Download New Molecular Based Methods of Diagnosis

Molecular Diagnostic
Methods
Recombinant DNA Technology
Nucleic Acid Test for
Infectious Disease
• Accurate to ID MO
– Specific – DNA, RNA
– Sensitive - detect low number
• Timely ID MO causing disease
• Rapid start proper clinical treatment
in patient
• Guide Public Health to prevent spread
of infection
Utility Nucleic Acid Test
• ID nonculturable agents:
– Human papilloma virus
– Hepatitis B virus
• ID fastidious, slow-growing agents:
– Mycobacterium tuberculosis
– Legionella pneumophilia
• ID highly infectious agents dangerous
to culture:
– Francisella tularensis
– Brucella melitensis
– Coccidioidis immitis
Utility Nucleic Acid Test
• In situ detection of MO:
– Helicobacter pylori (stomach)
– Toxoplasma gondii (intracellular protozoan)
• MO present low number:
– Human immunodeficiency virus (HIV) in antibody
negative patient
– Cytomegalovirus (CMV) in transplant organ
• Differentiate antigenically MO
• Detect specific virus genotypes associated with
human cancers (Papilloma virus 16, 18)
• MO in small volume specimens:
– Intra-ocular fluid
– Forensic sample
Utility Nucleic Acid Test
• Antiviral drug susceptibility testing:
– Decide therapy to use in HIV drugresistant strain
• Molecular epidemiology:
– Identify point source for hospital and
community-based outbreak
– Predict virulence
• Non-viable MO:
– In very labile, readily inactivated
– In immune complexes
• Confirm positive lab culture isolate
Nucleic Acid Molecular
Techniques
• Direct probe testing:
– Short specific nucleic acid sequence
– Better for ID than for detection
– Not as sensitive as amplification methods
• DNA Amplification methods:
– Make more copies of DNA
– Improve sensitivity of nucleic acid testing
– Amplification of: Target (DNA), Probe, Signal,
or combinations
DNA Structure: Double Helix
• Double strand linear DNA:
– 5’ carbon at one end
– 3’ OH group at other end
• Two strands “opposite”
orientation with respect to
each other (antiparallel,
complementary)
• Adenine base-pair with
thymine (2 hydrogen bonds)
• Guanine base-pair with
cytosine (3 hydrogen
bonds)
DNA Helix: Hydrogen Bonds
Hybridization: Direct Probe Testing
• Hybridization – to come
together through
complementary base-pairing
• Used in ID
• Colony hybridization – MO
colony treated to release
nucleic acid, denatured to
single strands
• Add single-stranded
fluorescent labeled
(ss)DNA (probe) unique to
test MO
• Hybridization allowed to
occur
• Unbound probe washed
away, bound probe detected
by presence of fluorescent
label
Target DNA Amplification:
Polymerase Chain Reaction
• Number of copies of target
DNA increased by in vitro
enzyme synthesis
• Use cell DNA polymerase to
copy target DNA
• DNA polymerase cannot initiate
synthesis on its own
• Need primer to start enzyme
reaction
• Primer - single strand piece of
DNA (or RNA) complementary
to unique region of sequence
copied
DNA
Synthesis
• Synthesis
occurs only in
5’ to 3’
direction
DNA Replication Semiconservative
• One ds parent strand copied
• Two ds parent:daughter strands
Polymerase Chain Reaction
(PCR)
• Enzyme reaction to amplify DNA:
– Two primers - one binds one strand of
dsDNA, other binds other strand
– Four nucleotide DNA precursors (dNTPs)
– Thermostable DNA polymerase
– Buffer (ions, salts) for enzyme reaction
• Primers (~15-20 bases) unique sequence
to DNA being amplified
• PCR reaction has three basic steps:
– 1) Denaturation
– 2) Primer Annealing
– 3) Primer Extension
PCR Assay: 1) Denaturation
• Separate dsDNA into ssDNA
• Heating 950 C, 15 seconds to 1 minute
• The two ssDNA strands serve as
templates for DNA synthesis
PCR Assay: 2) Primer Annealing
• DNA comes together through
complementary base-pairing (hybridization)
• Primers base-pair with their complementary
sequences on ssDNA template
• Primer concentration in excess of DNA
template concentration
• Excess primer ensures base-pairing with
complementary sequence on template DNA
instead of ssDNA templates base-pairing
back together
Primer Annealing
• Temperature used should ensure annealing will
occur only with DNA sequences that are
completely complementary. WHY?
• The annealing temperature depends upon
length and sequence of primers
• The longer the primer and the more Gs and Cs
in the sequence, the higher the annealing
temperature. WHY?
• Annealing temperature range from 45-65ºC
• Annealing time usually 15 seconds to 1 minute
PCR Assay: 3) Primer Extension
• DNA polymerase use dNTPs to synthesize
DNA complementary to template DNA
• Thermostable Taq DNA polymerase
(isolated from Thermus aquaticus)
extends annealed primers
• Temperature used is 720 C, optimum
reaction temperature for Taq DNA pol
• The extension time is usually 15 seconds
to 1 minute
• Why is this high temperature used?
• Why is a thermostable polymerase used?
PCR Assay
• The combination of
denaturation, primer
annealing, primer extension
constitute 1 cycle in a PCR
reaction
• Exponential increase of
DNA with each cycle (2, 4,
8, 16, 32, 64, 128, etc.)
• PCR assay uses 25 to 30
cycles to amplify target
DNA > 1 million copies
• Assay time ~5-6 hr.
PCR Product DNA Analysis
• DNA agarose gel electrophoresis
– ID DNA molecular weight by migration in gel
– Estimate size using DNA markers (100 bp
ladder; 100-1500 bp)
– Assay time ~1-2 hr.
• Real-Time PCR
– ID DNA during PCR assay in closed tube
– Use fluorescent reporter dye released during
enzyme reaction
– Thermal Cylcler with UV light to excite
reporter dye and camera for detection
– Assay time ~1 hr.
MICR 302 Lab PCR Exercise
• First PCR assay will amplify DNA sequence
in bacterial 16S ribosomal RNA (rRNA) gene
• Use primers for DNA consensus sequence
found in all bacteria
• Primers not unique to a specific bacteria,
but unique to a conserved region of 16S
rRNA gene of all bacteria
Bacterial 16S rRNA Gene:
ID Unknown Bacteria
• Amplify portion of 16S rRNA gene of unknown
bacteria
• DNA of all bacteria are amplified and yield
DNA product using the consensus primers
• Sequence of the amplified DNA will be
determined by Automated DNA Sequencer
• Identity of unknown found by searching and
matching your result in the National Institutes
of Health (NIH) DNA sequence database using
“BLAST” program
MICR 302 Lab PCR Exercise
• Second PCR assay will amplify DNA sequence in
Shiga Toxin gene
• Use two primer sets unique for the two different
Shiga toxin genes of EHEC
• Only E. coli that carry one or both of these toxin
genes yield DNA product using these primers
• Positive DNA product thus identifies bacteria
isolate as EHEC strain
• For lab diagnosis, only this second type of PCR
assay is of practical use for MO ID
Advantages of Molecular
DNA Test
• High sensitivity - theoretically detect
single MO
• High specificity – one MO species
– Detect specific genotypes
– Determine drug resistance
– Predict virulence
• Speed - more rapid than traditional MO
culture
• Simplicity - assays are automated; Realtime PCR
Disadvantages of Molecular
DNA Test
• Expensive
• Very specific, must have good clinical data to
support infection by MO before testing initiated
• Contaminating DNA give false positive
• Miss new MO unless sequencing is done (as for
your lab molecular unknown); not practical in
clinical lab
• Problem with mixed cultures – assay for all MOs
possibly causing infection
• Too sensitive? Are results clinically relevant?
• No isolated MO grown in culture
DNA Sequencing
• Determine actual DNA sequence of MO
• Using computer, based on nucleic acid
data:
– Identify MO
– Predict protein sequence and function
• The most commonly used sequencing
method is the dideoxy method
Dideoxy DNA Sequencing
• Use dideoxynucleotide
triphosphates (ddNTPs)
• H on 3’ carbon of the ribose
sugar instead of OH normally
found in deoxynucleotide
triphosphates (dNTPs)
• ddNTP is chain terminator DNA synthesis stops as 3’OH
necessary for addition of next
nucleotide
Dideoxy DNA
Sequencing
• Template DNA to be sequenced
mixed with primer, four normal
dNTPs (one radioactively
labeled)
• Mixture split into four tubes
labeled A, C, G,T
• Each tube “spiked” with
different ddNTP (ddGTP tube
1, ddATP tube 2, ddCTP tube 3,
ddTTP tube 4)
• Add DNA polymerase, synthesis
of new complementary strand
• Occasionally ddNTP added
instead of normal dNTP,
synthesis of strand terminated
Dideoxy DNA
Sequencing
• In ddATP tube, newly
synthesized DNA will get
ddATP in each place there is T
• Thus, set of DNA strands in
tube A, each of which ends in A
• Similar reaction occurs in other
tubes, result in DNA that ends
in C, G,T respectively
• After DNA synthesis, products
of four tubes loaded onto four
lanes of polyacrylamide gel
• DNA strands separated by size
• Sequencing gel able to resolve
DNA that differ in size by only
one base
Dideoxy DNA
Sequencing
• After electrophoresis to separate
by size, DNA strands visualized by
exposing gel to X-ray film (one
nucleotide was radioactive):
– Strands in lane A all end in A
– Strands in lane C all end in C
– Strands in lane G all end in G
– Strands in lane T all end in T
• The sequence of the DNA read
from the gel by starting at bottom
and reading upward
Automated DNA Sequencing
• All four ddNTP reactions done in single tube
• Each ddNTP labeled with a different
fluorescent dye
• Dye present in each synthesized strand
corresponds to ddNTP that terminate DNA
synthesis
• DNA strands in reaction tube analyzed by gel
electrophoresis
• The Sequencing reaction depends on quality and
purity of the amplified DNA product
Automated
DNA
Sequencing
• Flourimeter and
computer attach to
gel
• Detect, record dye
attached to DNA
strands as run off
bottom of gel
• DNA sequence
identified by order
of dyes coming off
gel
MICR 302 Lab Molecular
Unknown
• Culture of an isolated Unknown bacteria
• Isolate and purify DNA from Unknown bacteria
• Use PCR to amplify rRNA gene sequence of
Unknown bacteria DNA
• Do Automated Sequencing reaction on amplified
rRNA DNA product using fluorescent dyes
• Submit DNA to CSULA Sequencing Lab
• Use NIH DNA databank to search for matching
DNA sequence to ID Unknown
Class Assignment
• Textbook Reading: Chapter 11 Applications
of Molecular Diagnostics
– Introduction to Nucleic Acid Amplification
Procedures
– Polymerase Chain reaction
– Omit: Other Nucleic Acid Amplification
Reactions
• Key Terms – only from assigned reading
• Omit: Learning Assessment Questions
Identifying Fecal Unknown
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Gram Stain, Catalase, Oxidase
MO
TSI
Escherichia:
A/A
Shigella:
K/A
Enterobacter:
A/A
Klebsiella:
A/A
Salmonella:
K/A, H2S
Citrobacter:
K/A, H2S
Proteus:
K/A, H2S
Yersinia:
K/A
Aeromonas:
K/A
LIA
K/K
K/A
K/K
K/K
K/K, H2S
K/A, H2S
R/A, H2S
K/A
CIN
K/K
CIN,
Oxidase(+)
Lecture Exam II
Tue., Feb. 28, 2012
• Vibrio thru New Molecular Methods
• Lecture, Reading, Key Terms, Learning
Assessment Questions
• Case Study 4, 5, 6 (Pseudomonas,
Francisella, Bacillus)
• Review, Review, Review!
• Repetition is the key to retention
• Repetition is the key to retention
• Repetition is the key to retention
Case Study Reports
(Revised Due Date)
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March 1 - CS #7 Mycobacterium
March 6 – CS #8 Clostridium
March 8 – CS #9 Chlamydia
March 13 – CS #10 Legionella