Molecular Diagnostics
T. Mazzulli, MD, FRCPC
Department of Microbiology
UHN/Mount Sinai Hospital
October 19th, 2009
Objectives
Briefly review the concepts of DNA/RNA,
bases, etc.
Review the methodologies available for
molecular testing and describe some of
the advantages and disadvantages
Discuss the currently available commercial
assays that are available
The DNA/RNA Backbone
A ribose or a deoxyribose
A negatively charged
phosphate group
DNA has an H at C2 so
become “deoxy”
RNA has a hydroxyl at C2
C1 linked to a purine or
pyrimidine base
Counter-parallel strands
bind by H-bonding
between nucleotides
http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html
The Purines
The Pyrimidines
Components of DNA/RNA
Base
Nucleoside:
Base + Ribose
Nucleotide:
Base + Ribose + Phosphate
Cytosine
Cytidine (C)
Cytidine monophosphate (CMP)
Uracil
Uridine (U)
Uridine monophosphate (UMP)
Thymine
Thymidine (T)
Adenine
Adenosine (A)
Thymidine monophosphate
(TMP)
Adenosine monophosphate
(AMP)
Guanine
Guanosine (G)
Guanosine monophosphate
(GMP)
Basepairing is complementary
Chargaff's rule explains
the amount of adenine
(A) in the DNA of an
organism, is the same as
the amount of thymine (T)
and the amount of
guanine (G) is the same
as the amount of cytosine
(C)
The C+G:A+T ratio varies
from organism, especially
among the prokaryotes
http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html
What’s the point of all this? Genetic
information is grouped into codons
Codons are triplets of
nucleotides
Transcribed into anticodon mRNA
Translated into amino
acids which are the
building blocks of
proteins
http://www.accessexcellence.org/RC/VL/GG/genetic.html
Hybridization/Annealing
Probe
5’
3’----atacgtaataaatttccc----5’
tatgcattatttaaaggg
3’
Target
Sequences of DNA that anneal
to target DNA or RNA
– Must be complimentary
– Primers and probes are therefore
specific for the target
No nucleic acid amplification
Molecular Methods
Critical to all molecular tests is extraction of
RNA/DNA
Key steps include extraction, amplification, detection
May be used for:
– Diagnosis
– Monitoring
– Screening
Three Methods:
– Target amplification
– Signal amplification
– Probe amplification
Target (Nucleic Acid Amplification Tests)
Amplification Methods
1.
Polymerase Chain Reaction (PCR):
–
–
–
–
–
–
PCR
Reverse transcriptase (RT)-PCR
Real time (RT)-PCR
Nested PCR
Multiplex PCR
Qualitative/Quantitative PCR
Transcription-mediated amplification methods
(TMA, NASBA)
3. Strand displacement amplification (SDA)
2.
Polymerase Chain Reaction
Four key steps:
1.
2.
3.
4.
Denature (Melt) DNA
Anneal primers (20-25 nucleotides) to DNA target
Extension of DNA using target DNA template
Detection of amplified product (amplicon)
After n cycles target is amplified by a factor of 2n
(e.g. 35 cycles, 235 = 34 billion copies)
Need 30-40 cycles to be efficient
Requires excess of DNA polymerase, equimolar
dNTPs, deoxyribonuleotide triphosphates (dNTPs),
MgCl2 and buffer
PCR: Benefits
 Crude extracts and small amounts of DNA may suffice
 Detection of the smallest possible quantities of target




DNA in clinical samples
Can be automated and utilized in homogenous assay
methods
Can utilize in quantitative and high-throughput assays
Valuable for identifying cultured and non-cultivatable
organisms
Used in epidemiology: repetitive elements PCR spacer
typing, selective amplification of genome restriction
fragments, multilocus allelic sequence-based
PCR: Drawbacks
 False positives
 Chance of contamination
 Need equipment and training
 May not be validated for all samples
and populations
Reverse Transcriptase (RT)-PCR
Melt
RNA with
Secondary
structure
DNA primers
RNA
Reverse transcriptase
ssDNA
PCR step
Reverse transcriptase will
transcribe both single-stranded
RNA and single-stranded DNA
templates with equivalent
efficiency
RNA or DNA primer is required to
initiate synthesis
Generates DNA copies of RNAs
prior to amplifying that DNA by
polymerase chain reaction (PCR)
RNase H activity: RNase H is a
ribonuclease that degrades the
RNA from RNA-DNA hybrids and
functions as an endonuclease
and exonuclease
Problems with nonspecific primer
annealing and inefficient primer
extension due to secondary
structure can be overcome by
using Thermus thermophilus
reverse transcriptase
Kits available for HCV and HIV-1
Nested PCR
 Increases sensitivity due
Round I
PCR
15-30 cycles
Primer
set 1
Amplicon 1
Round II
PCR
20-30 cycles
Primer set 2
Amplicon 2
to high # cycles
 Increases specificity due
to annealing in 2nd
amplicon
 Contamination risk if tube
transfer-can overcome
 Often need nucleic acid
probe confirmation
Real Time PCR: SYBR Green
Real Time PCR: TaqMan probes
Real Time PCR
Multiplex PCR
 Two or more primer sets
ds DNA
 Different targets in same
reaction tubes produce
different amplicons
 Primers must have same
reaction kinetics and lack
complementarity
 Complicated to design
 Less sensitive than single
primer set PCR
Specimen Transport and Storage
Affect the Assay
Collection, transport, and assay setup must be
compatible with the assay
Different viruses are stable in different blood
components for different times
For HIV-1 viral load, HCV RNA & HBV DNA,
plasma must be separated from cells within 6
hrs and plasma can be stored at 4oC for several
days or -70oC for long-term
For CMV viral load testing virus is stable in blood
for 5 days at 4oC or -20oC to -70oC
Detection and Analysis of the
Amplicon
Open systems vs closed systems
Gel analysis
Colorimetric Microtiter Plate (CMP) system
Real-time (homogenous/kinetic) PCR
Allele-specific hybridization
Direct sequencing of product
DNA microarrays
How is the amplicon identified?
DNA amplicon
Probe or DNA-binding
chemical
Agarose gel
electrophoresis
Stain with EtBR
Image on UV lightbox
Denature
labeled amplicons
Hybridize with capture
Probe in 96 well plate
Detect bound product
i.e. biotin-streptavidin
Automated imaging
system
Type of sample effects amplification
yield…
May need to boil CSF to release nucleic
acids
Inhibitory substances in urine such as
hemoglobin, crystals, beta-human
gonadotropin, nitrates
Heparin and small volumes of whole blood
inhibit Taq polymerase
Acid citrate in vacutainers can inhibit HIV-1
viral load by 15%- volume effect
What are the sources of
contamination?
Contamination of specimens in NA
extraction step
Contamination with + control material
Carryover contamination of amplified
products
Commercially Available PCR-based
Assays
Viral:
– HCV RNA, HBV DNA, HIV-1 RNA, CMV DNA, HPV
DNA, WNV RNA
Bacterial:
– Chlamydia trachomatis/Neisseria gonorrheae,
Mycobacterium tuberculosis
Fungal:
– None
Parasites:
– None
Transcription Mediated Amplification
(TMA)
Transcription Mediated Amplification
(TMA)
 Reaction occurs isothermally at 41°C
 109 increase in target RNA in 2 hrs (produces 100 – 1000




copies per cycle)
Since this assay only amplifies RNA it can be used to
detect RNA genome and RNA from viable bacteria
Measures replication of DNA viruses by detecting late
mRNA expression
Transcription mediated amplification (TMA): RT with own
RNAse and T7 polymerase. Detection by hybridization
protection assay-2 fluorophors (Gen-Probe)
NA sequence based amplification (NASBA): RT, RNaseH,
T7 bacteriophage RNA polymerase. Detect with
hybridization with chemiluinescent probes (bioMeriieux)
Commercially Available TMA-based
Assays
Viral:
– CMV DNA, HCV RNA, HIV RNA
Bacterial:
– Chlamydia trachomatis/Neisseria gonorrheae,
Mycobacterium tuberculosis and others
Fungal:
– None
Parasitic:
– None
Strand Displacement Amplification (SDA)
Strand Displacement Amplification (SDA)
Isothermal nucleic acid amplification method that relies
on two concurrent polymerization steps and the
displacement of 1 nicked strand of genetic material
Primer containing a restriction site anneals to template
Amplification primers then annealed to 5' adjacent
sequences (form a nick) and start amplification at a
fixed temperature
Newly synthesized DNA is nicked by restriction enzyme,
polymerase starts amplification again, displacing the
newly synthesized strands.
 109 copies of DNA can be made in one reaction
 Alleviate non-specific reactions with organic solvents to
increase stringency of reactions
 If target is low with high background DNA, non-specific
amplification can swamp system and decrease
sensitivity
Walker, Linn and Nadeau, Nucleic Acid Research, 1995
Commercially Available SDA-based
Assays
Viral:
– CMV DNA
Bacterial:
– Chlamydia trachomatis/Neisseria
gonorrheae
Fungal:
– None
Parasitic:
– None
Nucleic Acid Amplification Tests (NAAT)
for Detection of RNA/DNA
Lower limit of detection (LLD)
– Based on probe-it analysis
– Set at amount of target DNA which is detected
>95% of the time
– In general, qualitative assays are more sensitive
than quantitative assays
Linearity (Dynamic range) of Quantitative
assays
– Range of DNA (or RNA) for which the amount can
be accurately extrapolated from a standard curve
using quantitative standards
Cycle Threshold for Detection of DNA
Measuring HBV DNA
Gish and Locarnini, Clin Gastro Hep 2006
Nucleic Acid Amplification Tests (NAAT)
for Detection of RNA/DNA
Quantitation of RNA or DNA may be reported as
copies/ml or IU/ml
Conversion factor for copies/ml to IU/ml is not
the same for different assays measuring the
same target or different targets
– HBV DNA: 5.82 copies/IU
– HCV RNA: PCR - 2.4 copies/IU; bDNA: 5.2 copies/IU
Coefficient of variation (COV) may range from
15 to 50%
Signal Amplification Methods
Branched chain DNA (bDNA)
Hybrid capture assay
Branched Chain DNA (bDNA) Assay
Multiple target probes capture
target nucleic acid on microtiter
well
Second set of target specific
probes bind to target
Preamplifier binds to 2nd set of
target probes and/or 8 amplifiers
Three alkaline phosphataselabelled probes hybridize to each
branch amplifier
Detect labelled probes by
incubating with dioxetane which
emits light in presence of AP
Remove non-specific hybridization
isoC and isoG in preamplifier and
label probes which recognize each
other but not native C and G
Commercially Available bDNA Assays
Viral:
– HBV DNA, HCV RNA, HIV-1 RNA
Bacterial:
Hybrid Capture Assay
ds DNA
denature
hybridize with
RNA probe
ss DNA
Capture hybrids
AP
AP
Multiple AP
conjugates bind to
each hybrid molecule
and amply the signal
Detect complex with
chemiluminescent
substrate
Intensity of emitted
light proportional to
amount of DNA in
reaction
Commercially Available Hybridization
Assays
Viral:
– HBV DNA, HPV DNA
Bacterial:
– Chlamydia trachomatis/Neisseria gonorrheae
Probe Amplification Assays
Ligase Chain Reaction (LCR)
Cleavase-Invader Technology
Cycling probe technology
Ligase Chain Reactions (LCR)
Thermostable DNA ligase to
ligate together perfectly
adjacent oligos.
Two sets of oligos anneal to
one strand of the gene
With a wild-type target
sequence, the oligo pairs ligate
together and become targets
for annealing other oligos in an
exponential amplification
At a point mutation the oligos
only completely anneal to the
mutant sequences and DNA
ligase will not ligate the two
oligos of each pair together
Withdrawn from Chlamydia
trachomatis testing
Cleavase Invader Technology
5’
Invader oligo
Probe oligo
5’
3’
3’
5’
Target sequence
Cleavase
5’
Cleaved probe
3’
5’
3’
5’
Produced by Third Wave
Technologies-bought by PEBIO
Invader displaces 5’ of bound
probe
FEN-1 family of themostable DNA
polymerases cleaves 5’ overlap
Can heat reaction to allow for
primer exchange equilibrium --new uncleaved probes bind
 Detect point mutations because
can overlap of invader probe can
be only 1 bp—track mutations
 Generate distinct fragments of
different genotypes
 Does not increase amount of
target sequence- fewer problems
of false + and contamination
Limitations of Molecular Assays
DNA/RNA extraction is critical
Specimen type: Plasma or serum may be
acceptable; tissue, fluids, etc. may not
Plasma/serum must be separated and
either frozen or tested within 4 to 6 hours
Expensive equipment is usually required
Genotyping
Used for:
– Detection of mutations that confer resistance to
antiviral agents
– Genotyping of isolates for epidemiological purposes;
categorizes patient isolates into 8 different HBV
genotypes (A to H) and 6 different HCV genotypes (1
to 6 with 24 subtypes)
Methods include:
– Sequencing
– Hybridization (e.g. Line Probe Assay, Trugene Assay,
Invader assay, etc.)
Genotyping Assays
Sequencing
Line Probe
Pros
Cons
Discovers
Labor-intensive
new mutations
Low sensitivity
(15-20% pop.)
High throughput
Detects known
High sensitivity
(5-10% pop.)
mutations only
InnoLiPA Principle
Chromogen
(NBT/BCIP)
Marker line
Conj.cont.
Amp.cont.
Purple
precipitate
Alkaline
Phosphatase
L180
Streptavidin
M180
Biotin
M204
V204
Amplified
target
I204
V207
L207
M207
I207
DNA-probe
Nitrocellulose
strip
InnoLIPA HBV Drug Resistance
Marker line
1 - Conj. Control
2 - Amp.Control
3 - L80 WT
4 - V80 Mutant
5 - I80 Mutant
6 - V173 WT
7 - G173 WT
8 - L173 Mutant
9 - L180 WT
10 - M 180 Mutant
11 - A181 WT
12 - T181 Mutant
13 - V181 Mutant
14 - M204 WT
15 - V204 Mutant
16 - I204 Mutant
17 - S204 Mutant
18 - N236 WT
19 - T236 Mutant
Molecular Diagnostics: Summary
NAAT is critical to patient management
Of the many NAAT tests available, PCR, bDNA
and TMA remain most popular
– Sensitivity and dynamic range varies between assays
– Standardization allows (to some degree)
interchangeability of the results with different assays
Resistance/Genotyping requires amplification
first
– Increasing role in making treatment decisions as
more drugs become available
Thank you for your attention!