1 / 25

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

odell
Download Presentation

Hepatitis C Genotyping using a Line Probe Assay

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related