RealLine HBV qualitative Uni-Format

Transcription

1 Instructions for use EXTRACTION AND REAL TIME PCR KIT FOR THE QUALITATIVE DETECTION OF DNA OF THE HEPATITIS B VIRUS Research Use Only (RUO) (Uni-format) incl. Extraction VBD Tests valid from: January 2019 Rev05_0119_EN Page 1 of 12

2 Explanation of symbols used in labeling RUO LOT REF For Research Use Only Batch code Catalogue number Contains sufficient for <n> tests Use-by-date Temperature limit Consult instructions for use Keep away from 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_0119_EN Page 2 of 12

3 Table of content: 1. INTRODUCTION 4 2. KIT CONTENTS 5 3. STORAGE AND TRANSPORTATION 5 4. PRINCIPLES OF THE PROCEDURE 5 5. PRECAUTIONS 6 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED 6 7. SPECIMENS TRANSPORT AND STORAGE 7 8. REAGENT PREPARATION 7 9. PROCEDURE PROTOCOL DATA ANALYSIS 11 VBD0598 Page 3 of 12

4 EXTRACTION AND REAL TIME PCR KIT FOR THE QUALITATIVE DETECTION OF DNA OF THE HEPATITIS B VIRUS Research Use Only 1. INTRODUCTION Assay kit RealLine HBV PCR (qualitative analysis) is intended for detection of hepatitis B virus (HBV) DNA in plasma and serum. The method is based on the amplification of target DNA 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 iq icycler, iq5 icycler, CFX96 (Bio-Rad, USA), Rotor-Gene 3000, 6000 and Q (Qiagen, Germany), RealLine Cycler (BIORON Diagnostics GmbH), DT-96 (DNA-Technology,Russia) or their analogues. Assay 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. The assay kit can be used with either of two specimen preparation procedures: In the Standard specimen preparation procedure: HBV DNA is isolated from 100 μl of serum (plasma). In the UltraSensitive specimen preparation procedure, HBV viral particles in serum (plasma) are concentrated by Concentrating Solution of 1 ml of serum (plasma). Specificity: The samples containing HBV DNA with concentration above the detection limit will be determined as positive. If specimen does not contain HBV DNA, analysis will give negative result (in 100% of cases). Sensitivity: Assay kit securely determines HBV DNA in concentration not less than 5 IU/ml for UltraSensitive specimen preparation procedure (or not less than 50 IU/ml for the Standard procedure). Rev05_0119_EN Page 4 of 12

5 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: Recovery Solution for Control samples (RSC) Positive Control (PC) Negative Control (NC) (Negative serum, human) Internal control (IC), freeze-dried 4 vials 14 ml each 4 vials 4 ml each 4 vials 7 ml each 1 vial 1 ml each 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 2 vials 12 ml each 2 vials Amplification reagents Ready Master Mix for PCR, freeze-dried (RMM) 48 test tubes 3. STORAGE AND TRANSPORTATION Store assay kit at (2-8) С in the manufacturer s packing. Transportation at 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. 4. PRINCIPLES OF THE PROCEDURE Principle of analysis is based on PCR amplification of target DNA 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. VBD0598 Page 5 of 12

6 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 side label of the box. 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED Real time PCR device iq/iq5 icycler, CFX96 (Bio-Rad, USA), Rotor-Gene 3000/6000 (Corbet Research, Australia), RealLine Cycler (BIORON Diagnostics GmbH), DT-96 (DNA-Technology, Russia); or equivalent; DNA Extraction Kit: RealLine Extraction 100 or RealLine Extraction 1000 RealLine Internal Control (VBC8881) and negative Control, if Extraction Kit from other supplier is used Microcentrifuge (min RCF rpm) Eppendorf MiniSpin, or equivalent; Vortex mixer; Thermo Shaker Disposable gloves, powder-free; Pipettes (capacity μl, μl) with filters (aerosol barriers); Disposable DNAse/RNase-free tips with filters Displacement tips 2.0 ml polypropylene tubes, sterile, non-siliconised Vacuum aspirator; 2.0 ml and 0.2 ml microtube racks; Magnetic Rack for nucleic acids isolation (BIORON Diagnostics GmbH, BI87000) Rev05_0119_EN Page 6 of 12

7 7. SPECIMENS 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 may 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. 8. REAGENT PREPARATION 8.1. Prior to use, warm reagents at room temperature (18-25) С for 30 minutes Add 1 ml of 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 after preparation Add 1 ml of Solution for Restoration of 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 after 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. VBD0598 Page 7 of 12

8 9. PROCEDURE PROTOCOL UltraSensitive procedure. DNA isolation from 1 ml of serum (plasma). Determine the appropriate number of reaction tubes needed for specimen and control testing. It is recommended to use оnе Positive Control sample and one Negative Control sample in each test run. Label each 2.0 ml tube for each specimen and control sample. Add 30 μl of IC to each tube. 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 specimen to the appropriately labeled tube. For each specimen or control tube, add 1 ml of Concentrating Solution. 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 of 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 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 of 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 C and 1300 rpm. Then centrifuge for 1 minute at rpm. Samples are ready for PCR. Attention! Isolated DNA can be stored at (2-8) С for 24 hours. Rev05_0119_EN Page 8 of 12

9 Standard protocol. DNA isolation from 100 μl of serum (plasma). Determine the appropriate number of reaction tubes needed for sample 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 sample 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 specimen to the appropriately labeled tube. Add 500 μl of 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 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 300 μl of Wash Solution 2 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 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 C and 1300 rpm. Then centrifuge for 1 minute at rpm. Samples are ready for PCR. Attention! Isolated DNA can be stored at (2-8) С for 24 hours. Run of Real time 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 sample and control. Attention! For blockcyclers put marks on the lateral part of a reaction tube. For Rotor-Gene mark on the cap of 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 sorbent particles! Place reaction tubes into the thermal block of real time PCR device. VBD0598 Page 9 of 12

10 Program real time PCR device as follows: For iq/iq5 icycler, CFX96, DT-96 and RealLine Cyclers Step 1: 94 C 1 min Step 2: 94 C 10 sec 50 Cycles 60 C* 20 sec * measurement of fluorescent at 60 C in FAM and ROX For Rotor-Gene 3000/6000 Step 1: 94 C 1 min Step 2: 94 C 10 sec 50 Cycles 60 C* 40 sec * measurement of fluorescent at 60 C in FAM and ROX Collect real-time PCR data through the FAM (Green) channel for detection of amplification of IC DNA. Collect real-time PCR data through the ROX (Orange) channel for detection of amplification of HBV DNA. For Rotor-Gene 3000/6000 In the New Run Wizard window click Calibrate. The window Auto Gain Calibration Setup opens. In the line Channel Settings choose ROX (Orange) and FAM (Green) 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 PCR program. 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. Rev05_0119_EN Page 10 of 12

11 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 (Green), 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. 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 0,04. In the result table (Quant. Results window) Ct will be displayed. Results for HBV DNA 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. 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 0,04. In the result table (Quant. Results window) Ct will be displayed. For all cyclers: 10.1 In Positive Control sample the program should detect: ROX (Orange) fluorescent signal increase and Сt value (HBV DNA amplification); FAM (Green) fluorescent signal increase and Сt value (IC DNA amplification) In Negative Control sample the program should detect: FAM (Green) fluorescent signal increase and Ct value, and no significant ROX (Orange) fluorescent increase should appear. If Ct value for NC along ROX (Orange) channel is less than 40, this indicates the presence of contamination The program should detect amplification signal increase for IC DNA (channel FAM /Green) 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. Calculate (IC Ct) av as the average Ct value of IC for all samples (including PC and NC). Samples with Ct of IC, that differ from (IC Ct) av by more than 2, should be ignored These samples should be repeated from the sample DNA extraction stage. After screening, recalculate (IC Ct) av for remaining samples. VBD0598 Page 11 of 12

12 10.4 A sample is considered negative, if Ct value along the ROX (Orange) channel exceeds 40 or is not determined The sample is considered positive, if in the ROX (Orange) channel the Ct value is not higher than The test results are considered reliable only when Positive and Negative controls perform as expected In the case of contamination (when NC is determined as positive) all positive results in this test should be repeated from the DNA extraction stage. Negative samples of such test run are considered reliable. For further questions: techsupport@bioron.de Rev05_0119_EN Page 12 of 12