RealLine HIV qualitative Str-Format

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1 Instructions for Use REAL TIME PCR DETECTION KIT FOR HUMAN IMMUNODEFICIENCY VIRUS RNA Research Use Only (RUO) RealLine HIV Qualitative (Str-format) VBD Tests valid from July 2016 Rev _EN Page 1 of 9

2 Explanation of symbols used in labeling RUO LOT REF Σ For research use only Batch code Catalogue number Content of number of tests Expiry date Temperature limitation Consult instructions for use Manufacturer Keep out of sunlight 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. VBD0196 Page 2 of 9

3 Table of content: 1. STORAGE AND TRANSPORTATION 4 2. KIT CONTENTS 4 3. INTRODUCTION 5 4. PRINCIPLES OF THE PROCEDURE 6 5. PRECAUTIONS 6 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED 7 7. REAGENT AND SAMPLE PREPARATION 7 8. PROCEDURE PROTOCOL 8 9. DATA ANALYSIS 9 VBD0196 Page 3 of 9

4 DETECTION OF HUMAN IMMUNODEFICIENCY VIRUS RNA BY REAL TIME PCR Research Use Only 1. STORAGE AND TRANSPORTATION Store assay kit at (2-8) С in the manufacturer s packing. Transportation at 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 for reverse transcription and PCR, freeze-dried (RMM) 96 test tubes (12 strips 8 tubes). Recovery Solution for Control samples (RSC) 1 vial 4 ml each; Weak Positive Control Sample (WPC HBV/HCV/HIV), freeze-dried 1 vial; VBD0196 Page 4 of 9

5 3. INTRODUCTION Assay kit RealLine HIV RT-PCR is intended for the detection of Human Immunodeficiency virus (HIV) RNA in patient 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. Assay kit RealLine HIV RT-PCR is intended for use in conjunction with clinical practice for diagnosis of AIDs C disease and highly sensitive screening of donor serum (plasma). Assay kit is adapted for real-time PCR detection systems like iq icycler, iq5 icycler, CFX96 (Bio- Rad, USA), DT-96 (DNA-technology, Russia) or their analogues. Assay 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. RealLine HIV RT-PCR is designed to detect HIV RNA isolated from serum (plasma) using RNA extraction kit: RealLine extraction 100 or RealLine extraction Specificity: The samples containing HIV RNA with concentration above the detection limit will be determined as positive. If specimen does not contain HIV RNA, analysis will give negative result (in 100% of cases). Sensitivity: Assay kit securely determines HIV RNA in concentration not less than 20 IU/ml for the RNA isolation from 1 ml of serum (plasma). VBD0196 Page 5 of 9

6 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 NA 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 NA 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. Do not use the kit after expiry. Note the date at the labels. VBD0196 Page 6 of 9

7 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED Real time PCR device iq/iq5 icycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-technology, Russia) or equivalent; RNA Extraction Kit: RealLine Extraction 100 or RealLine Extraction 1000 RealLine Internal Control (VBC8881) and negative Control, if Extraction Kit from other supplier is used Disposable gloves, powder-free; Pipettes (capacity μl) with filters (aerosol barriers); Disposable DNAse/RNase-free tips with filters); 0.2 ml microtube racks. 7. REAGENT AND SAMPLE PREPARATION 7.1. Sample preparation. Prepare specimens for the assay with Extraction kit RealLine Extraction 100 or RealLine Extraction 1000 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. We recommend to include the Internal Control IC, the Negative Control NC and Positive Control PC samples to the extraction procedure Reagent preparation Preparation of Control samples. Add 1 ml of Recovery Solution for Control samples (RSC) into a vial with Weak Positive Control sample (WPC), mix gently, keep for 15 minutes, 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 not used tubes for the test in the original bag. Try to squeeze excess of the air out of the bag before closing the clip. VBD0196 Page 7 of 9

8 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 patient specimen and control sample. Attention! 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 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 HIV cdna. VBD0196 Page 8 of 9

9 9. DATA ANALYSIS 9.1. In Positive Control sample and Weak Positive Control sample (also for CS1 and CS2) the program should detect: ROX fluorescent signal increase and Сt value (HIV 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 40. 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 differs from (IC Ct)m by more than 2, should be ignored. After screening, recalculate (IC Ct)m for remaining samples. The sample is considered 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 by more than 2, then result for this sample should be considered as equivocal. The test should be repeated from the sample RNA extraction stage The sample is considered positive 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. Note: Recommended setting for the Threshold is up to 20 % of the PC or the highest fluorescence signal, especially in not validated cyclers.. For further questions: VBD0196 Page 9 of 9

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