• Photoactivated chemical synthesis on silicon
  chips.

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What is cooking?

• Immunoliposomal technology
• Atto to zeptomole levels of detection
  (10-18-10-21).
• Combines antigen capture with real-time PCR and
  liposomes
• Excellent for toxin detection where direct
  DNA/RNA amplification is not possible.

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What is cooking?

• Automated Biological Agent Testing System (ABATS)
• Hybridization-based microchips Greene VrdB
  (Vertebral viral sequence database), Pan-viral
  DNA microarrays (ViroChip).
• Tissue-based biosensors
• B-cell amplifier (Canary).
• Nanotechnology Immunomagnetic discrimination,
  retroreflector-linked immunosorbent assay.
• Microfluidic systems.
• Microwave-Accelerated Metal-Enhanced Fluorescence
  (MAMEF)
• MassTag RT-PCR Primers labeled with low
  molecular weight tags. Dual signals identified.

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Techniques

• Amplification product inactivation
• Uracil-N-glycosylase (UNG)
• Photochemical cross-linkers
• Topical inactivation

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Current application of molecular diagnostics

• Detection of unculturable and fastidious
  organisms.
• ID of unusual bacteria by 16S rRNA sequencing
• Subtyping of microorganisms
• Organism quantitation
• Detection of microbial drug resistance
• Evaluation of epidemics and nosocomial infections
• Response to therapy and prognosis.

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Interpretation of results

• Dead versus live pathogens
• RNA targeting, DNA polymerase.
• Presence of nucleic acid versus disease
• Colonization, contamination, infection.
• Site of origin of specimen
• Causal or casual
• Modification of Kochs postulates to include
  molecular criteria