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 ID: 805307
Download The PPT/PDF document "Hepatitis C Genotyping using a Line Prob..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Hepatitis C Genotypingusing a Line Probe Assay
Carolyn A. Ragland
August 2008
Slide2Hepatitis 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
Slide3Hepatitis C
WHO - 3% have chronic HCV infections
In US:
3+ million become infected yearly
8000 – 10,000 die annually from complications
Cirrhosis
Liver cancer
Slide4Hepatitis C Virus
Positive sense, single stranded RNA
Linear genome of @ 9600 nucleotides
Produces viral core and envelope structures
Produces non-structural / operational products
Slide5Hepatitis 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.
Slide6Hepatitis 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
Slide7HCV Genotyping
TRUGENE HCV 5' Genotyping Kit
Abbott HCV Genotyping ASR Assay
Invader HCV Genotyping Assay
*VERSANT HCV Genotype 2.0 (
LiPA
)
Slide8Specimen & Handling
Acceptable
non-
hemolyzed
serum
EDTA plasma
Not acceptable
Lithium
anticoagulated
plasma samples
Hemolysis does have negative effects.
Slide9Specimen & Handling
Store at RT for 24 hours,
Refrigerate up to 5 days, or
Freeze -20 C to -80 C.
Slide10Viral Nucleic Acid Extraction
MagNA
Pure LC Total Nucleic Acid Isolation Kit
*a solid phase isolation methodology kit utilizing magnetic-bead technology
Slide11Viral Nucleic Acid Extraction
*Roche
MagnaPure
robotic nucleic acid isolation system.
Slide12RT of the HCV RNA & cDNA
Ampl
.
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
Slide13RT of the HCV RNA & cDNA
Ampl
.
Reaction tube has all reagents added
reverse transcriptase
Amplification polymerases
UNG
Slide14RT of the HCV RNA & cDNA
Ampl
.
GeneAmp
PCR 2700
Slide15*Reverse Transcription
Sensiscript
enzyme
RNA
amounts less than 50
ng
,
Omniscript
enzyme
RNA amounts greater than 50
ng
Slide16Amplification Phase
HotStar
Taq
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
Slide17Electrophoresis
Verification of an
amplicon
product
5uL sample
a pre-formed
polyacrylamide
gel
molecular weight ladder
Slide18HCV Genotyping using a Line Probe Assay (LiPA)
Slide19Line Probe Assay (LiPA)
Slide20Tecan Auto-LiPA 48
Capable of processing 48 strips / samples
All (proprietary) reagents kept at optimal temperatures and added in timed sequence
Slide21Interpretation of Results
Evaluate control lines
Position 1 – conjugate control
Position2 – amplification control
Position 23 - core products
Slide22Interpretation of Results
Evaluate control samples
Kit provides negative and positive controls
Must perform according to manufacturer
Slide23Interpretation of Results
Evaluate patient samples
Slide24Interpretation of Results
Banding patterns
Slide25