雅培RealTime HCV Genotype⒒(3)

the beginning of the sample preparation process to demonstrate that the process was completed correctly for each specimen and control.

Amplification Master Mix

The Abbott m2000sp instrument automates the assembly of three amplification master mixes (A, B, and C) by combining the respective Abbott RealTime HCV Genotype II Oligonucleotide Reagent (A, B, or C) with thermostable rTth DNA polymerase enzyme and Activation Reagent. The Abbott m2000sp dispenses the resulting master mixes into the Abbott 96-Well Optical Reaction Plate along with aliquots of the nucleic acid samples prepared by the Abbott m2000sp. Each processed sample is added to one well containing Master Mix A, one well containing Master Mix B, and one well containing Master Mix C.

Amplification

The Abbott RealTime HCV Genotype II assay uses four sets of PCR primers. One set of primers targets a

sequence within the 5′ untranslated region (UTR) of the HCV genome. This primer set is designed to amplify all HCV isolates. The second primer set is designed to amplify the non structural 5b (NS5b) region of genotype 1a. The third HCV primer set is designed to amplify the NS5b region of genotype 1b. By contrast, the IC primer set is designed to amplify a portion of the hydroxypyruvate reductase gene of the pumpkin plant, Cucurbita pepo and is delivered in an Armored RNA® particle that has been diluted in negative human plasma.

During the amplification reaction, the target RNA is converted to complementary DNA (cDNA) by the reverse transcriptase activity of the thermostable rTth DNA polymerase.7 The HCV and IC reverse primers anneal to their respective targets 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 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.

Detection

The assay requires three separate reactions to detect genotypes 1, 1a, 1b and 2 – 5:

Reaction A is designed to detect all HCV isolates, type 3 isolates, and subtype 1a isolates.

Reaction B is designed to detect type 1 isolates, type 2 isolates, and subtype 1b isolates.

Reaction C is designed to detect type 4 isolates and type 5 isolates. Reaction Probe A All HCV Isolates FAM

Genotype 3 Subtype 1a B Genotype 1 Genotype 2 Subtype 1b C Genotype Genotype 5