1. Virology – Diagnosis JU- 2 nd 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@yahoo.com

2. 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)

3. 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

4. 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.

5. 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

6. 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

7. 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.

8. 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.

9. Detection of viral genomes by nucleic acid amplification methods

11. ‘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.

12. 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

13. 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

16. 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

17. (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

19. 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

20. 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.

21. 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.

22. 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

23. Virus islation in cell culture Detection of antiviral antibodies

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