Only performed Monday - Friday
HepC Genotyping is determined using Roche cobas
4800 System. This is a highly sensitive real-time PCR-based test for the qualitative identification of HCV genotypes 1 to 6 and genotypes 1a and 1b subtypes in human plasma or serum.
The assay
uses three different target regions in the HCV genome (5'-UTR, Core, NS5B) to achieve optimal genotyping and subtyping concordance when compared to sequencing. In dual infections it has the capability to detect both genotypes, down to a ratio of 1:
100.
Hepatitis C genotyping involves two main procedures:
1. Sample preparation where hepatitis C viral RNA is extracted from human serum or plasma.
2. HCV amplification with reverse transcription followed by polymerase chain reaction using the cobas HCV GT assay. This is a real-time, reverse transcription-PCR (RT-PCR) assay using 3 pools of PCR primers. One set of primers targets the 5'untranslated region (5'UTR) for detection of all HCV types and additionally distinguishes GT 2,3 and 6. The second pool targets the core regions for GT 1,4 and 5. The third pool targets NS5b for subtyping of GT 1a and 1b.
HCV is an enveloped virion containing a single-stranded positive sense RNA genome of approximately 9,500 nucleotides. It has been identified as the major etiological agent for post-transfusion non-A and non-B hepatitis worldwide. Based on genetic similarity, HCV has been classified into six major genotypes (1-6) and numerous subtypes. HCV genotype is predictive of the response of HCV-infected patients to peginterferon/ ribavirin combination therapy and may influence response to other anvirals . Before starting combination therapy, it is recommended that the genotype be determined so that the patient can receive the most appropriate therapy regimen.
For further information contact the laboratory (contact via Lablink: 22000 or (09) 307-8995 or 0800 522 7587) ,or:
the Virology team
virology@adhb.govt.nz
SPECIMEN REQUIREMENTS
- Store whole blood at room temperature for no longer than 4 hours and do not refrigerate.
- Do not use haemolysed samples or heparinised plasma as haemoglobin and heparin inhibit PCR.
- Separate plasma or serum from whole blood within 4 hours of collection by centrifugation at 1500g for 5 minutes at room temperature. Transfer plasma or serum to sterile microcentrifuge tubes using a sterile transfer pipette. Store a minimum of 1.0mL for processing.
- Separated plasma or serum may be transported at 2-8 o C for up to 24 hours.
- Separated samples can be stored prior to processing at 2-8 o C for up to 48 hours, or at minus 20 o C for up to 72 hours, or at -70 o C for greater than 72 hours.
- Plasma and serum samples can be frozen and thawed no more than 4 times prior to specimen processing.
- Proper specimen handling is very important to protect RNA in the samples from degradation .