hepatitis c genotyping using a line probe assay
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Hepatitis C Genotyping using a Line Probe Assay. Carolyn A. Ragland August 2008. Hepatitis C. * Paraenterally transmitted Contact with infected blood / blood products Blood transfusions – no longer the culprit * US – most likely reason, sharing of needles

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hepatitis c
Hepatitis C
  • *Paraenterally transmitted
    • Contact with infected blood / blood products
      • Blood transfusions – no longer the culprit
      • * US – most likely reason, sharing of needles
      • Sexual and perinatal transmission possible
hepatitis c1
Hepatitis C
  • WHO - 3% have chronic HCV infections
    • In US:
    • 3+ million become infected yearly
    • 8000 – 10,000 die annually from complications
      • Cirrhosis
      • Liver cancer
hepatitis c virus
Hepatitis C Virus
  • Positive sense, single stranded RNA
  • Linear genome of @ 9600 nucleotides
    • Produces viral core and envelope structures
    • Produces non-structural / operational products
hepatitis c virus1
Hepatitis C Virus
  • The RNA 5’ end:
    • highly conserved un-translated /non-coding region
    • contains genotype specific arrangements
  • *Analysis of 5’ end and the core region
    • allows classification into one of six ‘clades’
    • and further sub-groupings.
hepatitis c treatment
Hepatitis C treatment
  • *Knowledge of the genotype provides insight to which treatment and length of treatment will work best.
    • Conventional - interferon alone (monotherapy)
    • Combined conventional - interferon and ribavirin
    • Pegylated interferon monotherapy
    • Combined pegylated interferon and ribavirin
hcv genotyping
HCV Genotyping
  • TRUGENE HCV 5\' Genotyping Kit
  • Abbott HCV Genotyping ASR Assay
  • Invader HCV Genotyping Assay
  • *VERSANT HCV Genotype 2.0 (LiPA)
specimen handling
Specimen & Handling
  • Acceptable
    • non-hemolyzed serum
    • EDTA plasma
  • Not acceptable
    • Lithium anticoagulated plasma samples
  • Hemolysis does have negative effects.
specimen handling1
Specimen & Handling
  • Store at RT for 24 hours,
  • Refrigerate up to 5 days, or
  • Freeze -20 C to -80 C.
viral nucleic acid extraction
Viral Nucleic Acid Extraction
  • MagNA Pure LC Total Nucleic Acid Isolation Kit
    • *a solid phase isolation methodology kit utilizing magnetic-bead technology
viral nucleic acid extraction1
Viral Nucleic Acid Extraction
  • *Roche MagnaPure robotic nucleic acid isolation system.
rt of the hcv rna cdna ampl
RT of the HCV RNA & cDNAAmpl.
  • Reverse Transcription of the HCV RNA and cDNA Amplification
    • VERSANT HCV ASR (analyte specific reagent) Amplification 2.0 Kit
      • reverse transcription and subsequent amplification of the target cDNA obtained
      • Produces biotinylated DNA product
rt of the hcv rna cdna ampl1
RT of the HCV RNA & cDNAAmpl.
  • Reaction tube has all reagents added
    • reverse transcriptase
    • Amplification polymerases
    • UNG
reverse transcription
*Reverse Transcription
  • Sensiscript enzyme
    • RNA amounts less than 50 ng,
  • Omniscript enzyme
    • RNA amounts greater than 50 ng
amplification phase
Amplification Phase
  • HotStarTaq polymerase
  • *Two pairs of primers
    • amplify portions of the 5\'UTR and core regions
    • producing biotinylated DNA fragments
      • of 240 base pairs of 5’ UTR
      • 270 base pairs of core
electrophoresis
Electrophoresis
  • Verification of an amplicon product
  • 5uL sample
  • a pre-formed polyacrylamide gel
  • molecular weight ladder
tecan auto lipa 48
Tecan Auto-LiPA 48
  • Capable of processing 48 strips / samples
  • All (proprietary) reagents kept at optimal temperatures and added in timed sequence
interpretation of results
Interpretation of Results
  • Evaluate control lines
    • Position 1 – conjugate control
    • Position2 – amplification control
    • Position 23 - core products
interpretation of results1
Interpretation of Results
  • Evaluate control samples
    • Kit provides negative and positive controls
    • Must perform according to manufacturer
interpretation of results2
Interpretation of Results
  • Evaluate patient samples
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