Method Description

Sample Preparation:

The Abbott mSample Preparation System kit is used with the Abbott m2000sp, an automated sample preparation system using the magnetic microparticle processes to extract and purify viral nucleic acids from human serum specimens. An internal control is incorporated in the nucleic acid extraction and purification procedure for processing the assay controls and clinical specimens. After capture of nucleic acids onto magnetic microparticles, the microparticles are washed to remove unbound sample components. Then, the bound nucleic acids are eluted from the microparticles, and the eluates are transferred to a 96-well microtiter plate containing polymerase chain reaction (PCR) mastermix reagents for amplification and detection.

Amplification, Detection, and Genotyping:

The Abbott RealTime HCV Genotype II assay is used to amplify the 5' noncoding (5' NC), nonstructural 5b (NS5b), and core regions of the hepatitis C virus (HCV) genome, with several PCR primer sets that are optimized to amplify all HCV isolates. An internal control primer set is included to amplify a portion of the hydroxypyruvate reductase gene of the pumpkin plant, Cucurbita pepo. The assays positive control is an armored RNA particle diluted in negative human plasma.

During the amplification reaction, complementary DNA (cDNA) sequences are generated from the target RNA sequences by the reverse transcriptase activity of the thermostable rTth DNA polymerase. First, the HCV and internal control reverse primers anneal to their respective target sequences and are extended during a prolonged incubation period. After a denaturation step, in which the temperature of the reaction is raised above the melting point of the double-stranded cDNA:RNA product, a second primer anneals to the cDNA strand and is extended by the DNA polymerase activity of the rTth enzyme to create a double-stranded DNA product. During each round of thermal cycling, amplification products dissociate to single strands at a high temperature, allowing primer annealing and extension as the temperature is lowered. Exponential amplification of the product is achieved through repeated cycling between high and low temperatures, resulting in a billion-fold or greater amplification of target sequences. Fluorescent probes specific for HCV genotypes 1 to 5 and subtypes 1a and 1b anneal to the amplified sequence products in 4 separate reaction wells for each specimen.(Package insert: RealTime HCV Genotype II. Abbott Molecular Inc; R1, 06/2013)