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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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Virology – Diagnosis JU- 2nd Year Medical Students
ByDr Hamed AlZoubi – Microbiology and Immunology
Department – Mutah University.MBBS (J.U.S.T)
MSc, PhD medical microbiology (UK).FRCPath (associate, medical microbiology).
Detection of viral genomes by nucleic acidamplification 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 acidamplification 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
Detection of viral genomes by nucleic acidamplification 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.
Detection of viral genomes by nucleic acidamplification 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 programmedin the device.
• Therefore one copy of viral DNA can be amplifieda 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 acidamplification 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.
• • 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
• OR• 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 ofplasma after antiviral drug treatment, compared with a typicalfigure of 10 000 RNA genome copies at the start of antiviraltherapy, 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 fordrug-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
END