A - C

aInternal Control (IC) Quasar 670a Reference Dye ROXb

b Quasar 670 dye contains the same spectral properties as Cy 5. ROX is a Passive Reference Dye in Oligonucleotide Reagents A, B, and C.

Amplification of both HCV and IC targets take place simultaneously in the same reaction. The HCV genotype-specific probes and IC-specific probe within each reaction are all labeled with different fluorophores, thus allowing for simultaneous detection of HCV genotype-specific and IC-specific amplified products.

During the annealing portion of each amplification cycle, the probes hybridize to their respective amplification target, if present. The 5′ end of each HCV-specific probe is labeled with a fluorescent moiety while the 3′

terminus is labeled with a quenching moiety and a Minor Groove Binder (MGB ) group. In the absence of the HCV target sequences, the probe fluorescence is quenched. In the presence of the HCV target sequences, the probe hybridizes to its complementary sequence. During PCR extension, the 5′ to 3′ exonuclease (or Taqman) activity of the rTth polymerase degrades the hybridized probe into constituent nucleotides thus separating the quencher and the fluorophore allowing fluorescent emission and detection.8

The Abbott m2000rt instrument detects the resultant fluorescence of the different fluorophores in each reaction well after each cycle.

PREVENTION OF NUCLEIC ACID CONTAMINATION

The possibility of nucleic acid contamination is minimized because:

Reverse transcription, PCR amplification, and oligonucleotide hybridization occur in a sealed 96-Well Optical Reaction Plate.

Detection is carried out automatically without the need to open the 96-Well Optical Reaction Plate. Aerosol barrier pipette tips are used for all pipetting. The pipette tips are discarded after use.

Separate dedicated areas are used to perform the Abbott RealTime HCV Genotype II assay. Refer to the

SPECIAL PRECAUTIONS section of this package insert.

REAGENTS

Abbott RealTime HCV Genotype II Amplification Reagent Kit (List No. 08L21-90)

1. Abbott RealTime HCV Genotype II Internal Control (List No. 8L21Y) (2 vials, 1.2 mL per vial)

Less than 0.01% noninfectious Armored RNA with internal control sequences in negative human

plasma. Negative human plasma tested and found to be nonreactive by FDA licensed tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material is also tested and found to be negative by FDA licensed PCR methods for HIV-1 RNA and HCV RNA. Preservatives: 0.1% ProClin® 300 and 0.15% ProClin 950.

2. Abbott RealTime HCV Genotype II Amplification Reagent Packs (ListNo.08L21)

(Reagent Packs A, B, C / 24 Tests)

(1) Reagent Pack A.

1 bottle (0.9 mL) HCV Genotype II Oligonucleotide Reagent A. Less than 0.1% synthetic

oligonucleotides (6 primers, 5 probes), less than 0.1% dNTPs, and 10.4% dimethylsulfoxide in a

buffered solution with a reference dye. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

1 bottle (0.141 mL) Thermostable rTth Polymerase Enzyme (2.9 to 3.5 Units/µL) in buffered solution. 1 bottle (0.400 mL) Activation Reagent, 30 mM manganese chloride solution.

Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.