RealLine Extraction 100
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1 Instructions for use EXTRACTION OF RNA AND DNA FROM CLINICAL SPECIMENS In vitro diagnosticum VBC Tests valid from October 2016 Rev _EN Page 1 of 12
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 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. VBC8896 Page 2 of 12
3 Table of content: 1. INTRODUCTION 4 2. PRINCIPLE OF THE METHOD 4 3. KIT COMPONENTS 5 4. SPECIFICATIONS 5 5. WARNING AND PRECAUTIONS 6 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED 6 7. PREPARATION OF REAGENTS AND SPECIMENS 7 8. EXTRACTION PROTOCOL STORAGE AND TRANSPORTATION 12 VBC8896 Page 3 of 12
4 EXTRACTION KIT FOR DNA AND RNA FROM CLINICAL SPECIMENS In vitro diagnosticum RealLine Diagnostic Kits 1. INTRODUCTION The kit is designed for DNA and RNA extraction from clinical samples: whole blood/dried blood spots (DBS) serum (plasma) biopsy material liquor urine epithelial cells specimens stool (feces) tick suspensions The kit can be applied in clinical practice during diagnostics of different human infections and diseases by the polymerase chain reaction (PCR) method and by reverse transcription with subsequent polymerase chain reaction (RT-PCR). The kit is designed for nucleic acids isolation from 48 samples including controls. The kit is intended for use with PCR kits of RealLine series designed for the detection of nucleic acids of infectious agents. 2. PRINCIPLE OF THE METHOD Nucleic acids isolation principle of the kit is a temperature treating of samples by multicomponent lysis Reagent that destructs the nucleoprotein complex, followed by precipitation of nucleic acids onto magnetic particles with alcohol, wash procedure and subsequent elution. Then the sample is ready for PCR or RT-PCR test run. The principle of the assay is based on the extraction of nucleic acids from samples with preliminarily added internal control sample and running the reaction of reverse transcription and amplification of selected RNA fragments or amplification of a selected DNA fragment with detection of PCR products in Real Time. For the convenience of working with a kit a use of a magnetic stand is recommended. Nucleic acids can be extracted from 100 µl and 200 µl of the sample. The PCR kits of the RealLine Pathogen series include a Positive Control (PC) sample corresponding to a given PCR assay kit. PC must undergo the isolation procedure together with biological samples and Negative Control (NC) sample. VBC8896 Page 4 of 12
5 3. KIT COMPONENTS Specimen preparation reagents Lysis Reagent Sorbent (suspension of magnetic particles) Solution for DNA/RNA Precipitation Wash Solution No.1 Wash Solution No.2 Specimen Diluent Control samples: Recovery Solution for Controls (RSC) Negative Control sample (NC) Internal Control sample (IC), lyophilized 8 vials, 4 ml each 1 vial, 1 ml 4 vials, 12 ml each 4 vials, 8 ml each 4 vials, 5 ml each 12 vials, 3 ml each 2 vials, 4 ml each 2 vials, 2 ml each 2 vials 4. SPECIFICATIONS I. Test for the absence of inhibition and RNA isolation efficiency: RNA isolation efficiency was determined on 5 samples with concentration of HCV RNA of 150 IU/ml prepared from HCV RNA SRS (attested against WHO standard, NIBSC code 96/798), as percentage of samples determined by the RealLine HCV PCR assay as positive. Isolation efficacy equaled 100%. The kit conformed to required quality specifications on criteria of lack of inhibition. II. Performance evaluation: Performance evaluation was performed on 45 clinical samples that were used for extraction: 20 samples from healthy individuals; 25 sera samples from patients with diagnosed infection of Hepatitis C. The test was performed on clinical samples with a CE-marked reference kit. The following PCR detection of extracted material was performed using RealLine HCV manufactured by BIORON Diagnostics GmbH. The coefficient of correlation between results of the PCR assay performed on material obtained using and the CE-marked reference kit achieved VBC8896 Page 5 of 12
6 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 Wear disposable latex gloves when working with the kit, since all human biological material should be treated as potentially infectious. 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. Do not use the kit after the expiration date. Treat all disposable materials with disinfectant before utilization. 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED laminar safety box; refrigerator; Eppendorf-type microcentrifuge with a maximum rotation speed of at least 13,000 rpm; vortex; thermoshaker; half-automatic variable-volume single-channel pipettes; disposable medical non-sterile powder-free gloves; disposable pipette tips with aerosol barrier; magnetic tube holder; biohazard waste container Additional necessary for isolation of DNA and RNA from ticks: 96 % Ethanol Liqud nitrogen SSP-Solution from RealLine TBEV, RealLine Borrelia or RealLine Anaplasma / Ehrlichia Or respectively: 0.15 M NaCl solution, TE Buffer Additional necessary for isolation of DNA and RNA from stool: 0.9 % Sodium Chloride Solution VBC8896 Page 6 of 12
7 7. PREPARATION OF REAGENTS AND SPECIMENS 7.1. Preparation of whole blood samples. Attention! Samples of heparinized blood are not allowed for the assay. RealLine Diagnostic Kits Blood is taken in a vacuum tube with EDTA or sodium citrate. After blood sampling, gently mix it with anticoagulant present in a test tube. Transportation and storage of samples: 6 hours at (18 25) C; up to 24 hours at (2 8) C. Do not freeze samples! 7.2. Preparation from dried blood spots. Attention! Please do not use Lysis solution already prepared with sorbent. Cut out the dried blood spot sample with a hole puncher or a sterile scissor and incubate it in a separate tube with 600 µl Lysis Solution without sorbent and lyse for 20 min at 65 C. Centrifuge the dried blood sample at rpm at room temperature. Transfer the supernatant without touching or pipetting the pieces of paper to a new tube. Add the sorbent, mix thoroughly and incubate for 5 min at room temperature Preparation of serum (plasma) samples. Attention! Samples of heparinized blood are not allowed for the assay. Serum (plasma) must be put into a separate tube within 6 hours after the blood sampling. Transportation and storage of samples: up to 24 hours at (2 8) C; up to 1 month at (- 20) C. Repeated freezing and thawing is not allowed! Centrifuge serum or plasma samples at rpm at (18 25) C for 5 min before use Preparation of tissue and cerebrospinal fluid samples. Tissue samples and cerebrospinal fluid are ready for analysis. Transportation and storage of samples: up to 2 hours at (18 25) C; up to 24 hours at (2 8) C; up to 2 weeks at (- 20) C. Repeated freezing and thawing is not allowed! VBC8896 Page 7 of 12
8 7.5. Preparation of urine samples. Collect the first portion of morning urine in a clean collection cup with leakproof lid. Transfer ml of urine into a test tube, then centrifuge at 3000 rpm for 3 min. Remove the supernatant carefully without touching the sediment. Use the resulting cell pellet for isolation of nucleic acids. Transportation and storage of samples of urine cell pellet: up to 2 hours at (18 25) C; up to 24 hours at (2 8) C; up to 2 weeks at minus (18-60) C Preparation of samples of epithelial cells. Spin the tube with a specimen briefly to collect all Transportation Solution containing the test material (epithelial cells specimens from the mucosa of cervical canal, urethra, vagina, etc.) from the walls of the tubes. Resuspend cell pellet formed by centrifugation. When using the Transportation Solution produced by BIORON Diagnostics GmbH, transport and store samples: up to 2 days at (18 25) C; up to 2 weeks at (2 8) C; up to 2 months at ( 20) C; Repeated freezing and thawing of samples is not allowed! When using the transportation/conservation solutions from other manufacturers, store and transport the samples as specified in the instructions for their use Preparation of samples from stool (feces) Prepare a homogeneous suspension of feces using 0.9 % sodium chloride solution: To 0.1 g feces add 0.8 ml sodium chloride (0.9 %) and mix it. Prepare a clarified fecal extract by centrifugation of this homogeneous suspension for 5 min at g. Then mix 50 µl of the supernatant obtained with 50 µl of NC. Use 100 µl obtained for DNA isolation for the extraction with kit. Transportation and storage of samples: up to 2 hours at (18 25) C; up to 24 hours at (2 8) C; up to 2 weeks at (-18 60) C. VBC8896 Page 8 of 12
9 7.8. Preparation of tick suspensions. Place ticks under study, or pools of ticks (up to 10 hungry individuals; up to 3 full ticks) of the Ixodes genus into the numbered 1.5 ml Eppendorf type tubes. Study the ticks of the Dermacentor genus individually. To clean the ticks from contamination by substances used for the removal of stuck individuals, wash them before preparation of suspension (see paragraph a). In case of analysis of free individuals the suspension can be prepared immediately (see paragraph b). If the following detection is conducted with RealLine TBEV, RealLine Borrelia or RealLine Anaplasma / Ehrlichia, please use the method in the Annex of the correspondent manual. 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 the tubes, shake the tubes on the shaker and discard the drops from the walls by centrifuging 15 sec at 7000 rpm; remove fluid from the tubes using a pipette or a vacuum pump. b) Preparation of tick suspensions: Freeze the tubes with ticks in liquid nitrogen (at least 5 min) or a freezing chamber at ( ) C, for 20 min. Take one frozen test tube and immediately carefully grind the tick (pool of ticks) pressing the material to the bottom of the tube with a separate sterile pestle. To prepare the suspension add TE buffer solution (ph ) to the powdered sample without removing the pestle: 250 µl - in case of a single tick of Ixodes genus; 500 µl - in case of a single tick of Dermacentor genus; 1 ml - to homogenize the pool of ticks. 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. Centrifuge 15 sec at rpm to dump the contents from the walls to the bottom of the tube. Without touching the sediment, take samples for isolation of nucleic acids: 100 µl suspension of the Ixodes genus ticks, or 50 µl suspension of the Dermacentor genus ticks. Storage of tick suspension samples: up to 24 hours at (2 8) C; up to 2 weeks at ( ) C; up to 1 year at temperatures below (- 60) C. Transportation of tick suspension samples: in special thermo-containers with refrigerant, thermos with thermo-packages, ice; transport refrigerated (2-8 C) and frozen ( ) C) samples within 1 day. VBC8896 Page 9 of 12
10 7.9. Reagent preparation Prior use, bring up reagents to room temperature (18-25) С for 30 minutes. Prepare a vial with Positive Control (PC) sample (a RealLine PCR kit component) according to PCR kit Instruction manual. Add 1 ml of Recovery Solution for Controls (RSC) into a vial with Internal Control (IC) sample, mix gently, keep for 15 minutes and then carefully mix once again. Store diluted IC at (2-8) С within 1 month of preparation. Negative Control (NC) sample is ready to use. After initial opening store NC at (2-8) С up to 1 month. Prior use, bring up Lysis Reagent to (50-60) С and mix thoroughly to dissolve the precipitated material. Shake the vial with Sorbent on Vortex to a condition of homogeneous suspension. Add 80 μl of Sorbent suspension into a vial with Lysis Reagent. Mix carefully. Attention! Once opened, any unused portion of Lysis Reagent with addition of Sorbent should be discarded. VBC8896 Page 10 of 12
11 8. EXTRACTION PROTOCOL 8.1. Determine the appropriate number of reaction tubes needed for patient specimen and control testing. Label each 2.0 ml tube for each patient specimen and control sample Add 30 μl of IC to each tube. Add 100 μl of Negative Control to the tube marked NC. Add 70 μl of Negative Control and 30 μl of Positive Control to the tube marked PC (the PC is part of the RealLine Detection Kit, be aware about the kind of PC) 8.3. Add 100 μl of each patient specimen to the appropriately labelled tube. For higher sensitivity add 200 μl of specimens. When performing extraction from whole blood, the recommended sample volume is 50 μl Add 300 μl of Lysis Reagent with Sorbent to each tube. Vortex for seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 65 C and 1300 rpm. Spin shortly to collect the drops. In case the specimen is not lysed completely transfer the contents to the other tube careful not to touch the pellet Add 400 μl of Solution for DNA/RNA Precipitation in each tube. Vortex for seconds Centrifuge at rpm for 5 minutes at room temperature Trying not to shake up a pellet, place the tubes into Magnetic Stand. 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 2 minutes Trying not to shake up a pellet, place the tubes into Magnetic Stand. 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 2 minutes Trying not to shake up a pellet, place the tubes to Magnetic Stand. 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. VBC8896 Page 11 of 12
12 8.13. Add 200 μl (up to 600 μl, if necessary) of Specimen Diluent to each tube. Vortex vigorously for seconds. Place the tubes into Thermo Shaker, and incubate for 5 minutes at 65 C and 1300 rpm. Then centrifuge for 1 minute at rpm Samples are ready for PCR or reverse transcription and PCR. For storage please transfer supernatant to sterile tubes without pipetting magnetic beads. Attention! Isolated RNA cannot be stored. Prepare on the day of RT-PCR run. Isolated DNA can be stored at (2-8) С for 24 hours. 9. 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. Deliver unfrozen samples to the laboratory for testing in adapted container within 3 to 4 hours. Samples must be transported following local and national instructions for the transport of pathogen material. 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: Internal Control sample: 1 month at 2 to 8 C after dilution. Negative Control. After initial opening up to 1 month at 2 to 8 C Technical support: techsupport@bioron.de VBC8896 Page 12 of 12
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