RealLine HCV Qualitative Str-Format

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1 Instructions for use REAL TIME PCR DETECTION KIT FOR THE HEPATITIS C VIRUS RNA (HCV) Research Use Only (RUO) (Str-format) VBD Tests valid from: October 2018 Rev05_1018_EN Page 1 of 8

2 Explanation of symbols used in labeling RUO LOT REF Research use only Batch code Catalogue number Amount of tests Expiry Date Temperature limitation Consult instructions for use Keep out of sunlight Manufacturer BIORON Diagnostics GmbH Rheinhorststr Ludwigshafen (Germany) Phone Fax: Legals: Limited Product Warranty: This warranty limits our liability for the replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. BIORON Diagnostics GmbH shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. Trademarks: FAM, HEX, JOE and ROX are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries. iq and CFX are trademarks of Bio-Rad Laboratories, Inc. Rotor-Gene is a registered trademark of Qiagen Group, Germany. Rev05_1018_EN Page 2 of 8

3 TABLE OF CONTENT: 1. STORAGE AND TRANSPORTATION 4 2. KIT CONTENTS 4 3. INTRODUCTION 4 4. PRINCIPLES OF THE PROCEDURE 5 5. PRECAUTIONS 5 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED 6 7. REAGENT AND SAMPLE PREPARATION 6 8. PROCEDURE PROTOCOL 7 9. DATA ANALYSIS 8 VBD0795 Page 3 of 8

4 REAL TIME PCR DETECTION KIT FOR HEPATITIS C VIRUS RNA (HCV) 1. STORAGE AND TRANSPORTATION Store assay kit at (2-8) С in the manufacturer s packing Transportation for up to 25 С for 10 days is allowed. Do not freeze reagents. Do not pool reagents from different lots or from different vials of the same lot. Strictly follow the Instruction manual for reliable results. 2. KIT CONTENTS Ready Master Mix (RMM) for reverse transcription and PCR, lyophilized, Weak Positive Control Sample (WPC HBV/HCV/HIV), lyophilized Solution for Restoration of Control samples (RSC) Adhesive Foils for PCR 96 test tubes (12 strips x 8 tubes) 1 vial 1 vials, 4 ml each; 3. INTRODUCTION Assay kit RealLine HCV qualitative kit is intended for the detection of hepatitis C virus (HCV) RNA in plasma and serum. The method is based on the reverse transcription of viral RNA to generate complementary DNA (cdna), with subsequent amplification of target cdna by Polymerase Chain Reaction (PCR) with fluorescent detection of amplified DNA in the real-time mode. The assay kit is adapted for real-time PCR detection systems like RealLine Cycler (BIORON Diagnostics GmbH), iq icycler, iq5 icycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-technology, Russia) or their analogues. The kit contains reagents sufficient for 96 test runs. It is strongly recommended to use one Weak Positive Control sample and one Negative Control sample in each test run. The RealLine HCV qualitative kit is designed to detect HCV RNA isolated from serum (plasma) using RNA extraction kits: RealLine Extraction 100 RealLine Extraction 1000 Rev05_1018_EN Page 4 of 8

5 Specificity Assay kit is designed for in vitro determination of HCV genotypes 1a, 1b, 2a, 2b, 2c, 2i, 3, 4, 5a, 6 regardless of subtype. The samples containing HCV RNA with concentration above the detection limit will be determined as positive. If specimen does not contain HCV RNA, analysis will give negative result (in 100 % of cases). Sensitivity Assay kit securely determines HCV RNA in concentration not less than 15 IU / ml for the RNA isolation from 1 ml of serum (plasma). 4. PRINCIPLES OF THE PROCEDURE Principle of analysis is based on the reverse transcription of viral RNA with subsequent PCR amplification of target cdna by PCR with fluorescent detection of amplified DNA in the real-time mode. Reliability of analysis is provided by application of Weak Positive control sample. Threshold cycle value Ct is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal rises significantly above the background fluorescence. The use of Internal Control (IC) prevents generation of false negative results associated with possible loss of RNA template during specimen preparation. IC indicates if PCR inhibitors occur in the reaction mix. IC template should be added in each single sample (including control samples) prior to NA extraction procedure. The amplification and detection of IC does not influence the sensitivity or specificity of the target RNA PCR. 5. PRECAUTIONS Wear protective disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents. Avoid microbial and ribonuclease contamination of reagents when removing aliquots from reagent vials. The use of sterile disposable pipettes and RNase-free pipette tips is recommended. Do not pool reagents from different lots or from different vials of the same lot. Dispose unused reagents and waste in accordance with country, federal, state and local regulations. No warranty for using kit after the expiry date at the side label of the box. VBD0795 Page 5 of 8

6 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED Real time PCR device RealLine Cycler (BIORON Diagnostics GmbH), iq/iq5 icycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-technology, Russia) or equivalent; Disposable gloves, powder-free; Pipettes (capacity μl) with filters (aerosol barriers); Disposable DNAse/RNase-free tips with filters 0.2 ml microtube racks; If the kit is used with another extraction kit from another supplier: Negative Control Sample (negative tested Plasma or Serum) Internal Control Sample (VBC8881, BIORON Diagnostics GmbH) 7. REAGENT AND SAMPLE PREPARATION 7.1. Sample preparation. Prepare specimens for the assay with Extraction kit RealLine Extraction 100 or RealLine Extraction 1000 or according to Extraction kit manual. It is strongly recommended to use one Weak Positive Control sample and one Negative Control sample in each test run and to implement the control to the Extraction procedure. Each group of samples undergoing the procedure of RNA isolation must include a positive control sample (PC) and negative control sample (NC), that are components of the RNA extraction kit. We strongly recommend the implementation of the Internal Control IC, the Negative Control NC and Positive Control PC samples to the extraction procedure. When using a kit of another supplier for the extraction of nucleic acids as recommended in chapter 1, add 20 μl of IC (VBC8881) to each tube. For the NC use 100 µl of the Negative Control Sample. For the PC use 70 μl of Negative Control Sample and 30 μl of Positive Control to the tube marked PC Reagent preparation Preparation of Control samples Add 1 ml of Solution for Recovery Solution for Control samples (RSC) into a vial with Weak Positive Control sample (WPC), mix gently, keep for 15 minutes, then carefully mix once again. WPC should be stored at (2 8) С and used within 1 month after preparation. Preparation of Ready Master Mix Prior to use, warm reagents (do not open!) at room temperature (18-25) С. Open the package, separate an appropriate number of reaction tubes with Ready Master Mix (RMM) using razor or scalpel. Keep the tubes which were not used for the test in the original bag. Try to squeeze excess of the air out of the bag before closing the clip. Rev05_1018_EN Page 6 of 8

7 8. PROCEDURE PROTOCOL 8.1. Place the tubes with processed specimens and controls to Magnetic Rack Prepare an appropriate number of reaction tubes with Ready Master Mix (RMM). Label each reaction tube for each specimen and control sample. Attention! For block-type cyclers put marks on the lateral part of a reaction tube Add 50 μl of each processed specimen and control to the appropriately labeled reaction tube using a new RNase-free tip with aerosol barrier for each sample. Do not grasp a sorbent particles! 8.4. Place reaction tubes into the thermal block of real time PCR device Program real time PCR device as follows: Stage 1: 45 С, 30 min; Stage 2: 94 С, 1 min; Stage 3: 94 C, 10 sec 60 C, 20 sec 50 cycles Fluorescence measurements should be done at 60 С Collect real-time PCR data through the FAM channel for detection of amplification of IC cdna Collect real-time PCR data through the ROX channel for detection of amplification of HCV cdna. NOTE: Settings for RealLine Cycler and DT96: for these cyclers the measurement exposure must be adjusted. Choose the Operation with the device mode in the Settings menu, select the item Measurement exposition: FAM to 250 ROX to 1000 Confirm that the current exposure value is saved by pressing YES Attention! The specified exposure values are applicable only for RealLine kits and, if necessary, must be changed for other purposes. VBD0795 Page 7 of 8

8 9. DATA ANALYSIS 9.1. In Weak Positive Control sample the program should detect: ROX fluorescent signal increase and Сt value (HCV cdna amplification); FAM fluorescent signal increase and Сt value (IC cdna amplification) In Negative Control sample the program should detect: FAM fluorescent signal increase and Ct value, and no significant ROX fluorescent increase should appear. If Ct value for NC along ROX channel is less than 40, this indicates the presence of contamination The program should detect amplification signal increase for IC cdna (channel FAM) in each sample and define Ct for IC. Probe analysis is valid if Ct of IC for this sample is equal to or less than In the case of contamination (when NC is determined as positive) all positive results in this test should be repeated from the RNA extraction stage. Negative samples of such test run are considered reliable Calculate (IC Ct) m as the average Ct value of IC for all samples (including WPC and NC). Samples with Ct of IC, that differ from (IC Ct) m more than 2 cycles should be ignored. After screening, recalculate (IC Ct) m for remaining samples The sample is negative if Ct value along the ROX channel exceeds 40 or is not determined If Ct of IC for this sample differs from (IC Ct) m more than 2 cycles, then the result for this sample should be considered as equivocal. The test should be repeated from the sample RNA extraction stage The sample is positive for HCV if Ct value along the ROX channel does not exceed The test results are considered reliable only when Weak Positive and Negative controls perform as expected. Questions and remarks: Rev05_1018_EN Page 8 of 8

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