Carolyn A. Ragland August 2008. Hepatitis C *Paraenterally transmitted Contact with infected blood /...
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Transcript of Carolyn A. Ragland August 2008. Hepatitis C *Paraenterally transmitted Contact with infected blood /...
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Hepatitis C Genotypingusing a Line Probe Assay
Carolyn A. RaglandAugust 2008
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Hepatitis C*Paraenterally transmitted
Contact with infected blood / blood productsBlood transfusions – no longer the culprit * US – most likely reason, sharing of needlesSexual and perinatal transmission possible
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Hepatitis CWHO - 3% have chronic HCV
infectionsIn US:3+ million become infected yearly8000 – 10,000 die annually from
complicationsCirrhosisLiver cancer
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Hepatitis C VirusPositive sense, single stranded RNA
Linear genome of @ 9600 nucleotidesProduces viral core and envelope
structuresProduces non-structural / operational
products
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Hepatitis C VirusThe 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.
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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
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HCV GenotypingTRUGENE HCV 5' Genotyping Kit
Abbott HCV Genotyping ASR Assay
Invader HCV Genotyping Assay *VERSANT HCV Genotype 2.0 (LiPA)
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Specimen & HandlingAcceptable
non-hemolyzed serumEDTA plasma
Not acceptable Lithium anticoagulated plasma samples
Hemolysis does have negative effects.
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Specimen & HandlingStore at RT for 24 hours,Refrigerate up to 5 days, orFreeze -20 C to -80 C.
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Viral Nucleic Acid ExtractionMagNA Pure LC Total Nucleic Acid
Isolation Kit*a solid phase isolation methodology kit
utilizing magnetic-bead technology
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Viral Nucleic Acid Extraction*Roche MagnaPure robotic nucleic acid isolation system.
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RT of the HCV RNA & cDNA Ampl.Reverse Transcription of the HCV RNA and cDNA AmplificationVERSANT HCV ASR (analyte specific
reagent) Amplification 2.0 Kit reverse transcription and subsequent
amplification of the target cDNA obtainedProduces biotinylated DNA product
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RT of the HCV RNA & cDNA Ampl.Reaction tube has all reagents addedreverse transcriptaseAmplification polymerasesUNG
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RT of the HCV RNA & cDNA Ampl.GeneAmp PCR 2700
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*Reverse TranscriptionSensiscript enzyme
RNA amounts less than 50 ng,Omniscript enzyme
RNA amounts greater than 50 ng
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Amplification PhaseHotStar 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
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ElectrophoresisVerification of an amplicon product 5uL samplea pre-formed polyacrylamide gelmolecular weight ladder
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HCV Genotyping using a Line Probe Assay (LiPA)
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Line Probe Assay (LiPA)
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Tecan Auto-LiPA 48 Capable of processing 48 strips / samples All (proprietary) reagents kept at optimal
temperatures and added in timed sequence
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Interpretation of ResultsEvaluate control lines
Position 1 – conjugate controlPosition2 – amplification controlPosition 23 - core products
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Interpretation of ResultsEvaluate control samples
Kit provides negative and positive controls Must perform according to manufacturer
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Interpretation of ResultsEvaluate patient samples
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Interpretation of ResultsBanding patterns
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