HCV by PCR

1. HCV by PCR Neelam Gajjar 7/26/2009

2. Objectives • Overview • Specimen type and handling • Procedure • Instruments Used • Treatment In this presentation:

3. Overview • HCV is a major, growing health problem worldwide • Serological tests are useful for screening, however molecular approaches are the mainstay of HCV tests • Qualitative Molecular Tests: • Confirmation of positive screening tests • Quantitative Molecular Tests: • Prognostic information regarding likelihood of response to therapy • Monitoring of treatment efficacy

4. Specimen Type and Handling • Human Serum or Plasma • Serum – Blood collected in SST tube [non-hemolysed serum only] • Plasma – EDTA as anticogulant [heparin is not acceptable] • Whole blood must be transported at 2-25o C OR frozen at 20-80o C. • Process within 6 hrs of collection • Stored at room temperature for 24 hrs • Refrigerate for up to 5 days

5. Procedure • Starts with Specimen and control preparation, extraction followed by PCR reagent preparation, amplification(real-time PCR), DETECTION 650 uL of vortexed and spun  and unclotted specimens are transferred in to Sample-tube which is loaded in to sample rack onto COBAS Ampliprep instrument  for processing with low and high pos control as well as negative control. •  Prepare K-carrier from your processed specimen • Prepare HCV-ASR  MMX  by adding Maganese AND then add MMX  to processed specimen in to K carrier. • Load the K-carrier in to Taq Man Analyzer • The COBAS Taq Man analyser automatically determines the HCV RNA titer for the sample .The HCV  RNA titer is expresse in IU/mL.

6. Procedural Information • Analytical reportable range for quantitative HCV ASR: • 10 – 5,000,000 IU/mL • Material and Process Information • Each CAP TNAI -Kit contains sufficient reagent for 24 specimen runs • Specimen preparation reagents  cassettes are packaged as 48 test runs and HCV ASR MMX COMES IN PACKAGE OF 2 x 24 TEST RUNS • One or Two Day Process

7. COBAS AmpliPrep Features and benefits • 72 samples can be loaded at once and processed within 4 hours to meet high-volume testing demands • Continuous sample and reagent loading without operation interruption minimizes downtime and maximizes hands-off time for the operator • Bar-coded sample racks and reagents provide positive sample identification and reagent tracking to reduce hands-on time • Dedicated and disposable sample processing units minimize the potential for cross-contamination • Ready-to-use, compact reagent cassettes are designed to eliminate manual preparation time and prevent manual preparation errors

8. COBAS TaqMan 48 System • Features and benefits • Real-time TaqMan PCR methodology provides unmatched accuracy, superior linear range, and the potential to run one test for quantitative and qualitative requirements • Run sizes of 6 to 48 samples provide flexibility and maximum efficiency • The utility channel adds flexibility and cost effectiveness by allowing the laboratory to run IVD and user-defined assays on one fast, easy, and accurate platform • Two independently operating thermal cyclers provide approximate run times of 90 to 120 minutes to increase workflow flexibility and minimize the need for multiple platforms • • AMPLILINK Software is Windows driven for easy navigation, and provides the security of positive sample ID throughout the run, as well as enhanced LIS capabilities

9. Treatment • HCV approx 170 million worldwide • Antiviral therapy with the combination of pegylated interferon alfa and ribavirin • HCV RNA testing is used to diagnose acute and chronic Hepatitis C • HCV genotype indicates: • Duration of treatment • Dose of ribavirin • Virological monitoring procedure