RealLine HBV quantitative - Uni-Format

Transcription

1 Instructions for Use - QUANTITATIVE ASSAY KIT FOR THE EXTRACTION AND REAL TIME PCR DETECTION OF THE HEPATITIS B VIRUS DNA Attention! Please read the information about quantification process carefully! Research Use Only (RUO) (Uni-format) incl. Extraction VBD Tests valid from: January 2019 Rev05_0119_EN Page 1 of 16

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 16

3 RealLine Pathogen Kits Table of content: 1. INTRODUCTION 4 2. KIT CONTENTS 5 3. STORAGE AND TRANSPORTATION 5 4. PRINCIPLES OF THE PROCEDURE 6 5. PRECAUTIONS 6 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED 7 7. SPECIMENS TRANSPORT AND STORAGE 7 8. REAGENT PREPARATION 8 9. PROCEDURE PROTOCOL DATA ANALYSIS 13 ATTACHMENT 1: CALCULATION OF VIRAL DNA CONCENTRATION 15 ATTACHMENT 2: ANALYTICAL SYSTEM VALIDATION. 15 VBD0599 Page 3 of 16

4 HEPATITIS B VIRUS DNA REAL TIME PCR DETECTION AND QUANTITATION KIT Research Use Only 1. INTRODUCTION The assay kit RealLine HBV PCR is intended for detection of hepatitis B virus (HBV) DNA in plasma and serum. The method is based on the amplification of viral 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), DT-96 (DNA-technology, Russia); Rotor-Gene 3000 and Rotor-Gene 6000 (Corbett Research, Australia), RealLine Cycler (BIORON Diagnostics GmbH) or their analogues. The assay kit contains reagents sufficient for 4 x 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 kit can be used with either of two specimen preparation procedures, the Standard procedure or the UltraSensitive procedure. 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: In samples containing HBV DNA above detection limit concentration of HBV DNA could be determined. If specimen does not contain HBV DNA, analysis must give negative result (in 100% of cases). Dynamic range of estimated concentration (linearity area): from 5 IU/ml to 10 8 IU/ml HBV DNA for the UltraSensitive procedure (or from 50 IU/ml to 10 8 IU/ml for the Standard procedure). 1 IU = 4,5 copies of HBV DNA (National Institute of Biological Standards and Control for WHO international standard for Hepatitis B virus NIBSC Code: 97/746). 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 16

5 RealLine Pathogen Kits 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 from human) Internal control (IC), freeze-dried CS1 and CS2: Samples for calibration, lyophilized - are used when the adequacy of analytical system has to be checked, see the Attachment 2 Passport with the specific amounts for the controls 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 2 vials 2 vials 12 ml each 2 vials 1 vial of each 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. Note the shelf life of the kit at the side label Do not pool reagents from different lots or from different vials of the same lot. Strictly follow the Instruction manual for reliable results. VBD0599 Page 5 of 16

6 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 with subsequent PCR amplification of target DNA by PCR with fluorescent detection of amplified DNA in the real-time mode. Quantification of Hepatitis B virus DNA is provided by application of the Positive Control (PC) in each test run. Positive Control is characterized in international units (IU/ml) by World Health Organization (WHO international standard for Hepatitis С virus NIBSC Code: 97/746), and serves as a Quantitation Standard for calculation of viral quantity. Threshold cycle value Ct is the cycle number at which fluorescence generated within a reaction crosses fluorescence threshold, a fluorescent signal significantly above the background fluorescence. Quantity of viral DNA in initial sample is calculated by comparison of the threshold cycle value of analyzed sample and Positive Control. Also efficiency of sample preparation should be considered. For convenience of the user it is recommended to use magnetic rack through workout. 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 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. Rev05_0119_EN Page 6 of 16

7 RealLine Pathogen Kits 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED Real time PCR device 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; 2.0 ml and 0.2 ml microtube racks; Magnetic Rack for nucleic acids isolation. 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. VBD0599 Page 7 of 16

8 8. REAGENT PREPARATION 8.1. Prior to use, bring up reagents to room temperature (18-25) С for 30 minutes Add 1 ml of Recovery Solution for Controls (RSC) into a vial with Internal Control (IC) sample, mix gently, keep for 15 minutes, and mix once again carefully. Attention! IC should be stored at (2-8) С and used within 1 month after preparation Add 1 ml of Recovery Solution for Controls (RSC) into a vial with Positive Control (PC) sample, mix gently, keep for 15 minutes, and mix once again carefully. Attention! PC should be stored at (2-8) С and used within 1 month of preparation Negative Control (NC) sample is ready to use. Attention! Once opened, NC should be stored at (2-8) С and used within 1 month of preparation 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. For the quantitative determination, the RNA extraction is conducted from 100 µl of serum (plasma) or whole blood using this kit. The Internal Control must be used with the Extraction, if another RNA Extraction kit I used, please add RealLine Internal Control (REF VBC8881) to the extraction procedure Each set of samples must contain 3 PC and 1 NC sample. The elution volume have to be 200 µl. Please use for the NC samples the Negative Control Sample provided with the kit and do not use water or buffer. Rev05_0119_EN Page 8 of 16

9 RealLine Pathogen Kits 9. PROCEDURE PROTOCOL UltraSensitive procedure for DNA isolation: from 1 ml of serum (plasma) 9.1. Determine the appropriate number of reaction tubes needed for specimen and control testing. It is recommended to use three replicas of Positive Control sample and one Negative Control sample in each test run and to proceed the controls to the extraction procedure 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 9.6. 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. VBD0599 Page 9 of 16

10 9.14. 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 10 of 16

11 RealLine Pathogen Kits Standard protocol for DNA isolation: from 100 μl of serum (plasma): Determine the appropriate number of reaction tubes needed for specimen and control testing It is recommended to use three replicas of 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, 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 500 μl of 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 of 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 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. VBD0599 Page 11 of 16

12 PCR Place the tubes with processed specimens and controls to the magnetic Rack Prepare an appropriate number of reaction tubes with Ready Master Mix (RMM). Label each reaction tube for each specimen and control. Attention! For blockcycler mark tubes on the lateral part, for Rotor-Gene mark on the cap 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 and program real time PCR device as follows: For iq/iq5 icycler, CFX96, DT-96, RealLine Cycler: 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 channel for detection of amplification of IC DNA Collect real-time PCR data through the ROX channel for detection of amplification of HBV DNA Settings: 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 and FAM channels. Make sure that of Tube position is 1. Tick off Perform Calibration before 1st Acquisition. Click Close button. 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 12 of 16

13 RealLine Pathogen Kits Record the positions of the controls and specimens According to the instruction to the used device Start the PCR program. 10. DATA ANALYSIS For Rotor-Gene 3000/ 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 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 In the result table (Quant. Results window) Ct will be displayed Results for HBV 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. 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 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 (HBV DNA amplification); FAM fluorescent signal increase and Сt value (IC DNA 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. VBD0599 Page 13 of 16

14 10.6. The program should detect amplification signal increase for IC DNA (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. 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. After screening, recalculate (IC Ct) av for remaining samples 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 The sample is considered negative if Ct value along the ROX channel exceeds 40 or is not determined The sample is considered positive if Ct value along the ROX channel does not exceed The test results are considered reliable only when Positive and Negative controls perform as expected. HBV quantitation analysis For quantitative analysis calculate the HBV DNA concentration in analyzed samples in accordance with Attachment 1. Attention! For DNA isolation from 100 μl result should be multiplied by If calculated HBV DNA concentration is in dynamic range from 100 IU/ml to 10 8 IU/ml result should be reported as positive with indication of calculated HBV DNA concentration in the sample (in IU/ml) Test results greater than 10 8 IU/ml are above the upper limit of quantitation of the Standard test and should be reported as Greater than 10 8 IU/ml Test results less than 100 IU/ml are below the lower limit of quantitation of the Standard test and should be reported as HBV DNA detected, less than 100 IU/ml Analyzed sample is considered as negative if obtains no Ct value along the ROX channel or Ct value exceed 40. Note: For precise calculation of viral NA concentration in analyzed samples, the validation of the analytical system should be done by comparison of Positive Control PC concentration, specified in the passport of the assay kit with PC concentration, calculated using calibration graph. For this purpose three independent Positive Control samples are the needed. For further information and help, ask us at techsupport@bioron.de. We can provide you a calculation sheet for an easy evaluation of your data. Rev05_0119_EN Page 14 of 16

15 ATTACHMENT 1: CALCULATION OF VIRAL DNA CONCENTRATION Calculate the viral DNA concentration using the following equation: RealLine Pathogen Kits С sampk = С PC 2 (Сt PC Сt SAMPk) (Сt ICk Сt ICPC) 2 Where: k sample number; С PC PC concentration, specified in the passport of the assay kit; Сt PC and Сt IC PC Сt value of the PC sample ROX and FAM channels, accordingly; Сt SAMPk and Сt ICk Сt value of the sample numbered k along ROX and FAM channels, accordingly, if amplification efficiency is (Еа) =100%. ATTACHMENT 2: ANALYTICAL SYSTEM VALIDATION. Calculation of viral DNA concentration for PC using calibration graph. For precise calculation of viral DNA concentration in analyzed samples, analytical system validation should be done by comparison of PC concentration, specified in the passport of the assay kit with PC concentration, calculated using calibration graph. Prior to use, warm reagents at room temperature (18-25) С for 30 minutes. Prepare NC, PC and IC as recommended in the instruction. Add 1 ml of Solution for Restoration of Control samples (SRC) into a vial with Calibration sample 1 (CS1) and Calibration sample 2 (CS2). Mix gently, keep for 15 minutes, then carefully mix once again. Procedure for the UltraSensitive procedure for DNA isolation: from 1 ml Prepare ml tubes one for NC, 9 for 3 repeats of PC, CS1 and CS2. Label the tubes. Add 30 μl of IC to each tube. For NC, add to the tube, marked NC, 1 ml of Negative Control. For PC, add to each of three tubes, marked PC, 970 μl of Negative Control, and 30 μl of Positive Control. For CS1 add to each of three tubes marked CS1 970 μl of Negative Control, and 30 μl of Calibration sample 1. For CS2 add to each of three tubes marked CS2 970 μl of Negative Control, and 30 μl of Calibration sample 2. For each specimen or control tube, add 1 ml of Concentrating Solution. Run the isolation of NA as recommended in the instruction. For Standard protocol for DNA isolation: from 100 μl: Prepare 10 tubes one for NC, 9 for 3 repeats of PC, CS1 and CS2. Label the tubes. Add 30 μl of IC to each tube. For NC, add to the tube, marked NC, 100µl of Negative Control. For PC, add to each of three tubes, marked PC, 70 μl of Negative Control, and 30 μl of Positive Control. VBD0599 Page 15 of 16

16 For CS1 add to each of three tubes marked CS1 70 μl of Negative Control, and 30 μl of Calibration sample 1. For CS2 add to each of three tubes marked CS2 70 μl of Negative Control, and 30 μl of Calibration sample 2. Run the isolation of NA as recommended in the instruction. Run the PCR (or reversed PCR). Insert concentration of CS1 and CS2, specified in the passport of Assay kit, as it recommended in the instruction of PCR instrument. For each sample calculate correct Ct value, using the following equation: Z = Сt SAMP (Сt IC m Сt IC). Calculate average Z value for PC, CS1 and CS2 (Z PCm, Z CS1m, Z CS2m). Samples with Z that differs from Z m more then 2, should be ignored. After screening, recalculate Z m for samples remained. Calculate B coefficient for calculating of specific viral DNA, using following equation: B = [Lg(C CS1) Lg(C CS2)] / (Z CS2m Z CS1m), Where: C CS1 and C CS2 specific viral DNA concentration in Calibration samples (specified in the passport of the assay kit); For amplification efficiency 100% B = Lg2 = 0,3. Using received results, calculate concentration of specific viral DNA for Positive Control sample, using the following analytical equation: С PC = 10 XPC (IU/ml)* Where: X PC = Lg (C CS1) + B (Z CS1m Z PCm); Z CS1m average Z value for Calibration Sample 1 repeats; Z PCm average Z value for Positive Control sample repeats; If the calculated value of specific virus DNA concentration in PC differs no more than in 2 times from the value specified in the passport of a set, PC can be used as the sample of comparison for the further calculations of specific virus DNA concentration in investigated samples. At performance of all specified conditions it is possible to spend calculation of concentration specific virus DNA (in IU/ml) in investigated samples under the formula (item *) using factor B value, calculated on calibration graph. Rev05_0119_EN Page 16 of 16