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).

dr_alzoubi@yahoo.com

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

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