Transcript Sojourn through the Land of Bad Bugs

Virology – Diagnosis
JU- 2nd Year Medical Students
By
Dr Hamed AlZoubi – Microbiology and Immunology
Department – Mutah University.
MBBS (J.U.S.T)
MSc, PhD medical microbiology (UK).
FRCPath (associate, medical microbiology).
[email protected]
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Detection of viral genomes by nucleic acid
amplification methods
Common and highly sensitive
Usedto detect and quantify
DNA viruses such as HIV provirus, Hepatitis B,
CMV and HPV in clinical samples
RNA viruses such as HIV, Hepatitis C and Influenza
viruses providing that an initial step of RT
included (to transcribe RNA into DNA)
Detection of viral genomes by nucleic acid
amplification methods
• Detect and quantify: helpful in diagnosis and
treatment follow up e.g reduction in viral load
following initiation of antivirals (e.g HIV, HBV,HCV)
• Prone to false positive results due to
contamination e.g plasmids
• To avoid false positive results strict control and
aseptic techniques are necessary
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Detection of viral genomes by nucleic acid
amplification methods
To avoid false positive results due to
contamination:
Separate places for DNA and mixture preparation
independent colour coded ventilated rooms, each
with its own gloves, gowns, pipettes, and other
equipment,
In the case of adjoining rooms, the direction of
flow of activities must always be from entrance to
exit.
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Detection of viral genomes by nucleic acid
amplification methods
PCR:
Components:
Primers, oligonucleotide bases, taq polymerase
enzyme (from thermophilus aquaticus), buffers
and genetic material
Step 1:
Treat DNA with a temperature (94 C for 1 minute)
and detergent to separate the 2 strands
Step 2:
• The oligonucleotide primers (forward and reverse)
specifically hybridize with the homologous
nucleotide stretches on each strand of the target
viral DNA genome.
• A DNA polymerase (open square) termed Taq
polymerase (from Thermophilus aquaticus), which
acts at high temperature,is also added.
• After 1 min the temperature is reduced to
• 52C for 20 s to allow annealing of primers
Step 3
• The temperature is then raised to 72C for 5 min to
allow DNA polymerization to occur
• multiple copies of the nucleotide stretch between
the two primers generated by the Taq polymerase.
• Multiple cycles of DNA denaturation, annealing of
primers, and polymerization can be programmed
in the device.
• Therefore one copy of viral DNA can be amplified
a million-fold in a few hours to give a quantity ofDNA
• DNA can be separated in a polyacrylamide gel, and
then visualized by addition to the gel of ethidium
bromide and exposure to UV light.
Detection of viral genomes by nucleic acid
amplification methods
‘Nested PCR’:
• more sensitive.
• Following initial amplification of a unique stretch
of viral DNA, a further set of ‘internal’ primers is
added that anneal to DNA within the original
fragment, allowing a smaller stretch to be
amplified.
Branched chain techniques (signal amplification):
• bDNA techniques Detect and quantify viral RNA
e.g HIV
• Highly sensitive
• Branched DNA Probe - based
• faster, less laborious and expensive, and requires
less technical ability than PCR
• bDNA Tecnique:
• Lyse virus and release RNA – Add lysate to microtitre plate
wells coated with oligonucleotide probes (capture
probes), which match conserved sequences in HIV.
• Add the HIV genome and it will be captured – then wash
• Add bDNA amplifier and it will hybridize to HIV RNA then
wash
• Add AP (alk. phosphatase) bound probe that binds to the
Bdna
• Add substrate and the AP enzyme will catalyse that to
produce chemiluminescent molecule which is
proportional to the quantity of viral genome
Nucleic acid sequence – based
amplification (NASBA)
• targets RNA viruses or mRNA transcripts of
DNA viruses
• uses three enzyme systems and 2 primers at
the same time to amplify a particular viral
genome sequence.
• It can be quantitative.
• The three enzymes are RT, T7 DNA-dependent
RNA polymerase, and RNase H.
• Isothermetic
(NASBA)
• A viral genome specific primer also incorporates
the T7 promoter and hybridizes to the viral
genome. This is extended by the RT enzyme.
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• The RNase degrades the RNA strand and the RT,
ulitizing a second primer, produces dDNA.
Multiple copies of
• RNA are produced from this DNA template by
the T7 DNAdependent RNA polymerase
Real time - PCR
• Detect and quantify while amplifying
• does not wait for an end quantitation i.e Faster
than conventional PCR
• A specific probe binds to the viral amplicon under
investigation, and is hydrolysed to produce
fluorescent molecules, which are immediately
detected and quantified
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• a dye is encouraged to intercalate into the dsDNA
being produced in the first reaction, and as more
• dye is trapped fluorescence increases
Uses
• Monitoring the effects of antivirals
• HIV:
• clinical care of AIDS patients being treated with drug
combinations; HAART .
• Patients should be monitored every 2–3 months
• target is to detect fewer than 50 HIV genome copies per ml of
plasma after antiviral drug treatment, compared with a typical
figure of 10 000 RNA genome copies at the start of antiviral
therapy, 3–4 months previously.
Uses
chronic hepatitis B:
• One hundred to 1000-fold reduction of viral DNA load would be
typical following antiviral therapy (lamivudine, famciclovir, and
adefovir)
HCV:
• a rapid and sustained reduction follwing starting Rx, IFN and
ribavirin indicate successful treatment
• identifying which of the five types has infected the patient,
because these respond differently to antiviral therapy.
Uses
• Analysis of hepatitis B and C and HIV genomes for
drug-resistant mutations
• resurgence of viraemia in a patient following longterm therapy
• In HIV patients on combination of therapy think of resistance due
to point mutation in RT or protease genes of HIV resistance
• Point mutation detection:
• point mutation assay utilizes PCR primers synthesized so as to
hybridize to the drug-sensitive or drug-resistant virus only
• Sequencing: automated method and it is the method of choice
• Chip technology
• Virus islation in cell culture
• Detection of antiviral antibodies
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