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Hepatitis C Genotyping using a Line Probe Assay

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

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  1. Hepatitis C Genotypingusing a Line Probe Assay Carolyn A. Ragland August 2008

  2. 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

  3. 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

  4. Hepatitis C Virus • Positive sense, single stranded RNA • Linear genome of @ 9600 nucleotides • Produces viral core and envelope structures • Produces non-structural / operational products

  5. 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.

  6. 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

  7. HCV Genotyping • TRUGENE HCV 5' Genotyping Kit • Abbott HCV Genotyping ASR Assay • Invader HCV Genotyping Assay • *VERSANT HCV Genotype 2.0 (LiPA)

  8. Specimen & Handling • Acceptable • non-hemolyzed serum • EDTA plasma • Not acceptable • Lithium anticoagulated plasma samples • Hemolysis does have negative effects.

  9. Specimen & Handling • Store at RT for 24 hours, • Refrigerate up to 5 days, or • Freeze -20 C to -80 C.

  10. Viral Nucleic Acid Extraction • MagNA Pure LC Total Nucleic Acid Isolation Kit • *a solid phase isolation methodology kit utilizing magnetic-bead technology

  11. Viral Nucleic Acid Extraction • *Roche MagnaPure robotic nucleic acid isolation system.

  12. 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

  13. RT of the HCV RNA & cDNAAmpl. • Reaction tube has all reagents added • reverse transcriptase • Amplification polymerases • UNG

  14. RT of the HCV RNA & cDNAAmpl. • GeneAmp PCR 2700

  15. *Reverse Transcription • Sensiscript enzyme • RNA amounts less than 50 ng, • Omniscript enzyme • RNA amounts greater than 50 ng

  16. 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

  17. Electrophoresis • Verification of an amplicon product • 5uL sample • a pre-formed polyacrylamide gel • molecular weight ladder

  18. HCV Genotyping using a Line Probe Assay (LiPA)

  19. Line Probe Assay (LiPA)

  20. Tecan Auto-LiPA 48 • Capable of processing 48 strips / samples • All (proprietary) reagents kept at optimal temperatures and added in timed sequence

  21. Interpretation of Results • Evaluate control lines • Position 1 – conjugate control • Position2 – amplification control • Position 23 - core products

  22. Interpretation of Results • Evaluate control samples • Kit provides negative and positive controls • Must perform according to manufacturer

  23. Interpretation of Results • Evaluate patient samples

  24. Interpretation of Results • Banding patterns

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