Virology%20

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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).
• dr_alzoubi_at_yahoo.com

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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)

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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

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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.

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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

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• 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

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• 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.

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• 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.
•  

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Detection of viral genomes by nucleic
acidamplification methods
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• 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.

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• 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

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• 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

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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

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(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

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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

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Uses

• Monitoring the effects of antivirals
• HIV
• clinical care of AIDS patients being treated with
  drug combinations HAART .
• Patients should be monitored every 23 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, 34 months previously.

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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.

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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

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• Virus islation in cell culture
• Detection of antiviral antibodies

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• END