RealLine HCV PCR Qualitative - Uni-Format

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1 Instructions for use PCR KIT FOR EXTRACTION OF RNA AND REAL TIME PCR DETECTION KIT FOR HEPATITIS C VIRUS RNA Research Use Only Qualitative Uni Format VBD tests valid from: December 2013 Rev Page 1 of 12

2 Explanation of symbols used in labeling RUO LOT REF For research use only Batch code Catalogue number Expiry Date Temperature limitation Consult instructions for use 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. Page 2 of 12

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 BUT NOT SUPPLIED 6 7. SPECIMEN TRANSPORT AND STORAGE 6 8. REAGENT PREPARATION 7 9. PROCEDURE PROTOCOL DATA ANALYSIS 11 Page 3 of 12

4 KIT FOR EXTRACTION OF RNA AND DETECTION OF HEPATITIS C 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 Specimen Preparation Reagents Concentrating Solution Lysis Reagent 1 Lysis Reagent 2 Sorbent (suspension of magnetic particles) Solution for DNA/RNA Precipitation Wash Solution 1 Wash Solution 2 Specimen Diluent Control Samples Restoration solution for Control samples (RSC) Positive Control (PC) sample Negative Control (NC) sample Internal control (IC) sample, lyophilized Amplification reagents Ready Master Mix for reverse transcription and PCR, freeze-dried (RMM) 4 vials 14 ml each; 4 vials 4 ml each; 4 vials 7 ml each; 1 vial 1ml; 4 vials 12 ml each; 4 vials 8 ml each; 4 vials 5 ml each; 4 vials 3 ml each. 2 vials 4 ml each; 1 vial; 1 vial 12 ml; 2 vials; 48 test tubes 3. INTRODUCTION Assay kit Qualitative is intended for detection of hepatitis C virus (HCV) 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 realtime mode. Assay kit Qualitative is intended for use in conjunction with clinical practice for diagnosis of hepatitis C disease and for highly sensitive screening of donor serum (plasma). Page 4 of 12

5 The assay kit is adapted for real-time PCR detection systems like iq icycler, iq5 icycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-technology, Russia); «Rotor-Gene 3000 и Rotor-Gene 6000 (Corbett Research, Australia) or their analogues. The kit contains reagents sufficient for 4 times 12 test runs, which may be performed separately or simultaneously. It is strongly recommended to use one Positive Control sample and one Negative Control sample in each test run. Assay kit can be used with either of two specimen preparation procedures, the Standard procedure or the UltraSensitive procedure. For the Standard specimen preparation procedure, HCV RNA is isolated from 100µl of serum (plasma). In the UltraSensitive specimen preparation procedure, HCV viral particles in serum (plasma) are concentrated by Concentrating Solution of 1 ml of serum (plasma). Specificity Assay kit is designed for in vitro determination of the HCV genotypes 1a, 1b, 2a, 2b, 2c, 2i, 3, 4, 5a, 6 regardless of subtype. The samples containing HIV RNA with concentration above the detection limit will be determined as positive. If the specimen does not contain HIV RNA, analysis will give negative result (in 100% of cases). Sensitivity The assay kit securely determines HCV RNA in concentration not less than 15 IU/ml for UltraSensitive specimen preparation procedure (or not less than 150 IU/ml for the Standard procedure). 4. PRINCIPLES OF THE PROCEDURE Principle of analysis is based on isolation of nucleic acids directly from serum (plasma) jointly with preliminarily added Internal Control, 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. For the convenience of the user it is recommended to use a magnetic rack through workout of the Extraction of RNA. Specimen preparation for 1ml or 100 µl of serum (plasma) is allowed 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. Page 5 of 12

6 Dispose unused reagents and waste in accordance with country, federal, state and local regulations. No warranty for using kit after the expiry date. 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED Real time PCR device iq/iq5 icycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-technology, Russia); Rotor-Gene 3000/6000 (Corbet Research, Australia) or equivalent; Microcentrifuge (min RCF rpm); Vortex mixer; Thermo Shaker Disposable gloves, powder-free; Pipettes (capacity µl, µl) with filters (aerosol barriers); Disposable DNAse/RNase-free tips with filters 2.0 ml polypropylene tubes, sterile, non-siliconised Vacuum aspirator as a recommendation; 2.0 ml and 0.2 ml microtube racks; Magnetic Rack for nucleic acids isolation; 7. SPECIMEN TRANSPORT AND STORAGE Attention! Specimens anticoagulated with heparin are unsuitable for this test. Blood should be collected in sterile tubes (or tubes, using EDTA or ACD as the anticoagulant). Separate serum (plasma) from whole blood within 6 hours of collection. Transfer serum (plasma) to a sterile polypropylene tube. Specimens can be transported and stored: At room temperature up to 2 hours; At (2-8) С up to 24 hours; Frozen at 20 С up to 2 weeks. Do not freeze - thaw samples repeatedly! Before use centrifuge specimens of serum (plasma) at rpm for 5 minutes at room temperature. Page 6 of 12

7 8. REAGENT PREPARATION 8.1. Prior to use, warm reagents at room temperature (18-25) С for 30 minutes. Add 1 ml of Solution for Recovery Solution for Control samples (RSC) into a vial with Internal Control (IC) sample, mix gently, keep for 15 minutes, then carefully mix once again. IC should be stored at (2-8) С and used within 1 month of preparation Add 1 ml of Solution for Recovery Solution for Control samples (RSC) into a vial with Positive Control (PC) sample, mix gently, keep for 15 minutes, then carefully mix once again. PC should be stored at (2-8) С and used within 1 month of preparation Negative Control (NC) sample is ready to use. Once opened, NC should be stored at (2-8) С and used within 1 month Prior to use, warm Lysis Reagents 1 and 2 at (50-60) С and mix thoroughly to dissolve the precipitated material. Vortex Sorbent to a condition of homogeneous suspension. Add 80 µl of Sorbent suspension into a vial with Lysis Reagent 2. Mix carefully. Attention! Once opened, any unused portion of Lysis Reagent 2 should be discarded. 9. PROCEDURE PROTOCOL UltraSensitive procedure. RNA isolation from 1 ml of serum (plasma) 9.1. Determine the appropriate number of reaction tubes needed for patient specimen and control testing. It is recommended to use one Positive Control sample and one Negative Control sample in each test run Label each 2.0 ml tube for each patient specimen and control sample Add 30 µl of IC to each tube 9.4. For NC, add to the tube, marked NC, 1000 µl of Negative Control sample For PC, add to the tube, marked PC, 970 µl of Negative Control sample, and 30 µl of Positive Control sample Add 1000 µl of each patient specimen to the appropriately labeled tube For each specimen or control tube, add 1 ml of Concentrating Solution. Page 7 of 12

8 9.8. Cap the tubes and vortex for 3-5 seconds. Leave the tubes for 10 minutes at room temperature, then centrifuge for 5 minutes, 3000 rpm Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. The pellet should be clearly visible at this step. Add 200 µl of Lysis Reagent 1 to each tube. Vortex vigorously for seconds. Some insoluble material may remain. Leave tubes for 5 minutes at room temperature Add 500 µl or Lysis Reagent 2 with Sorbent to each tube. Vortex for 5-10 seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 o C and 1300 rpm Add 750 µl of Solution for DNA/RNA Precipitation in each tube. Vortex for seconds. Incubate 5 minutes at room temperature Centrifuge at rpm for 5 minutes at room temperature Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet Add 500 µl of Wash Solution 1 in each tube. Vortex vigorously for seconds. Centrifuge at rpm for 5 minutes Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet Add 300 µl or Wash Solution 2 to each tube. Vortex vigorously for seconds. Centrifuge at rpm for 5 minutes Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet Dry the pellet with open caps for 2-3 minutes at room temperature (18-25) С Add 200 µl of Specimen Diluent to each tube. Vortex vigorously for seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 o C and 1300 rpm. Then centrifuge for 1 minute at rpm Samples are ready for reverse transcription and PCR. Attention! In the case of RNA isolation start reverse transcription and PCR within 2 hours of specimen and control preparation. Isolated DNA can be stored at (2-8) С for 24 hours. Page 8 of 12

9 Standard protocol. RNA isolation from 100 µl of serum (plasma) Determine the appropriate number of reaction tubes needed for patient specimen and control testing It is recommended to use one Positive Control sample and one Negative Control sample in each test run Label each 2.0 ml tube for each patient specimen and control sample Add 30 µl of IC to each tube For NC, add to the tube, marked NC, 100 µl of Negative Control sample For PC, add to the tube, marked PC, 70 µl of Negative Control sample, and 30 µl of Positive Control sample Add 100 µl of each patient specimen to the appropriately labeled tube Add 500 µl or Lysis Reagent 2 with Sorbent to each tube. Vortex for 5-10 seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 o C and 1300 rpm Add 600 µl of Solution for DNA/RNA Precipitation in each tube. Vortex for seconds. Incubate 5 minutes at room temperature Centrifuge at rpm for 5 minutes at room temperature Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet Add 500 µl or Wash Solution 1 in each tube. Vortex vigorously for seconds. Centrifuge at rpm for 3 minutes Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet Add 300 µl or Wash Solution 2 to each tube. Vortex vigorously for seconds. Centrifuge at rpm for 3 minutes Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet Dry the pellet with open caps for 2-3 minutes at room temperature (18-25) С. Page 9 of 12

10 9.36. Add 200 µl of Specimen Diluent to each tube. Vortex vigorously for seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 o C and 1300 rpm. Then centrifuge for 1 minute at rpm Samples are ready for reverse transcription and PCR. Attention! In the case of RNA isolation start reverse transcription and PCR within 2 hours of specimen and control preparation. Isolated DNA can be stored at (2-8) С for 24 hours. Reverse transcription and PCR 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. Attention! For iq/iq5 icycler and CFX96 put marks on the lateral part of a reaction tube. For Rotor-Gene 3000/6000 put marks on the cap on the 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! Place reaction tubes into the thermal block of real time PCR device Program real time PCR device as follows: For iq/iq5 icycler, CFX96, DT-96 Stage 1: 45 С, 30 min; Stage 2: 94 С, 1 min; Stage 3: 94 C, 10 sec 50 cycles 60 C, 20 sec Stage 4: 10 С, 10 min; Fluorescence measurements should be done at 60 С. For Rotor-Gene 3000/6000 Stage 1: 45 С, 30 min; Stage 2: 94 С, 1 min; Stage 3: 94 C, 10 sec 50 cycles 60 C, 40 sec 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. Page 10 of 12

11 For Rotor-Gene 3000/ In the «New Run Wizard» window click «Calibrate». The window Auto Gain Calibration Setup opens. In the line «Channel Settings» choose «ROX» and «FAM» channels. Make sure that of Tube position is 1. Tick off «Perform Calibration Before 1st Acquisition». Click «Close» button Record the positions of the controls and specimens According to the instruction to the used device Start the reverse transcription and PCR program. 10. DATA ANALYSIS For Rotor-Gene 3000 (6000) Results for Internal Control DNA amplification Click «Analysis» button, choose «Quantitation» from the list, choose «Cycling А. FAM», click «Show» button Click «ОK» button, and cancel automatic Threshold determination Click «Linear scale» button In the Quantitation menu buttons «Dynamic tube» and «Slope Correct» should be pressed Click «More Settings» (Outlier removal) button, determine NTC threshold value as 5% In the column «CT Calculation» (right part of the window) determine «Threshold» value as 0, In the result table («Quant. Results» window) Ct will be displayed. Results for HCV cdna amplification Click «Analysis» button, choose «Quantitation» from the list, choose «Cycling А. ROX» click, «Show» button Click «ОK» button, and cancel automatic Threshold determination Click «Linear scale» button In the Quantitation analysis menu buttons «Dynamic tube» and «Slope Correct» should be pressed. Page 11 of 12

12 Click «More Settings» (Outlier removal) button, determine NTC threshold value as 5% In the column «CT Calculation» (right part of the window) determine «Threshold» value as 0, In the result table («Quant. Results» window) Ct will be displayed In 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 PC and NC). Samples with Ct of IC, that differ from (IC Ct) m by more than 2 cycles, 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 cycles, then the result for this sample should be considered as equivocal. The test should be repeated from the RNA extraction stage. The sample is considered positive if Ct value along the ROX channel does not exceed 40. If Ct of IC for this sample differs from (IC Ct) m more then 2 cycles, the sample is considered as borderline and have to be checked properly The test results are considered reliable only when Positive and Negative controls perform as expected. Page 12 of 12

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