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HCV by PCR. Neelam Gajjar 7/26/2009. Objectives. Overview Specimen type and handling Procedure Instruments Used Treatment. In this presentation:. Overview. HCV is a major, growing health problem worldwide

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Hcv by pcr

HCV by PCR

Neelam Gajjar

7/26/2009


Objectives
Objectives

  • Overview

  • Specimen type and handling

  • Procedure

  • Instruments Used

  • Treatment

In this presentation:


Hcv by pcr

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


Specimen type and handling
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


Procedure
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.


Procedural information
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


Cobas ampliprep
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


Cobas taqman 48 system
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


Treatment
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


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