Wednesday October 01, 2008
The HCV Genotyping Kit from Sacace is a PCR-based kit for the detection of the 4 HCV genotypes (1a, 1b, 2 and 3) using an agarose gel. The kit is categorized into three parts - Ribosorb (Viral RNA extraction kit), Revert-R (cDNA conversion) and amplification detection kit.
The extraction kit includes all major components required for extracting viral RNA from plasma collected blood in EDTA/ACD. Components of extraction kit are lysis solution, sorbent, washing solution and elution solution; the only reagent required, but not provided in the kit is 70% acetone. The extraction process is a little tedious as compared to a spin protocol because it involves steps like centrifugation, vortexing and resuspension of the pellet. In the last step, the viral RNA can be eluted using 50 ul of elution solution. The total extraction procedure is nearly 45-50 minutes and the following items are required during the extraction protocol: 1.5 ml eppendorf tube, centrifuge (RCF 16,000 g), dry block, vortex mixer, pipettes, 70% acetone and absolute ethanol.
The second crucial component of this kit is the Revert-R for converting extracted viral RNA into cDNA. This conversion takes place on a thermal cycler by incubating at 37°C for 30 minutes. The kit contains all necessary reagents for conversion which are as follows: DTT (Dithiothreitol), RT Mix, RT Enzyme (MMLV Reverse Transcriptase) and TE buffer. The reagents are sufficient for 60 preparations. Reagent preparation (for 12 reactions), add 125 ul RT Mix into the tube containing lyophilized DTT and vortex for at least 5-10 seconds. Add 6 ul MMLV into the tube with Reagent Mix, (Reagent Mix = 125 ul RT Mix in DTT tube) vortex for 3 sec, centrifuge for 5-7 sec and dispense 10 ul to each tube. Pipette 10 ul RNA samples to the appropriate tube. Place tubes into thermal cycler and incubate at 37°C for 30 minutes.
In the last and final step: PCR amplification of the cDNA, requires preparation of a master mix followed by amplification (using a thermal cycler) and detection via agarose gel electrophoresis and a gel documentation system. Reagents provided for the preparation of the master mix are sufficient for 55 reactions: PCR-mix-1 genotypes 1a/1b - 55 ready-to-use, single-dose test tubes (contain primers for both genotypes, i.e. 1a and 1b); PCR-mix-1 genotypes 2/3a - 55 ready-to-use, single-dose test tubes (contain primers for both genotypes, i.e. 2 and 3a); PCR-mix-2 (1.2 ml); mineral oil (4.0 ml); HCV cDNA 1a C+ (0.1 ml); HCV cDNA 1b C+ (0.1 ml); HCV cDNA 2 C+ (0.1 ml); HCV cDNA 3a C+ (0.1 ml); DNA buffer (0.5 ml); negative control C (1.6 mL). Kit comes with negative controls (extraction and amplification) and individual positive control for all four genotypes. Genotypes can be differentiated on the basis of product size (base pair) for example genotype 1a gives band at 338 bp; genotype 1b at 395; genotype 2 at 286 and genotype 3 at 227 bp.
The complete process takes nearly 4 hours which includes starting from sample preparation up to detection on agarose. We usually use this kit to differentiate the genotype pattern of a positive HCV sample from patients. This genotype pattern helps in therapeutics and prognosis.
In terms of sensitivity, this kit is up to the mark; because we have reported results even for less positive samples (less than 33 copies/ul). I strongly recommend this state of the art kit for those who are associated with patient screening and working with HCV genotyping.
Akshat Mathur
Research Associate
Molecular Diagnostic
Gene Diagnostic
India