Viral DNA/RNA Purification Kit
Contents
1. Viral DNA RNA Purification Kit Y 50 DK0011 For isolation and purification of viral nucleic acids from human plasma or serum samples for in vitro diagnostic purposes For in vitro diagnostic use wal CE Fermentas UAB A wholly owned subsidiary of Thermo Fisher Scientific Inc V A Grai i no 8 LT 02241 Vilnius Lithuania 370 700 55131 PA DK001_EN Rev 05 01 2013 CONTENTS page Components of TG WAU ssavescccsve stent centeys crate veciaivecewide dent a duidenlenistdeneaeauvels 3 VETO e E neem saceece tate E 3 Storage and SADM R AAAA S 4 GUAN COMMON aean a E EAE 4 Naaa RIS S E A E AE ees E E N E E E EA AE 4 Product USC MIANO sieis ua tals cet un teat td tatriedectar duct nahentaitatnalena tata ataltnat male 4 DESCNDUON wistavenlavutatinlatielainlanialatalainlavalatiulanlanleinl aateneteauenesien 5 FL sse 5 HMMS NOO S earnan tak tee ies eee iattdawinttece ta teaw inten tabteevie Hen eateiaaeees 6 SAIC NINOI eioi ads 6 Preparing reagents and DUuffers c ccecsssssescsscssscsssssssesssesseecseesscessessceesasaseesacsesavsseesaserses 7 Carner RNAi desea cheat stadt aaa a T Preparation Of Gamer RNA scena A E E T Calculating the required quantity of carrier RNA c ccecsssecssscessssessesssessssecesseeesssesesaterses 8 HATER AVC OUR ses east She or SA a Spd Seat Syd A ae Os oa el es 8 Additional materials and equipment required cccecccccscscssssssssssessssessecsecsseessessesseseteeseess 82. FVOIOCOlOVEIVIEW Auton tcanluthad alia de tet htt E A E leita d lan tamale it ale 9 Viral DNA RNA isolation and purification protocol c cccccesecscesscsssecsecsssessesecsesessesesseees 10 gale Ly MONI MOISES encie d atcst ance ctacet th encuo l aa 13 RECE AE E o ENEE E EAE A tect acuta cata uate EETA 14 COMPONENTS OF THE KIT DK0011 Viral DNA RNA Purification Kit W 50 CPL Column Preparation Liquid red cap COL PREP LIQ 2x1 4mL LS Lysis Solution 12 mL WB1 Wash Buffer 1 concentrated 25 mL WB2 Wash Buffer 2 concentrated WASH BUF 2 CONC 11 mL EL Eluent white cap 3x 1 25 mL PK Proteinase K green cap PROTK 2x1 3mL CR Carrier RNA blue cap 1 vial LT Lysis Tubes 1 5 mL 50 Spin Columns preassembled Be with Wash Tubes oo WT Wash Tubes 2 mL 4x 50 ET Elution Tubes 1 5 mL 50 T m a E ollz mils m HB Handbook contains guanidine hydrochloride guanidinium chloride 1 x SYMBOLS A Attention see instructions for Ada indicated eormoncnt use P Batch code COMP Components gt lt Use by CONT Contains In vitro diagnostic medical device Ethanol eal Manufacturer Material identification number Consult instructions for use VOL Volume or quantity Catalogue number gt Leads to y Temperature limitation After addition of ethanol mark the EO completed step Kit contains reagents SARJANA Remove Carrier RNA N3 50 sufficient for 50 sample y immediate
3. acids are ready for subsequent use in downstream NAT applications IMPORTANT NOTES e Ensure the integrity of the kit components upon the delivery Contact technical service or your local distributor in case of damage Do not use damaged kit components e The Lysis Solution LS and Wash Buffer 1 WB1 contain irritants Always wear gloves and follow standard safety precautions when handling these reagents For more information refer to SAFETY INFORMATION page 13 and Material Safety Data Sheets available by request e All sample material and waste should be regarded as potentially infectious Wear the proper protection when handling samples and waste Avoid any skin or eye contact Work under laminar air flow conditions if possible until samples are lysed Disinfect all work surfaces thoroughly after the procedure Follow proper waste disposal guidelines as recommended by local authorities e The following steps should be taken in order to avoid cross contamination always change pipette tips between liquid transfers aerosol barrier pipette tips recommended open only one tube at a time use disposable gloves and discard if contaminated e Always use RNase free equipment e Use only a freshly prepared mixture of Carrier RNA CR and Lysis Solution LS when beginning a new extraction procedure e Before beginning the procedure a new Spin Column SC must be prepared by adding 50 uL of Column Preparation Liquid CPL into the cent
4. purified nucleic acids immediately or store at 20 C VIRAL DNA RNA ISOLATION AND PURIFICATION PROTOCOL For isolation and purification of viral DNA RNA from 200 uL of EDTA or citrate treated human plasma or serum Do not use heparin treated samples The following procedure provides instruction for processing one sample Before starting Read the user manual make sure all the directions are followed as indicated e Make sure all working solutions and samples have been prepared according to recommendations page 5 7 e Ensure all necessary equipment and additional materials are available before beginning the procedure page 7 Organize proper handling of potentially infectious waste before beginning the procedure Follow recommended safety instructions as you will be working with potentially infectious material Attention and care must be taken during the entire process All centrifugation steps must be performed at room temperature Procedure 1 Spin Column preparation Before starting the procedure each new Spin Column SC must be prepared by treating it with Column Preparation Liquid CPL Column treatment maximizes binding of the nucleic acids to the membrane resulting in more consistent yields a Add 50 uL of Column Preparation Liquid CPL to the center of Spin Column SC membrane so that the membrane is entirely moistened Do not centrifuge the prepared column The prepared column should be store
5. alimans C L Jansen P M E W Dillen and J van der Noordaa 1990 Rapid and simple method for purification of nucleic acids J Clin Microbiol 28 495 503
6. ane while impurities are effectively removed during subsequent washing and centrifugation steps Finally ready to use nucleic acids are eluted from the column The purified viral nucleic acids are free of proteins nucleases and other contaminants or inhibitors of downstream applications Isolated DNA RNA can be directly used in PCR qPCR or other nucleic acid based assays PRINCIPLE The Viral DNA RNA Purification Kit uses a well established nucleic acid isolation and purification technique comprised of following steps 1 Sample lysis The sample is lysed by incubation with Lysis Solution LS and Proteinase K PK under denaturating conditions at elevated temperatures 56 C The Lysis Solution and Proteinase K inactivate both RNases and DNases ensuring protection of viral nucleic acids against degradation Binding viral nucleic acids to the spin column membrane The lysed sample is transferred to a spin column SC where released viral nucleic acids immediately bind to the silica based filter in the presence of chaotropic salts The remaining lysate is removed by centrifugation Removing remaining contaminants The remaining contaminants are removed during three wash steps using Wash Buffer 1 WB1 and Wash Buffer 2 WB2 whereas pure nucleic acids remain bound to the membrane Elution of pure viral nucleic acids Pure viral nucleic acids are released from the spin column filter using Eluent EL The resulting purified nucleic
7. assembled within the wash tube b Centrifuge the column for 1 min at 6000 x g c Discard the Wash Tube containing flow through d Place the Spin Column SC into a new 2 mL Wash Tube WT 5 Washing with Wash Buffer 1 WB1 Supplement concentrated Wash Buffer 1 WB1 with ethanol prior to the first use Page 7 a Add 700 uL of Wash Buffer 1 WB1 supplemented with ethanol to the Spin Column SC b Centrifuge the column for 1 min at 6000 x g c Discard the Wash Tube containing flow through d Place the Spin Column SC into a new 2 mL Wash Tube WT 6 lt Washing with Wash Buffer 2 WB2 Supplement concentrated Wash Buffer 2 WB2 with ethanol prior to the first use Page 7 a Add 500 uL of Wash Buffer 2 WB2 supplemented with ethanol to the Spin Column SC b Centrifuge the column for 1 min at 6000 x g c Discard the Wash Tube containing flow through d Place the Spin Column SC into a new 2 mL Wash Tube WT 11 User supplied Ethanol 96 100 Kit components used sc cor WT WASH TUBE Kit components used WB WASH BUFF 1 CONC WT wash TUB Kit components used WB2 WASH BUFF 2 CONC WT WASH TUBE 7 Repeated washing with Wash Buffer 2 WB2 a Add 500 uL of Wash Buffer 2 WB2 supplemented with ethanol to the Spin Column SC a b Centrifuge the column for 1 min at 6000 x g cP c Discard the Wash Tube containing flow through d Pla
8. ce the Spin Column SC into a new 2 mL Wash Tube WT 8 Spin dry te Centrifuge the column for 3 min at 16000 x g b Discard the Wash Tube WT containing remaining flow through 9 Elution of pure nucleic acids a Place the Spin Column SC into a new 1 5 lt gt d Centrifuge the column for 1 min at 13000 x g e Discard the Spin Column SC s mL Elution Tube ET b Add 50 uL of Eluent EL preheated to 56 C to the center of Spin Column SC membrane c Incubate for 2 min at room temperature Kit components used WB2 WASH BUFF 2ICONC WT WASH TUBE Kit components used ET eL E Storage and use in downstream applications a Keep the Elution Tube ET containing pure UJ viral nucleic acids b Use the purified nucleic acids immediately or store at 20 C For further use in downstream qPCR applications use 10 uL of viral DNA per 25 uL reaction volume For reverse transcription RT use up to 10 uL of viral RNA per 20 uL cDNA synthesis reaction volume SAFETY INFORMATION x Lysis Solution LS Xn Harmful Hazard determining component of labeling Guanidinium chloride Risk phrases R22 Harmful if swallowed R38 Irritating to skin R41 Risk of serious damage to eyes R52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Safety phrases S23 Do not breathe gas fumes vapour spray S26 In case of contact w
9. d at room temperature until it is used for sample processing Kit components used CPL COL PREP LIQ SC COL 2 Sample lysis Supplement Lysis Solution LS with Carrier RNA CR prior to use page 8 Use the appropriate internal control as required in a downstream assay user s manual Do not add the internal control directly to plasma samples Do not add Proteinase K PK directly to Lysis Solution LS a Load 200 uL of serum or plasma sample to an empty 1 5 mL Lysis Tube LT b Add 200 uL of Lysis Solution LS supplemented with Carrier RNA and 50 uL of Proteinase K PK mix thoroughly by vortexing or pipetting c Incubate the sample for 15 min at 56 C ina thermomixer Leave thermomixer turned on for Eluent EL preheating later during procedure d Centrifuge for 3 5 s at full speed to collect any sample solution from the inside of the lid 10 Kit components used CR CAR RNA LS LT PK User supplied Internal controls for a downstream application 3 Adjusting binding conditions a Add 300 uL of ethanol 96 100 and mix by pipetting or vortexing b Incubate the sample at room temperature for 3 min c Centrifuge for 3 5 s at full speed to collect drops from the inside of the lid 4 Binding nucleic acids to the spin column Ensure that a new Spin Column SC has been prepared as described in step 1 a Transfer the lysate to the prepared Spin Column SC pre
10. e following table Supplement Lysis Solution with the required quantity of Carrier RNA CR and mix by pulse vortexing or pipetting No Vol Lysis Vol Carrier No Vol Lysis Vol Carrier samples Solution LS mL RNA CR uL samples Solution LS mL RNA CR uL 1 0 22 5 9 13 2 86 71 5 2 0 44 11 0 14 3 08 77 0 3 0 66 16 5 15 3 30 82 5 4 0 88 22 0 16 3 02 88 0 5 1 10 27 9 17 3 74 93 5 6 1 32 33 0 18 3 96 99 0 7 1 54 38 5 19 4 18 104 5 8 1 76 44 0 20 4 40 110 0 9 1 98 49 5 21 4 62 159 10 2 20 55 0 22 4 84 121 0 11 2 42 60 5 23 5 06 126 5 12 2 64 66 0 24 5 28 132 0 Note Required volumes uL of Carrier RNA CR are calculated using following formula N x 0 22 mL Y mL Y mL x 25 0 uL mL Z uL Where N number of samples to be processed Y calculated volume mL of Lysis Solution LS Z volume uL of Carrier RNA CR to add to Y mL of Lysis Solution LS INTERNAL CONTROL The presence of an internal control throughout the extraction and purification procedure is necessary when using the kit in combination with diagnostic amplification systems Please refer to the user manual provided with the downstream diagnostic assay for further directions on how to use an internal control ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED Pipettes and sterile nuclease free pipette tips with aerosol barrier Vortex Ethanol 96 100 Microcentrifuge Thermomixer Disposable gloves Measuring cylinder Nuclease free m
11. er RNA CR is important for efficient recovery of viral nucleic acids for two reasons First CR facilitates binding of viral nucleic acids to the silica membrane especially when there are only a small number of viral nucleic acid molecules in the sample Additionally in the rare event when there are a small number of active RNase molecules large amounts of Carrier RNA reduce the probability of viral RNA being degraded under chaotropic conditions If Carrier RNA is not added to the Lysis Solution LS reduced viral nucleic acid yields may result PREPARATION OF CARRIER RNA Carrier RNA CR is provided in a dried state packed in a moisture impermeable aluminum bag Prior to the first use reconstitute the dried Carrier RNA CR by adding 300 uL of Eluent EL Allow the freshly reconstituted Carrier RNA CR to incubate for 5 min at room temperature then mix thoroughly and briefly centrifuge the vial Use immediately or store at 20 C Do not freeze thaw the reconstituted Carrier RNA CR more than 10 times If only few samples will be processed at a time divide the Carrier RNA CR solution into 50 uL aliquots using nuclease free tubes and store at 20 C CALCULATING THE REQUIRED QUANTITY OF CARRIER RNA When starting a new procedure always use a freshly prepared mixture of Carrier RNA CR and Lysis Solution LS To calculate the correct quantity of Carrier RNA CR and Lysis Solution LS required to process multiple samples use th
12. er of the column membrane Do not centrifuge the column after addition of CPL e The kit should be only used by professional personnel trained in IVD practices SAMPLE HANDLING e After collection and centrifugation plasma serum samples can be stored at 2 8 C for up to 6 hours or frozen at 20 C or 70 C for long term storage e Do not freeze thaw samples more than once e Equilibrate samples to room temperature 20 5 C before use Remove precipitates if any by centrifugation for 5 min at 3000 x g e Use EDTA or citrate treated samples Heparin treated samples are not suitable for use with the kit PREPARING REAGENTS AND BUFFERS Add the indicated volume of ethanol 96 100 to the concentrated Wash Buffer 1 WB1 and concentrated Wash Buffer 2 WB2 prior to first use DK0011 Y 50 Wash Buffer 1 WB1 Wash Buffer 2 WB2 Concentrated wash buffer 25 mL 11 mL Ethanol 96 100 15 mL 44 mL Total volume 40 mL 55 ML e Ensure all working solutions are prepared according to the recommendations in the protocol e After preparing each solution mark the bottle to indicate that this step has been completed e Check all solutions in the kit for any salt precipitation before each use Re dissolve any precipitate by warming the solution to 37 C and then equilibrate to room temperature 205 C e tis the user s responsibility to use appropriate controls during the procedure CARRIER RNA Usage of Carri
13. icrocentrifuge tubes of an appropriate size for preparing mixtures of Carrier RNA CR and Lysis Solution LS PROTOCOL OVERVIEW Read the protocol page 10 carefully before beginning Ensure that you are familiar with the symbols and abbreviations of each component of the kit page 3 Procedure timing for processing one sample 30 min an a lt Fi a a y a 4 E E Timing Column Add 50 uL CPL on to SC membrane P preparation Do not centrifuge Lyse Load 200 uL of a sample into LT 15 min Add 200 uL LS supplemented with CR Add 50 uL PK Mix Incubate 15 min at 56 C in a thermomixer Adjust Add 300 uL of ethanol 96 100 3 min binding Mix conditions Incubate 3 min at room temperature Bind Transfer sample into SC 1 min Centrifuge 1 min 6000 x g Discard wash tube Place SC into anew WT Wash Add 700 uL WB1 into SC 1 min WB1 Centrifuge 1 min 6000 x g Discard wash tube Place SC into anew WT Wash Add 500 uL WB2 into SC 1 min WB2 Centrifuge 1 min 6000 x g Discard WT Place SC into anew WT Wash Add 500 uL WB2 into SC 1 min WB2 Centrifuge 1 min 6000 x g Discard WT Place SC into anew WT Spin dry Centrifuge 3 min 16000 x g 3 min Discard WT Elute Place the SC into ET 2 1 min Add 50 uL EL preheated to 56 C onto SC membrane Incubate 2 min at room temperature Centrifuge 1 min at 13000 x g Discard SC Store Keep ET containing pure viral nucleic acids Use the
14. ith eyes rinse immediately with plenty of water and seek medical advice S36 37 39 Wear suitable protective clothing gloves and eye face protection S60 This material and its container must be disposed of as hazardous waste S61 Avoid release to the environment Refer to special instructions safety data sheets x Wash Buffer 1 WB1 Xn Harmful Hazard determining component of labeling Guanidinium chloride Risk phrases R22 Harmful if swallowed R36 38 Irritating to eyes and skin Safety phrases 23 Do not breathe gas fumes vapour spray S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 37 Wear suitable protective clothing and gloves S60 This material and its container must be disposed of as hazardous waste 13 Proteinase K PK Xn Harmful Hazard determining component of labeling Proteinase Tritirachium album serine Risk phrases R42 May cause sensitization by inhalation Safety phrases S23 Do not breathe gas fumes vapor spray S36 Wear suitable protective clothing S45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible S60 This material and its container must be disposed of as hazardous waste Material Safety Data Sheet of the product is available upon the request Field Safety Notice Fax 370 5 2602142 Email ts molbio eu thermofisher com REFERENCES 1 Boom R C J A Sol M M M S
15. ly upon the arrival and preparations store at 20 C 3 STORAGE AND STABILITY When the kit is delivered remove the Carrier RNA CR from the package and store in the original aluminum bag at 20 C Other components of the kit should be stored at room temperature 15 25 C All components are stable until the listed expiration date Wash Buffer 1 WB1 and Wash Buffer 2 WB2 are stable until the listed expiration date after addition of ethanol QUALITY CONTROL The Viral DNA RNA Purification Kit meets the essential requirements as outlined on Directive 98 79 EC Annex and is in compliance with the requirements of Directive 98 79 EC Annex Ill The quality of the product is controlled using validated procedures In accordance with our Company Quality Management System each lot of the Viral DNA RNA Purification Kit is tested against predetermined specifications to ensure consistent product quality A certificate of analysis of the product is available upon request INTENDED USE The Viral DNA RNA Purification Kit is a general purpose device intended for isolation and purification of viral nucleic acids from human plasma or serum samples for in vitro diagnostic purposes Any diagnostic results generated using the sample preparation procedure in conjunction with any downstream diagnostic nucleic acid technology NAT assay should be interpreted with regard to other clinical or laboratory findings The product is intended for use by
16. professionals such as technicians and physicians trained in in vitro molecular diagnostic techniques The Viral DNA RNA Purification Kit is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modification of DNA or RNA followed by signal detection The isolated and purified viral nucleic acids can be used in qualitative e g blood screening as well as quantitative e g viral load monitoring diagnostic NAT assays To minimize irregularities in diagnostic results the product must be used with an appropriate internal control as well as positive and negative controls throughout the process of sample preparation amplification and detection PRODUCT USE LIMITATION The Viral DNA RNA Purification Kit is not for use with whole blood tissue or cultured cells The performance of the kit in isolating and purifying viral nucleic acids from other cell free body fluids such as urine and CSF has not been evaluated The yields of viral nucleic acids may depend on the type of virus Plasma or serum samples from heparin treated blood are not suitable for use The user is responsible for validating the performance characteristics necessary for downstream diagnostic applications DESCRIPTION The Viral DNA RNA Purification Kit utilizes a silica based membrane technology in the form of a convenient spin column Viral nucleic acids from lysed plasma or serum samples bind to the column membr
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