RealLine Borrelia burgdorferi s.l. Fla-Format
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1 Instructions for use AN ASSAY KIT FOR THE QUALITATIVE DETECTION OF DNA OF BORRELIA BURGDORFERI SENSU LATO COMPLEX USING REAL-TIME PCR METHOD In vitro diagnosticum () VBD Tests valid from May 2016 Rev _EN Page 1 of 15
2 Explanation of symbols used in labeling IVD LOT REF Σ For in vitro diagnostic use 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. VBD1499 Page 2 of 15
3 Table of content 1. INTRODUCTION 4 2. KIT CONTENTS 5 3. PRINCIPLE OF THE METHOD 5 4. SPECIFICATIONS 6 5. WARNING AND PRECAUTIONS 7 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED 8 7. PREPARATION OF THE ANALYSED SAMPLES AND REAGENTS 9 8. PROCEDURE DATA ANALYSIS AND INTERPRETATION STORAGE AND TRANSPORTATION 14 ANNEX I 15 VBD1499 Page 3 of 15
4 ASSAY KIT FOR THE QUALITATIVE DETECTION OF DNA OF BORRELIA BURGDORFERI SENSU LATO COMPLEX USING REAL-TIME PCR METHOD In vitro diagnosticum 1. INTRODUCTION Clinical Information: Lyme disease or borreliosis is considered to be one of the fastest spreading illnesses in the world. Gram negative spirochetal bacteria from the genus Borrelia, which are collectively known as Borrelia burgdorferi sensu lato group cause Lyme disease and are transmitted by infected ticks. Early symptoms may include fever, headache, fatigue, depression, and a characteristic circular skin rash called erythema migrans (EM). Left untreated, later symptoms may involve the joints, heart, and central nervous system. In most cases, the infection and its symptoms are eliminated by antibiotics, especially if the illness is treated early. Delayed or inadequate treatment can lead to the more serious symptoms, which can be disabling and difficult to treat. (Fla-format) assay kit is designed to detect DNA of pathogenic to human Borrelia species of Borrelia burgdorferi sensu lato complex (B. afzelii, B. garinii, B. burgdorferi sensu stricto), isolated from clinical specimens using extraction kits: RealLine DNA-Extraction 2 (REF VBC8897) RealLine DNA-Extraction 3 (REF VBC8889) RealLine Extraction 100 (REF VBC8896) RealLine Extraction 1000 (REF VBC8895) (Fla-format) kit is designed for the analysis of blood serum (plasma), cerebrospinal fluid and tick suspension samples The assay is based on the real-time polymerase chain reaction (PCR) method with fluorescent detection of the amplified product. The Fla-format Kit contains 5 vials with the lyophilized Mastermix, each vial with 10 reactions, for volume of 50 µl per reaction. The kit contains reagents required for 50 tests, including the positive control samples. The kit is designed for use with block cyclers iq icycler, iq5 icycler, CFX96 (Bio-Rad, USA), DT96 (DNA-Technology Research and Production Company ZAO, Russia); and rotor type cyclers Rotor-Gene 3000 and Rotor-Gene 6000 (Qiagen, Germany). For the Eco 48 Realtime PCR System (PCRmax, UK) das RealLine Fla-format kits can be recommended. The practice with this cycler to use 10 µl of the diluted Mastemix and 10 µl of extracted DNA, was validated. The protocol for using and cycling can be provided. VBD1499 Page 4 of 15
5 The use of:! Extraction Kits for nucleic acids from clinical specimen from other supplier! other real-time PCR devices! appropriate reaction volumes, other than 50 µl have to be validated in the lab by the user. The special notes regarding the internal control IC have to be strongly followed. 2. KIT CONTENTS Positive Control sample (PC) Master Mix (MM), lyophilized Recovery Solution (RS) 1 vial, 1 ml 5 tubes (10 tests each); 1vial, 2ml 3. PRINCIPLE OF THE METHOD The Real time PCR is based on the detection of the fluorescence, produced by a reporter molecule, which increases as the reaction proceeds. Reporter molecule is dual-labeled DNAprobe, which specifically binds to the target region of pathogen DNA. Fluorescent signal increases due to the fluorescent dye and quencher separating by Taq DNA-polymerase exonuclease activity during amplification. PCR process consists of repeated cycles: temperature denaturation of DNA, primer annealing and complementary chain synthesis. 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. Ct depends on initial quantity of pathogen DNA template. The use of Internal Control (IC) prevents generation of false negative results associated with possible loss of DNA template during specimen preparation. IC indicates if PCR inhibitors occur in the reaction mixture. IC template should be added in each single sample (including control samples) prior to DNA extraction procedure. The amplification and detection of IC does not influence the sensitivity or specificity of the target DNA PCR. Note: IC is a component of the NA extraction kits of RealLine series. Internal Control is added to the sample during NA isolation step and is used throughout the whole process of NA extraction, amplification, detection. VBD1499 Page 5 of 15
6 4. SPECIFICATIONS I. Sensitivity: Sensitivity control was performed on 5 samples containing 100 Borrelia burgdorferi s.l. DNA copies per sample, prepared from SRS (Standard Reference Sample containing Borrelia burgdorferi s.l. DNA), reg. No The sensitivity equals 100%. II. Specificity: Specificity of Borrelia burgdorferi s.l. DNA detection was determined using Standard Reference Panel of control sera samples not containing HBV DNA, Borrelia burgdorferi sensu lato DNA, Toxoplasma gondii DNA, Helicobacter pylori DNA, HCV RNA, HIV RNA, TBE RNA, Rubella RNA, Herpes virus infections DNA (reg. No ). Specificity of Borrelia burgdorferi s.l. DNA detection equals 100%. III. Diagnostic evaluation: Diagnostic evaluation was performed on 50 samples: 20 samples (No. 1-20), positive samples of nucleic material obtained from Borrelia burgdorferi s.l. infected ticks; 20 samples (No.21-40), negative samples; 10 samples (No ), nucleic material obtained from ticks infected with tick-borne encephalitis virus. Determination of specificity was performed on 20 negative samples and 10 samples obtained from ticks infected with tick-borne encephalitis virus, with a CE-marked reference kit. Studying samples from healthy ticks by (Fla-format), negative results were recorded for all 20 samples. Analysis of similar samples by the reference kit confirms the results obtained in all cases. Studying samples obtained from ticks infected with tick-borne encephalitis virus using RealLine Borrelia burgdorferi s.l. (Fla-format) negative results were recorded for all 10 samples. Analysis of similar samples by the CE-marked reference kit confirms the results obtained in all cases. Thus, (Fla-format) and the CE-marked reference kit show a 100% agreement in results. Specificity equals 100%. Determination of sensitivity was performed on 20 nucleic samples obtained from the ticks containing Borrelia burgdorferi s.l. DNA with the reference kit. (Fla-format) kit determined all 20 samples as positive. Analysis by the reference kit proved all 20 samples containing Borrelia burgdorferi s.l. DNA to be positive. Diagnostic sensitivity equals 100%. VBD1499 Page 6 of 15
7 5. WARNING AND PRECAUTIONS For in vitro use only. The kits must be used by skilled personnel only. When handling the kit, follow the national safety requirements for working with pathogens. To prevent contamination, the stages of DNA isolation and PCR test run must be spatially separated. Avoid microbial and nuclease contamination of reagents when removing aliquots from reagent vials. Wear protective disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents. Every workplace must be provided with its own set of variable-volume pipettes, necessary auxiliary materials and equipment. It is prohibited to relocate them to other workplaces. The use of sterile disposable pipette tips is recommended. Never use the same tips for different samples. 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. Do not use the kit after the expiration date. VBD1499 Page 7 of 15
8 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED real time PCR system, like described in paragraph 1 DNA-Extraction Kit, RealLine Extraction 100 or see p.1 Extractions Kits with Internal control reagent; Internal Control reagent (VBC8881), if the kit is used with the extraction kits of other supplier; Negative Control Sample or H 2O (molecular biology grade), if the kit is used with the extraction kits of other supplier; plates or tubes suitable for the used device with caps or a sealing foil for PCR laminar safety box; refrigerator; microcentrifuge for ml tubes; vortex mixer with adjustable rotation speed; half-automatic variable-volume single-channel pipettes with disposable tips; disposable medical non-sterile powder-free gloves; disposable pipette tips with filters; biohazard waste container. For preparation of tick suspension samples: Safety laminar cabinet Tube rack for 1.5 ml microtubes Iso Freeze-type refrigerator Refrigerant or reservoir with ice Metallic pestles for crushing ticks 96 % Ethanol 0.15 M NaCl VBD1499 Page 8 of 15
9 7. PREPARATION OF THE ANALYSED SAMPLES AND REAGENTS Each group of samples undergoing the procedure of DNA isolation must include a Positive Control sample (PC) and Negative Control sample (NC), that are components of the DNA extraction kit. We 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 Negative Control NC use 100 µl of the Negative Control Sample or H2O (molecular biology grade). For the PC use 70 μl of Negative Control Sample or H2O (molecular biology grade) and 30 μl of Positive Control to the tube marked PC Sample preparation Prepare the samples for the assay using RealLine DNA-Extraction 2, RealLine DNA- Extraction 3, RealLine Extraction 100 or RealLine Extraction 1000 extraction kits according their instruction manuals. If samples of isolated DNA were stored frozen prior the assay, thaw them and keep at least 30 minutes at a temperature of (18 25) C. The isolated DNA can be stored at (2 8) C for 2 days. After initial opening shelf life of Positive Control sample (CP) at (2 8) C is 1 month Preparation of tick suspension samples is described in Annex I Preparation of the reagents. Prior the test take the kit out of the refrigerator and keep the Master Mix (MM) closed in the package at (18 25) C for at least 30 minutes. Then open the package and take the necessary number of tubes with MM (including prepared samples and controls: 1 NC and 1 PC). Attention! 1 test tube with MM is intended for 10 assays. Put the remaining strips immediately back into the foil pouch, squeeze the air out and tightly close with the clip. Store the unused MM at (2 8) C for the entire shelf life of the kit. 7.3 Add 300 µl of Recovery Solution (RSC) to the vial with MM. Mix thoroughly and keep at room temperature for 15 min. Mix again. Store the recovered MM at (2 8) C for no longer than 7 days. After initial opening shelf life of Recovery Solution of Controls at (2 8) C is 3 months. VBD1499 Page 9 of 15
10 8. PROCEDURE 8.1. Prepare an appropriate number of 0.2 ml tubes, or a plae for PCR. Label each tube for each specimen and control. Attention! Labels should be placed on the caps of tubes for Rotor-Gene 3000/6000 devices. For iq icycler, iq 5 icycler, CFX 96, DT-96 PCR devices labels should be placed on the lateral side of the tubes Add 25 µl of recovered MM to all test tubes Add 25 µl of corresponding isolated DNA solution to each tube using a separate pipette tip with filter. Add 25 µl of NC and PC to the corresponding tubes. Tightly close the tubes with caps or the plate with PCR foil Place the tubes into the real-time PCR instrument Program real time PCR system as follows: For Rotor-Gene 3000 (6000): Press «New» in the main menu of the program. In the window that opens, select «Advanced» and check «Dual Labeled Probe». Click «New». Choose a rotor for 36 wells «36-Well Rotor» and confirm that tubes used are no domed. Click «Next». In the next window select the volume of the reaction mixture 50 µl. Click «Next». In the new window specify the temperature profile of the experiment. Press «Edit Profile» and set the following parameters: Stage 1: 50 C 2min Stage 2: 95 C 2min Stage 3: 94 C 10 sec C* 40 sec cycles * Measure the fluorescence at 60 C using the FAM and ROX channels (Green and orange) Once the temperature profile of the experiment is selected, click «OK». In the «New Run Wizard» window click «Calibrate» («Gain Optimizations»). In the «Channel Settings» of the new window select channels ROX (Orange) and FAM (Green). Set Tube Position 1, Min Reading 5, Max Reading 10. Click on the box against «Perform Calibration Before 1st Acquisition». Press «Close». Click «Next», run the amplification by pressing «Start run». VBD1499 Page 10 of 15
11 Save the file in a folder called Rotor-Gene /templates under the name RealLine with extension *.ret. During further work the RealLine program will be represented in the «New» list of the program s main menu. Recreation of temperature protocol or calibration protocol will not be required. The results of the run are saved as a file with extension *.rex after pressing the «Start run» button. For iq icycler, iq 5 icycler, CFX 96, DT-96: 8.6. Program real time PCR device according the instruction manual as follows: Stage 1: 50 С, 2 min; Stage 2: 95 С, 2 min; Stage 3: 94 C, 10 sec 60 C*, 20 sec 50 cycles * Measure the fluorescence at 60 C Select the amplification detection channels: 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 Borrelia burgdorferi s.l. DNA Program the positions of test tubes with Samples, positive and negative controls according to the instruction manual for the real time PCR system in use Run the program. VBD1499 Page 11 of 15
12 9. DATA ANALYSIS AND INTERPRETATION RealLine Pathogen Diagnostic Kits For Rotor-Gene 3000 (6000): 9.1 Analysis of the IC amplification results: Press the menu button «Analysis», select the «Quantitation» mode, choose «Cycling А. FAM» («Cycling А. Green»), click «Show» button. Cancel automatic selection Threshold by clicking «OK» in the popup window. Press the «Linear scale». In the main window («Quantitation analysis») two buttons «Dynamic tube» and «Slope Correct» must be pressed. In the menu «Quant. Settings» («More Settings») set the NTC threshold value as 5%. In the menu «CT Calculation» (in the right pane) set Threshold value as Ct values will appear in the output table («Quant. Results» window). 9.2 Analysis of the Borrelia burgdorferi DNA amplification results: Press the menu button «Analysis», select the «Quantitation» mode, choose «Cycling А. ROX» («Cycling А. Orange»), click «Show» button. Cancel automatic selection Threshold, by clicking «OK» in the popup window. Press the «Linear scale». In the main window («Quantitation analysis») two buttons «Dynamic tube» and «Slope Correct» must be pressed. In the menu «Quant. Settings» («More Settings») set the NTC threshold value as 15%. In the menu «CT Calculation» set Threshold value as Ct values will appear in the output table («Quant. Results» window). 9.3 For NC the program should detect the increase of the amplification signal of IC DNA in channel FAM (Green) and determine the Ct IC. The program must not detect the signal rise of the Borrelia burgdorferi s.l. DNA amplification via ROX (Orange) channel. 9.4 For PC the program should detect: increase of the amplification signal of IC DNA in channel FAM (Green) and determine the threshold cycle, Ct IC; increase of the signal of a specific amplification product of Borrelia burgdorferi s.l. DNA via channel ROX (Orange) and determine the threshold cycle, Ct PC. 9.5 For each sample the program should detect the increase of the amplification signal of IC DNA and determine Ct IC. 9.6 Calculate (Ct IC)av as an average Ct IC of all analyzed samples (including PC and NC). Ct IC values that differ by more than 2 from the (Ct IC)av should be ignored. Recalculate the (Ct IC)av for the remaining values after the screening. VBD1499 Page 12 of 15
13 9.7 The sample is considered negative (not containing Borrelia burgdorferi s.l. DNA), if Ct value via ROX (Orange) channel for this sample is above 40 or is not determined. When Ct IC value for such sample differs from the (Ct IC)av value by more than 2, the result is not regarded as negative. A repeated analysis of the sample, starting with the isolation step is necessary. 9.8 The sample is considered positive (containing Borrelia burgdorferi s.l. DNA) when Ct value via ROX (Orange) channel for this sample is less than or equal to When Ct NC value via the ROX (Orange) channel is less than or equal to 40, this indicates the presence of contamination. In this case all positive results of the individual PCR run are considered unreliable. Actions are required to identify and eliminate the source of contamination, and repeat the analysis of all samples of this run that were identified as positive. Samples that showed negative results in this run should be considered as negative. For iq icycler, iq 5 icycler, CFX 96, DT-96: 9.10 For PC the program should detect: increase of the IC DNA amplification signal (channel FAM) and determine the threshold cycle, IC Ct; increase of the Borrelia burgdorferi s.l. DNA amplification signal (channel ROX) and determine the Ct value; 9.11 For NC the program should detect the increase of the amplification signal of IC DNA (channel FAM) and determine the threshold cycle, IC Ct. No ROX fluorescent increase should appear (no Borrelia burgdorferi s.l. DNA amplification). When Ct value for NC through ROX channel is less than or equal to 40, this indicates the presence of contamination (see paragraph 9.16.) For each sample the program should detect the increase of the amplification signal of IC DNA (channel FAM) and determine IC Ct Calculate (IC Ct)av as an average IC Ct of all analyzed samples (including PC and NC). IC Ct values that differ by more than 2 from the (IC Ct)av should be ignored. Recalculate the (IC Ct)av for the remaining values after the screening The sample is considered negative (not containing Borrelia burgdorferi s.l. DNA), if Ct value via ROX channel for this sample is above 40 or is not determined. When IC Ct value for such sample differs from the (IC Ct)av value by more than 2, the result is regarded as equivocal. A repeated analysis of the sample, starting with the DNA isolation step is necessary. VBD1499 Page 13 of 15
14 9.15 The sample is considered positive (containing Borrelia burgdorferi s.l. DNA) when Ct value via ROX channel for this sample is less than or equals to In case of contamination all positive results of this individual PCR run are considered equivocal. Actions are required to identify and eliminate the source of contamination, and repeat the analysis of all samples of this run that were identified as positive. Samples that showed negative results in this run should be considered as negative. 10. STORAGE AND TRANSPORTATION Store the assay kit at (2-8) С in the manufacturer s packing. Transportation at 25 С for up to 10 days is allowed. Do not freeze the kit! Do not pool reagents from different lots or from different vials of the same lot. Strictly follow the Instruction manual for reliable results. Do not use kits with damaged inner packages and get in contact with BIORON Diagnostics GmbH. Storage and shelf life of solutions and components of the kit after initial opening: Positive Control sample: 1 month at (2 8) C. Ready Master Mix (MM): unused MM at (2 8) C for the entire shelf life of the kit. Diluted MM: at (2 8) C for 2 weeks. Recovery Solution: at (2 8) C for 3 months. Technical Support: techsupport@bioron.de VBD1499 Page 14 of 15
15 ANNEX I Preparation of tick suspension samples. Place ticks under study into the numbered 1.5 ml Eppendorf type tubes. To clean the ticks from contamination by substances used for the removal of stuck individuals, wash them before preparation of suspension (see p. a). In case of analysis of free individuals the suspension can be prepared immediately (see p. b). a) Preliminary washing of ticks: Add 300 µl (500 µl when analyzing pools) of 96% ethanol to each tube with tick, shake the tubes on the shaker, remove the alcohol after short centrifugation using a pipette or a vacuum pump without touching the tick, using separate tips for each sample. During the next step add 500 µl of 0.15 M sodium chloride solution to tubes, shake the tubes on the shaker and discard the drops from the walls by centrifuging 5 sec at 5000 rpm; remove fluid from the tubes using a pipette or a vacuum pump. b) Preparation of tick suspensions: Attention! To prevent degradation of nucleic acids (NA) isolated from the ticks, avoid heating of the sample sample to a temperature above 8 C at all stages of preparation of ticks suspension samples. When preparing the ticks for the analysis use 0.15 M NaCl pre-cooled to a temperature of (2-8) C. Method 1: Freeze the tubes with ticks in liquid nitrogen (at least 5 min) or freezing chamber (at ( ) C, 20 min). Take one frozen test tube and immediately carefully grind the tick pressing the material to the bottom of the tube with a separate sterile pestle. To prepare the suspension add 250 µl of 0.15 M NaCl to the powdered sample without removing the pestle: Carefully rinse the pestle in the contents of the tube, pull it out and put in a disinfectant solution. Mix the tube contents on a shaker (5-10 seconds). Grind the rest of the samples. Remove the contents from the walls to the bottom of the tube by short centrifugation. Without touching the sediment, take100 µl of suspension samples for isolation of nucleic acids and further assay. Method 2: Add 30 µl of 0.15 M NaCl to the tubes with ticks (in the case of analysis of full or large ticks add 50 µl of 0.15 M NaCl). Place the tubes into a chilled to minus (20-30) C stand-fridge and keep for at least 20 minutes in the freezer at minus (20-30) C. Next, place the rack with the analyzed samples into a container with ice or refrigerant. Take a tube with the tick frozen in the 0.15 M NACL, and as quickly as possible, without waiting for the thawing of the solution, thoroughly crush the tick with separate sterile pestle. Not removing the pestle, put a tube with crushed tick into the rack, placed in ice. Add 200 µl of chilled 0.15 M NaCl to the tube. Gently rinse the pestle in the tube, pull it out and put in a disinfectant solution. Stir the contents of the tube on a shaker (5-10 seconds). Perform the grinding procedure with other samples. Collect the contents from the walls of the tube with brief centrifugation and without touching the sediment carefully take 100 µl of the suspension for NA isolation and further analysis. Carry out NA isolation from ticks using RealLine Extraction 100 according to the instruction manual. Storage of ticks and tick suspension samples: up to 24 hours at (2 8) C; up to 2 weeks at minus (18-60) C; up to 1 year at temperatures below (-60) C. Transportation of ticks and tick suspension samples: in special thermo-containers with refrigerant, thermos with thermo-packages, ice VBD1499 Page 15 of 15
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