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 genotyping using a line probe assay

Hepatitis C Genotypingusing a Line Probe Assay

Carolyn A. Ragland

August 2008


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


Rt of the hcv rna cdna ampl2
RT of the HCV RNA & cDNAAmpl.

  • GeneAmp PCR 2700


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


Interpretation of results3
Interpretation of Results

  • Banding patterns


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