HCV PCR By Henrietta Orji July 31 st, 2010 Hepatitis C Virus by Polymerase Chain Reaction

HCV is highly asymptomatic and it affects people of all ages, races, and ethnicity leading to chronic liver disease. Transmission of HCV is through blood and body fluids, and also through sexual contact. Nucleic acid testing for HCV is performed using the fully automated instrument called the COBAS Ampliprep / COBAS Taqman 96 HCV PCR test is performed to determine the response to and duration of antiviral therapy. COBAS Ampliprep / COBAS Taqman is sensitive, reliable, fast, has low cross contamination, and increased productivity. The real time PCR technology utilizes dual oligonucleotide probes for Probe Capture technology during amplification, while flourescence emission is used for detection of HCV RNA. Objectives

HCV is highly asymptomatic and it affects people of all ages, races, and ethnicity leading to chronic liver disease Transmission of HCV is through blood and body fluids, and also through sexual contact Nuclueic acid testing for HCV is performed using the fully automated instrument called the COBAS Ampliprep / COBAS Taqman 96 HCV PCR test is performed to determine response to and duration of antiviral therapy COBAS Ampliprep / COBAS Taqman is sensitive, reliable, fast, has low cross contamination, and increased productivity The real time PCR technology utilizes dual oligonucleotide probes for Probe Capture technology during amplification, while fluorescence emission is used for detection of HCV RNA Objectives

Any of THEM could have the Hepatitis C Virus…

Clinical Reasons for the Test Aids in the management of HCV – infected individuals a. Monitoring the rate of virological response b. Determining the duration of therapy

COBAS Ampliprep / COBAS Taqman 96 Analyzer Fully automated instrument Improves laboratory work flow Leads to increased productivity Robust, sensitive, and reliable

Principles of the Procedure The principles are based on: Automated sample preparation (extraction) Automated reverse transcription, PCR amplification, and detection Quantitation

Automated Sample Preparation

Utilizes the Probe Capture Technology Specimen Preparation is based on the following key principles: Load of 850µL serum or plasma specimen Lysis with chaotrope (a reagent that causes a change in protein conformation to release nucleic acid) at an elevated temperature in presence of Quantification Standard (QS) or Internal Control (IC) Hybridization of biotinylated oligonucleotide probes to target Capture of biotinylated probe: target by streptavidin magnetic particle Magnetic removal and washing of particles Resuspension of particles

Automated Reverse Transcription Extracted specimens are added to the K tubes (amplification tubes) Heat mixture Downstream primer anneal specifically to the HCV target RNA and to the HCV QS RNA In the presence of Mn 2+ and excess dNTPs the ZO5 polymerase extends the annealed primer forming a DNA strand complimentary to the RNA target (cDNA)

PCR Amplification PCR Amplification can be divided into Target Amplification and Selective Amplification. The amplification takes place in the Thermocycler that is inside the COBAS Taqman 96 analyzer. Target Amplification The steps for amplification include: Denaturation: Thermocycler hits the mixture at 90°C to denature the RNA: cDNA hybrid. Specific primer target sequence is exposed

PCR Amplification, cont’d Annealing: Here the primer anneals to the target DNA at a temperature of 40 – 65°C Extension: The enzyme thermos specie DNA polymerase (ZO5), in the presence of Mn 2+ and excess dNTPs extend the annealed primer along the target template to produce a double-stranded DNA molecule called an amplicon.

PCR Amplification, cont’d The picture shows what happens at the end of the first PCR cycle – results in two copies of target sequence This cycle is then repeated several times. The cycle only occurs in the region of HCV genome between the primers

PCR Amplification, cont’d Selective Amplification The enzyme AmpErase (Uracil-N-glycosylase) catalyzes and destroys all the DNA containing deoxyuridine and also removes nonspecific product formed after initial activation of the mastermix by Mn 2+ – Naturally occuring DNA does not contain deoxyuridine – Deoxyuridine is in master mix and DNA containing it is removed before amplification

Detection of PCR Product Instrument utilizes real time PCR technology Uses dual labeled fluorescent probes Probes monitor the emission intensity of fluorescent reporter dyes Fluorescent reporter dyes are released during the amplification process Amplification of HCV RNA & HCV QS RNA are measured independently at different wavelengths This process is repeated for the designated number of cycles The higher the HCV titre of a specimen, the earlier the fluorescence of the reporter dye of the HCV rises above the baseline fluorescence level

Limitations of the Procedure The COBAS Ampliprep / COBAS Taqman 96 comes with its own limitations, which include: – Only human serum or plasma collected in EDTA anticoagulant is suited for the test. – Only personnel trained in PCR technique can use this instrument. – Reliable results are dependent on adequate specimen collection, transport, storage, and processing procedure. – The quantity of HCV RNA is dependent on the number of virus particles present in the specimen and may be affected by specimen collection methods and patient factors. – Mutation in the region of the viral genome covered by the test primers and or probe may result in the under-quantitation of or failure to detect the presence of the virus. – The AmpErase enzyme reduces the risk of amplicon contamination; but contamination from HCV positive controls and clinical specimens can be avoided only by good laboratory practices and adherence to procedures. – Switching from one assay that performs HCV RNA quantitation to the COBAS Ampliprep / COBAS Taqman 96 HCV test requires users to perform method correlation studies in order to quantify assay differences.

Causes of Erroneous Results Putting reagents in the wrong cassettes Using expired reagents Mixing reagents with different lot numbers / different cassetes Using controls from different lots / different kits Using specimen other than serum or EDTA plasma Using contaminated reagents