Molecular Diagnostics in the Clinical Microbiology Laboratory

Molecular Diagnostics in the
Clinical Microbiology Laboratory
Patrick Tang, MD, PhD, FRCPC
B.C. Centre for Disease Control
University of British Columbia
BCCDC Public Health Microbiology & Reference Laboratory

Molecular Diagnostics in the Clinical
Microbiology Laboratory
Introduction to molecular microbiology
Overview of PCR
Molecular genotyping methods
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Traditional Microbiology
Culture
Microscopy
Serology
PCR
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Detection of Organism
Microscopy
Culture
Antigens
Nucleic Acids
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Detection of Host Response
Serology
(Host
Immune
Response)
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Detection of Host Response (Future)
Metabolomics
Gene Expression
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Nucleic Acid Detection
Detection of organism-specific DNA or RNA
Must know the sequence of the target region
ATGATTTCGAGAACGGGACCTATTGCTAGTTGCGTACATGCTCTTCGAGTCACTGGCT
Non-amplified Nucleic Acid Probe
labelled 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, not from target
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Non-Amplified Nucleic Acid Probe
Liquid-phase hybridization protection assay
e.g., 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
DNA probe
target sequence
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Signal Amplification
Branched DNA (Bayer)
sandwich hybridization assay
with multiple sets of probes
bDNA has 15 identical
branches, each can bind 3
labeled probes
bDNA
target sequence
enzyme-labeled probes
microwell with capture probes
target probes
capture probes
Hybrid capture assay (Digene)
target DNA is hybridized to RNA
probe
DNA:RNA hybrids are captured by
immobilized antibodies
soluble enzyme-conjugated
antibodies then bind to the hybrids
tube with capture antibodies
enzyme-conjugated
antibody
RNA probe
target DNA
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Target Amplification
Polymerase Chain Reaction (PCR)
reverse transcriptase PCR (RT-PCR)
nested PCR
multiplex PCR
real-time PCR (qPCR)
Transcription-mediated amplification (TMA) /
Nucleic acid sequence-based amplification (NASBA)
isothermic amplification of RNA target
Strand displacement amplification (SDA)
isothermic amplification of DNA or RNA target
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Polymerase Chain Reaction
95°C
denaturation
72°C
primer extension
50°C
primer annealing
95°C
50°C
72°C
3’
5’
5’ 3’
3’
5’
exponential
amplification
5’ 3’
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Probe Amplification
Ligase Chain Reaction
employs two sets of labeled probes that bind to adjacent target
regions
ligase enzyme joins the two contiguous probes into a linear
product that can be captured and detected
biotinylated probe 1
enzyme-labeled probe 2
ligase
target DNA
ligated probe
streptavidin matrix
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Probe Amplification
Multiplex Ligation-dependent Probe Amplification
employs two probes that bind to adjacent target regions
one probe contains forward primer site, other probe contains
reverse primer site
multiple sets of probes bind to different targets
each probe set has a different length linker region
ligase enzyme joins the contiguous probes
PCR with the forward and reverse primers is used to generate
amplicons of variable length corresponding to each target
probe A1
probe A2
probe B1
probe B2
target A
target B
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Polymerase Chain Reaction
DNA Extraction
Polymerase Chain Reaction
Thermal cycling
Components
Primers
Controls
Detection of PCR amplicons
Amplicon Contamination
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DNA Extraction
Sample homogenization
tissues, viscous fluids, formalin-fixed tissue
Mechanical lysis
boiling, sonication, freeze/thaw, mortar/pestle
Enzymatic lysis
proteinase K
Chemical lysis
detergents
guanidinium thiocyanate +/- phenol/chloroform
DNA precipitation
ethanol, isopropanol
adsorption to silica matrix
columns, silica beads/resin, silica-coated magnetic beads
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Selecting a Method of Extraction
Automated versus manual extraction
cost per extraction, cost of equipment
ease of use, hands-on time, turn-around time
throughput (number of samples per run)
Type of specimens being extracted
Volume (mass) of specimen being extracted
Efficiency of DNA recovery
Quality of extracted DNA
Extraction of DNA and/or RNA
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Polymerase Chain Reaction
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Stages of PCR
Denaturation (90-95 ° C)
separate the two strands in double stranded DNA
Annealing (50-55 ° C)
temperature depends upon primers
Extension (65-72 ° C)
depends upon enzyme and size of targeted region
Final extension (65-72 ° C)
fill in partially completed PCR products
Cooling (4-10 ° C)
keep cold to maintain DNA amplicons
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PCR Thermal Cycling
Temperature
denaturation
extension
annealing
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PCR Target Amplification
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Choosing a Thermal Cycler
Ramp rate
rate of heating and cooling
Temperature stability
Block uniformity
Single temperature or gradient
Capacity
Compatibility with PCR tubes and plates
Cost
Conventional or real-time PCR
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PCR Reaction Components
Master mix (typically 10-50 µL)
Target (DNA/RNA) – typically 5-10% of total volume
Polymerase
Primers (~0.2 µM)
Nucleotides – dNTPs (50-200 µM)
KCl (≤ 50 mM)
salts affect stability of dsDNA stringency of reaction
Mg 2+ (0.5 to 2.5 mM greater than [dNTP])
binds to DNA and dNTP
required for activity of polymerase
Buffer
Water
Available as commercial pre-mixes
just add water, primers and sample
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PCR Primers
Proper PCR primer design is crucial in the success of
the assay
requires access to database of sequences
sensitivity, specificity
Typically 20bp and 50-60% GC content
GC clamp at 3’ end of primer
too many G and C at 3’ end may lead to mispriming
Typical melting temperature (Tm) 50-70°C
Must pick sequences to avoid:
self dimerization
secondary structures (hairpins)
dimerization with other primer
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Degenerate Primers
Mix of primers with variable bases at one or
more sites
GCATCTATATAACGTACGT
GCATCTATATAACGTCCGT
GCATCTATATAACGTGCGT
Inosine can be used as a base instead
universal base (found in tRNA)
more expensive
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Controls for PCR
Positive control (low titer)
ensure reagents were added and working properly
Negative control(s)
detect amplicon contamination, non-specific
amplification
Extraction control
ensure that DNA was efficiently extracted
Internal positive control
ensure that amplification is occurring in each sample
i.e., no PCR inhibitors are present in sample
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Variations of PCR
Reverse-transcriptase PCR (RT-PCR)
use reverse transcriptase to convert RNA to cDNA
other steps are identical to regular PCR
Nested PCR
amplify a larger target region in first PCR reaction
amplify a sub-region of the initial target in second PCR
Multiplex PCR
detect multiple targets in a single reaction
multiple sets of primers
Real-time PCR (rt-PCR or qPCR)
real-time PCR can be quantitative
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Detection of PCR product
Agarose gel electrophoresis
Polyacrylamide gel electrophoresis
DNA separated by size
Visualized with intercalating dye
Ethidium bromide
SYBR green
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Agarose Gel Electrophoresis
-
larger fragments
slower migration
+
smaller fragments
faster migration
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pos extraction control
neg extraction control
water extraction control
strong pos PCR control
weak pos PCR control
water PCR control
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PCR Contamination
PCR clean room/area vs. “dirty area”
one-way flow of samples
amplicons from previous PCR reactions
highly positive samples or controls
laminar flow hood
gloves
filtered pipette tips
change lab coats
careful technique
open one tube at a time, avoid aerosols
bleach, high concentration NaOH, ultraviolet, commercial products
uracil N-glycosylase
use dUTP instead of dTTP in PCR reactions
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Conventional PCR vs. Real-Time PCR
Real-time PCR
detection of amplification
during exponential and
linear phase
measure amount of PCR
product at each PCR cycle
Conventional PCR
detection of amplification with
ethidium bromide when
reaction complete
results are not quantifiable
because of variability of the
endpoint yield of PCR product
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Amplification Plot
Fluorescence
Cycle Number
C t (cycle threshold)
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Intercalating Dyes
SYBR Green, SYBR Gold,
Yo-Yo-1, Yo-Pro-1
Dye binds to double stranded
DNA once extension is
complete
Only fluorescent when bound
to the dsDNA
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SYBR Green PCR
Standard PCR
reaction with SYBR
Green added to detect
total DNA amplification
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Melting Curve Analysis
Different sizes of DNA
molecules melt at a different
temperatures
Melting curves are needed to
confirm amplification of
desired DNA target
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SYBR Green Disadvantages
Binds to all the double stranded DNA in
the reaction including non-specific
amplification products and primer-dimers
Melt-curve analysis is required to resolve
amplification products
Real-time (quantitative) PCR reactions
that use non-specific dyes must be VERY
well optimized to give good results
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DNA Probes
5’ exonuclease probes (TaqMan)
Molecular beacons
Hybridizes to specific region of PCR
product
Based on the principle of fluorescent
resonant energy transfer (FRET)
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Fluorescent Resonant Energy
Transfer (FRET)
distance-dependent interaction between the
electronic excited states of two dye molecules in
which excitation is transferred from a donor
molecule to an acceptor molecule
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Anatomy of a TaqMan Probe
Fluorescent reporter dye
(donor)
R
Quencher molecule
(acceptor)
Q
Target-specific single stranded DNA
(13-24 base pairs in length)
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Polymerization
Primers and probe anneal to target DNA
5’
3’
Forward
Primer
R
TaqMan
Probe
Q
5’
5’
Reverse
Primer
3’
5’
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Displacement
Taq polymerase displaces the probe
strand
5’
3’
5’
Forward
Primer
R
Q
Reverse
Primer
5’
3’
5’
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Cleavage
5’exonuclease activity of Taq polymerase
cleaves reporter molecule
R
5’
3’
5’
Q
5’
3’
5’
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Polymerization Completed
R
Q
5’
3’
5’
5’
3’
5’
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Molecular Beacons
Target specific DNA in loop
Probe forms hairpin loop
when not hybridized to
template
Reporter and quencher in
proximity when loop closed
Hybridized
to
template
Hairpin
loop
Melted
Annealing exactly to correct
template favored over stemloop
formation
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Quantitative PCR
There is a quantitative relationship between the amount of target
nucleic acid present at the start of PCR and the amount of product
amplified during its exponential (geometric) phase
Exponential
phase
Threshold
Baseline
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Relationship Between Initial Copy
Number and Cycle Number
Cycle number
Threshold
28 29 30
Copy No.
1000
500
250
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Advantages of real-time PCR
Faster than conventional PCR
Faster cycling and no need to run samples on
agarose gels
Higher analytical sensitivity
Detection limit at 1-10 copies versus 10-100 copies
Results available during testing/thermocycling
Reduced chance for PCR contamination
Closed system
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Molecular Typing
Methods
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Laboratory Methods for Epidemiological
Analysis of Microorganisms
Biotyping (Phenotyping)
Biochemicals
Assimilation of different biochemicals
Can combine with antibiotic susceptibility profile
Serotyping
Recognition by type-specific antibodies
Phage typing
Susceptibility to different bacteriophage
Multilocus enzyme electrophoresis (MLEE)
Compare electrophoretic mobility of a set of proteins
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Laboratory Methods for Epidemiological
Analysis of Microorganisms
Genotyping
Restriction fragment-length polymorphism (RFLP)
Pulsed-field gel electrophoresis (PFGE)
Random amplification of polymorphic DNA
(RAPD)
Amplified fragment length polymorphism (AFLP)
Variable number of tandem repeats (VNTR)
Multilocus sequence typing (MLST)
Single nucleotide polymorphism (SNP) typing
Microarray typing
Whole genome sequencing
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RFLP
1. AMPLIFY
ORGANISM
3. RUN GEL
1
2
3
2. FRAGMENT
GENOME
3
Restriction
Endonucleases
1 2
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IS6110-based RFLP for Genotyping of
Mycobacterium tuberculosis
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PFGE
Restriction enzyme
Voltage gradient
Switch interval
Reorientation angle
Agarose content of gel
Temperature
Run time
A-
B+
B-
A+
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PFGE of Shigella flexneri (BlnI)
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RAPD
1. AMPLIFY
GENOME
3
Short Primers
1 2
2. GENERATE
PCR FRAGMENTS
1
2
3
3. RUN GEL
1
2
3
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AFLP
1. FRAGMENT
GENOME
2. LIGATE
ADAPTERS
3. PCR
3
Restriction
Endonucleases
Adapters
1 2
4. RUN GEL
1
2
Primers
3
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Laboratory Methods for Epidemiological
Analysis of Microorganisms
Restriction fragment-length polymorphism (RFLP)
Amplify DNA by culturing the organism
Restriction endonuclease digestion of DNA into small pieces
Resolve DNA fragments on agarose gel
Pulsed-field gel electrophoresis (PFGE)
PFGE is a type of RFLP
DNA is cut into large fragments that can only be resolved using pulsedfield
gel apparatus
Random amplification of polymorphic DNA (RAPD)
A defined set of short primers are used to PCR amplify the genomic DNA
The short primers bind at multiple locations creating a band pattern when
resolved by agarose gel electrophoresis
Amplified fragment-length polymorphism (AFLP)
Restriction digest DNA first
Amplify by PCR and resolve with gel electrophoresis
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VNTR
1. AMPLIFY
TARGET
REGIONS
1 2 3
3 different PCR reactions
2. GENERATE
PCR FRAGMENTS
1
2
Determine number
of tandem repeats
3
3. CAPILLARY
ELECTROPHORESIS
5
VNTR pattern = 5 - 9 - 6
6
9
1
2
3
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MLST
1. AMPLIFY
MULTIPLE
LOCI
2. GENERATE
PCR FRAGMENTS
1
2
3
1 2 3
3 different PCR reactions
3. DNA SEQUENCING
ATCGTTAGGAAGCAT
TTACAACCAGTAGCACCC
GAGCTTACCAATCGGAC
1
2
3
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SNP Genotyping
DETERMINE
SNPs @
MULTIPLE LOCI
1 2 3
T
G
A
1. qPCR 2. PYROSEQUENCING
3. MICROARRAY
A
T
A
C
G
C
P
SNP1 = T SNP2 = G SNP3 = A
G
A
A
T
C
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Microarray Genotyping
Oligonucleotide
probes targeting
different regions of
the genome
different alleles of
the same gene
different SNPs
unique genes
A1 A2 A3 A4 A5 A6 A7
B1 B2 B3 B4 B5 B6 B7
C1 C2 C3 C4 D1 D2 D3
E1 E2 E3 F G1 G2 G3
H1 H2 I J1 J2 K L
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Microarray Genotyping
Isolate No.1
A1 A2 A3 A4 A5 A6 A7
B1 B2 B3 B4 B5 B6 B7
C1 C2 C3 C4 D1 D2 D3
Isolate No.2
A1 A2 A3 A4 A5 A6 A7
B1 B2 B3 B4 B5 B6 B7
C1 C2 C3 C4 D1 D2 D3
E1
E2 E3 F G1 G2
G3
E1 E2 E3 F G1 G2 G3
H1 H2 I J1 J2 K L
H1 H2 I J1 J2 K L
Isolates 1 and 2 are related but not identical
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Laboratory Methods for Epidemiological
Analysis of Microorganisms
Variable number of tandem repeats (VNTR)
PCR amplify genetic regions containing tandem repeats
Electrophoresis to resolve size of PCR products
Multilocus sequence typing (MLST)
Sequence a defined set of loci within the genome
Single nucleotide polymorphism (SNP) typing
Determine the SNP at a set of defined positions in the
genome by PCR, sequencing or microarray
Microarray typing
Determine presence or absence of a defined set of markers
within the genome
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Laboratory Methods for Epidemiological
Analysis of Microorganisms
Whole genome sequencing
Fragment genome into smaller overlapping
pieces
PCR, restriction digest, sonication, etc.
Sequence fragments
Assemble contigs
Bioinformatics analysis
Whole genome alignments
Detection of mutations
Genomic islands
Insertions/deletions
Point mutations, SNPs
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Phylogenetics
Compare the genetic
relatedness between
organisms
Distance-based methods
are used to create trees
which approximate
phylogenetic relationships
Based on genotyping data
RFLP, MLST, etc.
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The End
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