carolyn a. ragland august 2008. hepatitis c *paraenterally transmitted contact with infected blood /...

Hepatitis C Genotypingusing a Line Probe Assay

Carolyn A. RaglandAugust 2008

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

Hepatitis CWHO - 3% have chronic HCV

infectionsIn US:3+ million become infected yearly8000 – 10,000 die annually from

complicationsCirrhosisLiver cancer

Hepatitis C VirusPositive sense, single stranded RNA

Linear genome of @ 9600 nucleotidesProduces viral core and envelope

structuresProduces non-structural / operational

products

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.

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 GenotypingTRUGENE HCV 5' Genotyping Kit

Abbott HCV Genotyping ASR Assay

Invader HCV Genotyping Assay *VERSANT HCV Genotype 2.0 (LiPA)

Specimen & HandlingAcceptable

non-hemolyzed serumEDTA plasma

Not acceptable Lithium anticoagulated plasma samples

Hemolysis does have negative effects.

Specimen & HandlingStore at RT for 24 hours,Refrigerate up to 5 days, orFreeze -20 C to -80 C.

Viral Nucleic Acid ExtractionMagNA Pure LC Total Nucleic Acid

Isolation Kit*a solid phase isolation methodology kit

utilizing magnetic-bead technology

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

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

RT of the HCV RNA & cDNA Ampl.Reaction tube has all reagents addedreverse transcriptaseAmplification polymerasesUNG

RT of the HCV RNA & cDNA Ampl.GeneAmp PCR 2700

*Reverse TranscriptionSensiscript enzyme

RNA amounts less than 50 ng,Omniscript enzyme

RNA amounts greater than 50 ng

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

ElectrophoresisVerification of an amplicon product 5uL samplea pre-formed polyacrylamide gelmolecular weight ladder

HCV Genotyping using a Line Probe Assay (LiPA)

Line Probe Assay (LiPA)

Tecan Auto-LiPA 48 Capable of processing 48 strips / samples All (proprietary) reagents kept at optimal

temperatures and added in timed sequence

Interpretation of ResultsEvaluate control lines

Position 1 – conjugate controlPosition2 – amplification controlPosition 23 - core products

Interpretation of ResultsEvaluate control samples

Kit provides negative and positive controls Must perform according to manufacturer

Interpretation of ResultsEvaluate patient samples

Interpretation of ResultsBanding patterns