NucleoSpin® Dx Virus - MACHEREY
Contents
1. Do not warm up Buffer RAV1 containing Carrier RNA more than 4 times Frequent warming temperatures gt 80 C and extended heat incubation will accelerate the degradation of Carrier RNA MACHEREY NAGEL 07 2014 Rev 04 11 Viral nucleic acid isolation Wash Buffer RAV3 Add the indicated volume see table below or on the bottle of ethanol 96 100 to Wash Buffer RAV3 Concentrate Mark the label of the bottle to indicate that the ethanol is added Store Wash Buffer RAV3 at room temperature Wash Buffer RAV3 can be stored at room temperature 18 25 C for up to one year but only until the expiration date NucleoSpin Dx Virus 50 preps REF 740895 50 Wash Buffer RAV3 Concentrate 12 mL Add 48 mL ethanol Proteinase K 30 mg Add 1 35 mL Proteinase Buffer PB 12 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation 4 Safety instructions The following components of the NucleoSpin Dx Virus kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze RAV1 Guanidinium thiocyanate Warni2. This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or spec
3. RNase free H O HO 13 mL 2 Elution Buffer RE BUF RE 13 mL Carrier RNA lyophilized Carrier RNA img Proteinase Buffer PB BUF PB 1 8mL Proteinase K lyophilized Proteinase K 30 mg NucleoSpin Dx Virus Dx Virus Columns 50 Columns dark blue rings plus Collection Tubes Collection Tubes 2 mL Collection Tubes 4 x 50 Lysis Tubes 1 5 mL Lysis Tubes 50 Elution Tubes 1 5 mL Elution Tubes 50 User manual 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer RE 5 mM Tris HCI pH 8 5 4 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation w LOT REF 2 wl Ivo TU A EN Do not Use by Batch Item Contains Manufac n vitro Please Permitted reuse identifica number sufficient turer diagnos readin storage tion for lt n gt tic pro structions tempe tests ducts for use rature range DE Nicht Verwend Chargen Artikel Ausrei Hersteller n vitro Ge Tempe wieder bar bis code nummer chend f r Diagnos brauchs raturbe verwen lt n gt Pr tikum anwei grenzung den fungen sung beachten ES Producto Fecha de C digo Referen Con Fabri Diagn s Obser Limites de un caduci de lote cia tenido cante tico in vense
4. Viral nucleic acid isolation User manual NucleoSpin Dx Virus A M In Vitro Diagnostic Medical Device S 740895 50 J m T MACHEREY NAGEL GmbH amp Co KG D 52355 D ren Tel 49 0 2421 969 0 JE July 2014 Rev 04 MACHEREY NAGEL www mn net com MN Viral nucleic acid isolation Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 6 1 3 About this user manual 6 2 Product description 7 2 1 Intended use 7 2 2 Product use limitations 7 2 3 Quality control 8 2 4 Introduction and kit specifications 8 2 5 Remarks regarding sample quality and preparation 10 2 6 Remarks regarding elution 10 3 Storage conditions and preparation of working solutions 11 4 Safety instructions 13 5 Viral nucleic acid purification with NucleoSpin Dx Virus 16 5 1 Protocol at a glance 17 5 2 Viral RNA isolation procedure 19 5 3 Viral DNA isolation procedure 21 5 4 Simultaneous viral RNA and DNA isolation procedure 23 6 Appendix 25 6 1 Troubleshooting 25 6 2 Ordering information 26 6 3 Product use restriction warranty 27 MACHEREY NAGEL 07 2014 Rev 04 3 Viral nucleic acid isolation 1 Components 1 1 Kit contents NucleoSpin Dx Virus 50 preps REF Symbol 740895 50 Lysis Buffer RAV1 BUF RAV1 35 mL Wash Buffer RAW BUF RAW 30 mL Wash Buffer RAV3 BUF RAV3 Conc 12mL Concentrate
5. 5 4 Simultaneous viral RNA and DNA isolation procedure Provide 150 uL sample in a Lysis Tube 1 5 mL provided 2 Add 600 pL Buffer RAV1 containing Carrier RNA to the Lysis Tube 3 Add 20 uL Proteinase K solution to the Lysis Tube Note Proteinase K is necesary for lysis of DNA viruses 4 Pipette mixture up and down and vortex well Note Make sure that the mixture incubates at least 1 min at room temperature before starting the heat incubation 5 Incubate for 5 min at 70 C 6 Briefly centrifuge Lysis Tube approx 1 s at 2 000 x g to remove drops from the lid short spin only 7 Add 600 pL ethanol 96 100 to the clear lysate 8 Mix by vortexing 10 15 s 9 Carefully load 700 uL of the lysate onto the NucleoSpin Dx Virus Column placed in a Collection Tube and close the lid 10 Centrifuge 1 min at 8 000 x g 11 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 12 Load the residual lysate approx 650 uL onto the NucleoSpin Dx Virus Column and close the lid 13 Centrifuge 1 min at 8 000 x g 14 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 15 Add 500 uL Buffer RAW to the NucleoSpin Dx Virus Column 16 Centrifuge 1 min at 8 000 x g 17 Place the NucleoSpin Dx Virus Column into
6. Dx Virus Column Centrifuge 3 min at 11 000 x g 23 24 25 Place the NucleoSpin Dx Virus Column into an Elution Tube 1 5 mL provided and discard the Collection Tube with flow through from the previous step Add 50 uL RNase free H O preheated to 70 C and incubate for 1 2 min Centrifuge 1 min at 11 000 x g to elute nucleic acid from the column 20 MACHEREY NAGEL 07 2014 Rev 04 NucleoSpin Dx Virus viral DNA isolation procedure 5 3 Viral DNA isolation procedure Provide 150 pL sample in a Lysis Tube 1 5 mL provided 2 Add 600 uL Buffer RAV1 containing Carrier RNA to the Lysis Tube 3 Add 20 uL Proteinase K solution to the Lysis Tube Note Proteinase K is necesary for lysis of DNA viruses 4 Pipette mixture up and down and vortex well Note Make sure that the mixture incubates at least 1 min at room temperature before starting the heat incubation 5 Incubate for 5 min at 70 C 6 Briefly centrifuge Lysis Tube approx 1 s at 2 000 x g to remove drops from the lid short spin only 7 Add 600 pL ethanol 96 100 to the clear lysate 8 Mix by vortexing 10 15 s 9 Carefully load 700 uL of the lysate onto the NucleoSpin Dx Virus Column placed in a Collection Tube and close the lid 10 Centrifuge 1 min at 8 000 x g 11 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the pre
7. P 280 P 3014312 P 302 352 P 3044340 P 305 351 338 P 312 P 330 P 3324313 P 337 313 P 3424311 P 363 P 403 233 P 403 235 Keep away from heat hot surfaces sparks open flames and other ignition Sources No smoking Von Hitze heiBen Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe vapours Dampf nicht einatmen Avoid breathing dust Einatmen von Staub vermeiden Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove victim to fresh air and keep at rest in a position com fortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len Call a POISON CENTER doctor if you feel
8. 0 996 100000 10000 LL _ 1000 100 HBV IU mL ee in 10 1 5 T T T 1 0 0001 0 001 0 01 0 1 1 Sample 10fold dilution Figure 1 Serial dilution of a plasma sample with high HBV viral load Real time PCR of HBV DNA Artus RealArt HBV DNA quantification in Roche LightCycler 480 8 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation 100990 J F 0 9917 10000 3 700 3 gt 5 100 rI J 10 1 T T 1 0 001 0 01 0 1 1 Sample 10fold dilution Figure 2 Serial dilution of a plasma sample with high HCV viral load Real time RT PCR of HCV RNA Artus RealArt HCV RNA quantification in Roche LightCycler 480 Kit specifications NucleoSpin Dx Virus is designed for the rapid preparation of highly pure viral RNA and DNA e g HCV HIV HBV CMV H1N1 from plasma and serum NucleoSpin Dx Virus is suitable for 150 uL serum or plasma samples The viral nucleic acids isolated and purified with NucleoSpin Dx Virus can be used in qualitative applications e g RT PCR or PCR for blood screening as well as in quantitative applications e g detection of viral load by qPCR employing diagnostic nucleic acid amplification techniques Protocols for isolation of viral RNA viral DNA and simultaneous isolation of viral RNA and DNA are included in the user manual The prepared nuc
9. Suficiente Fabri Diagn Observar Limites reutilizar vencime de lote de artigo para n cante stico in as in de tem testes vitro strug es peratura de uso SV teranv Anv nd Lot num Kata R cker till Tillver Medicin Se hand Tempera nd ej f r mer lognum n antal kare tekniska havande turbegra mer tester produkter beskriv nsning f r in ningen vitro dia gnostik MACHEREY NAGEL 07 2014 Rev 04 5 Viral nucleic acid isolation x x9 Irritant Harmful Flammable Reizend Gesundheitssch dlich Entz ndbar Irritante Nocivo Inflamable Irritant Nocivo Infiammabile Irritante Nocif Inflammable Irritierend Schadelijk Ontvlambaar Lokalirriterende Skadelig Brandfarlig AuaBoo tix EmfAapfic Evohexta Irritante Nocivo Inflam vel Irritanderende Skadligt Brandfarliga 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol to adjust nucleic acid binding conditions and to prepare Wash Buffer RAV3 Consumables Disposable pipet tips aerosol barrier pipet tips are recommended to avoid cross contamination Equipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Heating block or water bath for 70 C incubation Personal protection equipment e g lab coat gloves goggles 1 3 About this user manual It is strongly recommended to read the detailed protocol sectio
10. a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 18 Add 600 uL Buffer RAV3 to the NucleoSpin Dx Virus Column 19 Centrifuge 1 min at 8 000 x g MACHEREY NAGEL 07 2014 Rev 04 23 NucleoSpin Dx Virus simultaneuos viral RNA and DNA isolation procedure 20 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 21 Add 200 uL Buffer RAV3 to the NucleoSpin Dx Virus Column 22 Centrifuge 3 min at 11 000 x g 23 Place the NucleoSpin Dx Virus Column into an Elution Tube 1 5 mL provided and discard the Collection Tube with flow through from the previous step 24 Add 50 uL RNase free H O preheated to 70 C and incubate for 1 2 min 25 Centrifuge 1 min at 11 000 x gto elute nucleic acid from the column 24 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Low viral load in the sample The nucleic acid yield depends on the viral load in the sample Problems with Carrier RNA Carrier RNA not added See remarks concerning storage of Buffer RAV1 with Carrier Small RNA section 3 amounts or no viral Proteinase K digestion may be necessary nucleic acids e Choose the appropriate protocol for viral RNA or viral DNA in the eluate isolation see sec
11. controls positive negative controls should be used The NucleoSpin Dx Virus kit is intended for use by professional users such as technicians and physicians experienced and trained in molecular biological techniques including experience with serum and plasma samples and viral nucleic acid isolation The NucleoSpin Dx Virus kit does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay Besides human samples also fresh and frozen animal samples can readily be used together with the NucleoSpin Dx Virus kit Samples include but are not limited to serum plasma or swabs It has to be noted that CE IVD labeling of the kit does not apply for animal samples but is limited to human diagnostic use only 2 2 Product use limitations The NucleoSpin Dx Virus kit is not for use with human whole blood tissue stool samples or cultured cells The kit performance has not been evaluated with other cell free fluid samples like urine or cerebrospinal fluid The kit is also neither specified for the isolation and purification of bacterial fungal or parasite nucleic acids from human samples nor for the isolation of viral nucleic acids from human swab samples or other sample collections systems MACHEREY NAGEL 07 2014 Rev 04 7 Viral nucleic acid isolation 2 3 Quality control In accordance with MACHEREY NAGEL s Quality Managem
12. Column Check all samples especially old or frozen samples for precipitates It is very important to avoid clearing samples by centrifugation filtration before the RAV1 lysis step because viruses may be associated with particles or aggregates Incubation with Buffer RAV1 can be prolonged to dissolve and digest residual cell structures precipitates and virus particles However RNA is sensitive and prolonged incubation may cause decreased yields Remarks regarding elution Pure nucleic acids are finally eluted under low ionic strength conditions with RNase free H O pH about 7 8 or slightly alkaline Buffer RE 5 mM Tris HCl pH 8 5 both are supplied with NucleoSpin Dx Virus RNA should be eluted with the RNase free H O and DNA with Elution Buffer RE To elute both types of nucleic acids together use RNase free H O provided with the kit preheated to 70 C 10 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation 3 Storage conditions and preparation of working solutions Attention Buffer RAV1 contains guanidinium thiocyanate and Buffer RAW contains guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste Check all components for damages after receiving the kit If kit contents like buffer bottles or blister packages are damaged contact MACHEREY NAGEL technical support
13. Lysis Tube approx 1 s at 2 000 x g to remove drops from the lid short spin only 7 Add 600 pL ethanol 96 100 to the clear lysate 8 Mix by vortexing 10 15 s 9 Carefully load 700 pL of the lysate onto the NucleoSpin Dx Virus Column placed in a Collection Tube and close the lid 10 Centrifuge 1 min at 8 000 x g 11 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 12 Load the residual lysate approx 650 pL onto the NucleoSpin Dx Virus Column and close the lid 13 Centrifuge 1 min at 8 000 x g 14 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 15 Add 500 uL Buffer RAW to the NucleoSpin Dx Virus Column 16 Centrifuge 1 min at 8 000 x g 17 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 18 Add 600 uL Buffer RAV3 to the NucleoSpin Dx Virus Column 19 Centrifuge 1 min at 8 000 x g 20 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step MACHEREY NAGEL 07 2014 Rev 04 19 NucleoSpin Dx Virus viral RNA isolation procedure 21 22 Add 200 uL Buffer RAV3 to the NucleoSpin
14. MACHEREY NAGEL products MACHEREYNAGEL does not warrant the correctness of any of those applications Please contact Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 07 2010 Rev 03 Trademarks LightCycler is a registres trademark of the Roche Group NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 28 MACHEREY NAGEL 07 2014 Rev 04
15. and customer service or your local distributor Do not use damaged kit components Upon arrivalthe NucleoSpin Dx Virus kit should be stored atroom temperature 18 25 C It is NOT required to open the kit on delivery and remove individual components for separate storage NucleoSpin Dx Virus Columns can be used until the expiration date on the kit box Use RNase free equipment Before starting the NucleoSpin Dx Virus protocol prepare the following Lyophilized Proteinase K can be stored at room temperature 18 25 C until the expiration date without decrease in performance Before first use of the kit add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Reconstituted Proteinase K should be stored at 20 C for up to 6 months but only until the expiration date Carrier RNA Before first use add 1 mL Lysis Buffer RAV1 to the Carrier RNA vial Dissolve the Carrier RNA and pipette the solution back to the RAV1 bottle Note Due to the production procedure and the small amount of Carrier RNA contained in the vial the Carrier RNA may haraly be visible Lysis Buffer RAV1 including Carrier RNA can be stored at 4 C for up to 4 weeks Storage at 4 C or below may cause salt precipitation If precipitates are visible make sure to dissolve all precipitates before use by heating at 40 60 C for a maximum of 5 min Carrier RNA dissolved in Buffer RAV1 and stored at 20 C is stable for at least one year
16. compounds when combined with bleach Thus do not add bleach or acidic solutions directly to the sample preparation waste The waste generated with the NucleoSpin Dx Virus kit has not been tested for residual infectious material A contamination of the liquid waste with residual infectious material is highly unlikely due to strong denaturing lysis buffer and Proteinase K treatment but it cannot be excluded completely Therefore liquid waste must be considered infectious and should be handled and discarded according local safety regulations MACHEREY NAGEL 07 2014 Rev 04 15 NucleoSpin Dx Virus 5 Viral nucleic acid purification with NucleoSpin Dx Virus The procedures below provide instructions for processing a single plasma or serum sample However several samples can be processed at the same time the number depends on the capacity of the microcentrifuge used Before starting the preparation Check that Wash Buffer RAV3 and Proteinase K were prepared according to section 3 Check that Carrier RNA has been dissolved in Lysis Buffer RAV1 according to section 3 Check that 96 100 ethanol denatured or non denatured is available to adjust nucleic acid binding conditions Set an incubator e g heating block or water bath to 70 C Equilibrate the plasma serum samples to room temperature 18 25 C Make sure that the samples are mixed well If a precipitate has formed in Lysis Buffer RAV1 or Buffer RAW incuba
17. ent System each lot of NucleoSpin Dx Virus kit is tested against predetermined specifications to ensure consistent product quality 2 4 Introduction and kit specifications NucleoSpin Dx Virus is based on well established NucleoSpin silica membrane technology and provides an easy way to isolate viral RNA and viral DNA simultaneously from 150 uL of serum or plasma samples Purified RNA and DNA are ready to use for downstream amplifications like RT PCR or PCR The NucleoSpin Dx Virus procedure is based on a series of simple steps First the serum or plasma samples are lysed in the presence of chaotropic salts For the purification of viral DNA Proteinase K is added to the lysis reaction Lysis buffer and ethanol create appropriate conditions for binding of nucleic acids to the silica membrane of the NucleoSpin Dx Virus Columns Carrier RNA improves binding and recovery of low concentrated viral RNA and DNA Contaminations potential PCR inhibitors like salts metabolites and soluble macromolecular cellular components are removed in washing steps with ethanolic buffers RAW and RAV3 The nucleic acids are finally eluted in 50 uL low salt buffer or water The linear range of the NucleoSpin Dx Virus procedure has been determined for HCV RNA and HBV DNA in downstream diagnostic assays Figures 1 and 2 The kit shows linearity over several orders of magnitude comprising relevant viral titer for diagnostic purposes 1000000 F
18. ial including but not limited to loss of use revenue or profit whether based upon warranty contract tort MACHEREY NAGEL 07 2014 Rev 04 27 Viral nucleic acid isolation including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using
19. las de tem solo uso dad suficiente vitro instruc peratura para lt n gt ciones de tests uso IT Non riuti Utilizzare Codice Numero Contenu Fabbri Dispo Consul Limiti di lizzare entro del lotto dicata to suf cante sitivo tare le tempera logo ficiente medico istruzioni tura per lt n gt diagno per l uso test Stico in vitro FR Ne pas utiliser Num ro R f Contenu Fabricant Diagnos Respec Limites reutiliser avant de lot rence suffisant tic in ter les de tem pour n vitro instruc p rature tests tions d utilisa tion NL Niet Te ge Produc Artikel Vol Fabrikant In vitro Leesde Tempe herge bruiken tienum nummer doende diagnosti bijsluiter ratuur bruiken tot mer voor lt n gt cum begren tests zing DA M ikke Holdbar Batch Artikel Tilsttr k Produ in vitro F lg Tempe bruges til kode nummer keli gt cent diagnosti brugs raturbe igen til lt n lt kum anvisni gr ns kontroller ngen ning EL Heoiov Hyueoo AQ AQ Enaoxe Kata Atayva Tnoeite Ooa ma unv a nagtidag s ovc yia n OREVO OUx in ri Osouox yorons AnEng doxuues otic vitro o nyies oao ac xehons PL Nie Przydat Numer Numer Wy Produ Diagnos Pr Ograni u ywa no do partii artyku u starcza cent ty ka zestrzeg cze nie ponownie u ycia j co dla in vitro a tempera kontroli instrukcj tur y lt n gt u ycia PT Nao Data de N mero N mero
20. leic acids are suitable for applications like automated fluorescent DNA sequencing RT PCR or any kind of enzymatic reaction The detection limit for certain viruses depends on the individual procedures e g in house nested RT PCR To minimize irregularities in diagnostic results suitable controls for downstream applications e g extraction controls positive negative controls should be used to monitor the purification amplification and detection process Besides human samples also fresh and frozen animal samples can readily be used together with the NucleoSpin Dx Virus kit Samples include but are not limited to serum plasma or swabs It has to be noted that CE IVD labeling of the kit does not apply for animal samples but is limited to human diagnostic use only MACHEREY NAGEL 07 2014 Rev 04 9 Viral nucleic acid isolation 2 5 2 6 Table 1 Kit specifications at a glance Parameter NucleoSpin Dx Virus Technology Silica membrane technology Sample material Serum or plasma Sample volume 150 uL Elution volume 50 uL Preparation time 30 min 4 6 preps Processing Centrifugation Remarks regarding sample quality and preparation NucleoSpin Dx Virus is suitable for human serum or plasma samples For successful nucleic acid purification it is important to obtain a homogeneous clear and non viscous sample lysate before adjusting binding conditions and loading the sample onto the NucleoSpin Dx Virus
21. n of this user manual The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure MACHEREY NAGEL user manuals are available on the internet at www mn net com 6 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation 2 Product description 2 1 Intended use The NucleoSpin Dx Virus kit is a generic system for the isolation and purification of viral nucleic acids from human serum or plasma samples for subsequent in vitro diagnostic purposes The kit can be used with fresh and frozen human serum and plasma stabilized with either EDTA or citrate from common blood collection systems The kit is designed to be used with any downstream application employing enzymatic amplification and detection of RNA and DNA e g RT PCR PCR The viral nucleic acids isolated and purified with NucleoSpin Dx Virus can be used in qualitative applications e g RT PCR or PCR for blood screening as well as in quantitative applications e g detection of viral load by qPCR employing diagnostic nucleic acid amplification techniques Any diagnostic results generated using nucleic acids isolated with the NucleoSpin Dx Virus kit in conjunction with an in vitro diagnostic assay should be interpreted with regard to additional clinical or laboratory findings To minimize irregularities in diagnostic results suitable controls for downstream applications e g extraction
22. ng 302 412 260 273 3014312 30 60 96 EUHOS1 330 Guanidiniumthiocyanat Achtung 30 60 RAW Guanidine hydrochloride Warning 226 302 210 233 301 312 24 36 ethanol 35 lt gt 330 403 235 55 Guanidinhydrochlorid 24 36 96 4 Achtung Ethanol 35 55 Proteinase K Proteinase K Iyophilized Danger 315 317 261 280 302 352 Proteinase K Iyophilisiert Gefahr 319 334 304 340 335 305 351 338 312 lt gt 332 313 337 313 342 311 363 403 233 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen H 335 May cause respiratory irritation Kann die Atemwege reizen H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung MACHEREY NAGEL 07 2014 Rev 04 13 Viral nucleic acid isolation EUHOS1 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase Precaution phrases P 210 P 233 P 260 P 261 P 273
23. ransfer the Transfer the Transfer the NucleoSpin NucleoSpin NucleoSpin Dx Virus Column Dx Virus Column Dx Virus Column to a new Collection to a new Collection to a new Collection Tube Tube Tube 18 600 uL RAV3 600 uL RAV3 600 uL RAV3 19 8 000 xg 1 min 8 000 x g 1 min 8 000 x g 1 min 20 Transfer the Transfer the Transfer the NucleoSpin NucleoSpin NucleoSpin Dx Virus Column Dx Virus Column Dx Virus Column to a new Collection to a new Collection to a new Collection Tube Tube Tube 21 200 uL RAV3 200 uL RAV3 200 uL RAV3 22 11 000 x g 3 min 11 000 x g 3 min 11 000 x g 3 min Elute RNA 23 Transfer the Transfer the Transfer the DNA NucleoSpin NucleoSpin NucleoSpin Dx Virus Column to Dx Virus Column to Dx Virus Column to an Elution Tube an Elution Tube an Elution Tube 24 50 uL RNase free 50 uL Buffer RE 50 pL RNase H O 70 C 70 C free H O 70 C Incubate 1 2 min Incubate 1 2 min Incubate 1 2 min 25 11 000 x g 1 min 11 000 x g 1 min 11 000 x g 1 min 18 MACHEREY NAGEL 07 2014 Rev 04 NucleoSpin Dx Virus viral RNA isolation procedure 5 2 Viral RNA isolation procedure Provide 150 pL sample in a Lysis Tube 1 5 mL provided 2 Add 600 pL Buffer RAV1 containing Carrier RNA to the Lysis Tube 3 Note No Proteinase K is used for the isolation of viral RNA only 4 Pipette mixture up and down and vortex well 5 Incubate for 5 min at 70 C 6 Briefly centrifuge
24. te the buffer at 40 60 C until the precipitate is dissolved Generally do not mix reagents and columns from different kits and lots Heat RNase free H O Elution Buffer RE to 70 C for final elution of nucleic acids Do not add Proteinase K solution directly to Lysis Buffer RAV1 The sample has to be combined with the Lysis Buffer RAV1 before addition of Proteinase K All centrifugation steps should be carried out at room temperature 18 25 C 16 MACHEREY NAGEL 07 2014 Rev 04 NucleoSpin Dx Virus 5 1 Protocol at a glance Supplemental protocol overview Carefully read the detailed protocol section 5 2 5 4 before starting the procedure Note The protocols differ in Proteinase K Iysis step step 3 and elution step step 24 only Viral RN isolation procedure section 5 2 Viral DN isolation procedure section 5 3 Viral RNA DNA isolation procedure section 5 4 Provide 1 150 pL sample 150 pL sample 150 pL sample sample in Lysis Tubes in Lysis Tubes in Lysis Tubes lyse viruses clear lysate 2 600 uL 600 uL 600 uL Buffer RAV1 Buffer RAV1 Buffer RAV1 containing containing containing Carrier RNA Carrier RNA Carrier RNA 3 Note 20 uL Proteinase K 20 pL Proteinase K No Proteinase K is Incubate at least Incubate at least used for the isolation 1 min at room 1 min at room of viral RNA only temperature temperature 4 Pipette mixture Pipette mixture Pipette mixture
25. tion 5 1 Viral nucleic acids degraded Samples should be processed immediately Ensure appropriate storage conditions up to the processing Check that all buffers have been prepared and stored correctly If in doubt use new aliquots of Buffer RAV1 Carrier RNA and Elution Buffer RE Reduced sensitivity Problems Change the volume of eluate added to the PCR RT PCR with subsequent Ethanol carry over detection Prolong centrifugation step step 22 in order to remove Buffer RAV3 completely MACHEREY NAGEL 07 2014 Rev 04 25 Viral nucleic acid isolation 6 2 Ordering information Product REF Pack of CE IVD marked kits NucleoSpin Dx Virus 740895 50 50 NucleoSpin Dx Blood 740899 50 250 50 250 Kits for research purposes NucleoSpin Virus 740983 10 50 250 10 50 250 NucleoSpin RNA Virus F 740958 25 NucleoSpin totalRNA FFPE XS 740969 10 50 250 10 50 250 NucleoSpin totalRNA FFPE 740982 10 50 250 10 50 250 NucleoSpin DNA FFPE XS 740980 10 50 250 10 50 250 NucleoSpin Blood 740951 10 50 250 10 50 250 NucleoSpin Tissue 740952 10 50 250 10 50 250 NucleoSpin Tissue XS 740901 10 50 250 10 50 250 NucleoSpin miRNA 740971 10 50 250 10 50 250 Proteinase K 740506 100 mg Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 26 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation 6 3 Product use restriction warranty The NucleoSpin D
26. unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen Rinse mouth Mund aussp len If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltende Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen an einem gut bel fteten Ort aufbewahren Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com 14 MACHEREY NAGEL 07 2014 Rev 04 Viral nucleic acid isolation When working with the NucleoSpin Dx Virus kit wear suitable protective clothing e g lab coat disposable gloves and protective goggles For more information consult the appropriate Material Safety Data Sheets MSDS available online at http www mn net com msds Caution Guanidinium thiocyanate in Lysis Buffer RAV1 and guanidine hydrochloride in Wash Buffer RAW can form highly reactive
27. up and down and up and down and up and down and vortex well vortex well vortex well 5 Incubate at Incubate at Incubate at 70 C for 5 min 70 C for 5 min 70 C for 5 min 6 Short spin to Short spin to Short spin to clean the lid clean the lid clean the lid Adjust 600 uL ethanol 600 uL ethanol 600 uL ethanol iai DR Mix by vortexing Mix by vortexing Mix by vortexing 10 15 s 10 15 s 10 15 s Bind RNA 9 Load 700 pL Load 700 uL Load 700 uL DNA lysate onto the lysate onto the lysate onto the NucleoSpin NucleoSpin NucleoSpin Dx Virus Column Dx Virus Column Dx Virus Column 10 8 000 xg 1 min 8 000 x g 1 min 8 000 x g 1 min MACHEREY NAGEL 07 2014 Rev 04 17 NucleoSpin Dx Virus 11 Transfer the Transfer the Transfer the NucleoSpin NucleoSpin NucleoSpin Dx Virus Column Dx Virus Column Dx Virus Column to a new Collection to a new Collection to a new Collection Tube Tube Tube 12 Load the residual Load the residual Load the residual lysate ca 650 uL lysate ca 650 uL lysate ca 650 uL onto the column onto the column onto the column 13 8 000 xg 1 min 8 000 x g 1 min 8 000 x g 1 min 14 Transfer the Transfer the Transfer the NucleoSpin NucleoSpin NucleoSpin Dx Virus Column Dx Virus Column Dx Virus Column to a new Collection to a new Collection to a new Collection Tube Tube Tube Washsilica 15 500 uL RAW 500 uL RAW 500 uL RAW membrane 16 8 000 x g 1 min 8 000 x g 1 min 8 000 x g 1 min 17 T
28. vious step 12 Load the residual lysate approx 650 uL onto the NucleoSpin Dx Virus Column and close the lid 13 Centrifuge 1 min at 8 000 x g 14 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 15 Add 500 uL Buffer RAW to the NucleoSpin Dx Virus Column 16 Centrifuge 1 min at 8 000 x g 17 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 18 Add 600 uL Buffer RAV3 to the NucleoSpin Dx Virus Column 19 Centrifuge 1 min at 8 000 x g MACHEREY NAGEL 07 2014 Rev 04 21 NucleoSpin Dx Virus viral DNA isolation procedure 20 Place the NucleoSpin Dx Virus Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step 21 Add 200 uL Buffer RAV3 to the NucleoSpin Dx Virus Column 22 Centrifuge 3 min at 11 000 x g 23 Place the NucleoSpin Dx Virus Column into an Elution Tube 1 5 mL provided and discard the Collection Tube with flow through from the previous step 24 Add 50 uL Buffer RE preheated to 70 C and incubate for 1 2 min 25 Centrifuge 1 min at 11 000 x gto elute nucleic acid from the column 22 MACHEREY NAGEL 07 2014 Rev 04 NucleoSpin Dx Virus simultaneuos viral RNA and DNA isolation procedure
29. x Virus kit is a generic system for the isolation and purification of viral nucleic acids from human plasma or serum samples for subsequent in vitro diagnostic purposes The kit is designed to be used with any downstream application employing enzymatic amplification and detection of RNA and DNA e g RT PCR PCR Any and all diagnostic results generated using nucleic acids isolated with the NucleoSpin Dx Virus kit in conjunction with a diagnostic assay should be interpreted with regard to additional clinical or laboratory findings The NucleoSpin Dx Virus kit does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay ONLY MACHEREY NAGEL products specially labeled as IVD are suitable for N VITRO diagnostic use The NucleoSpin Dx Virus kit is intended for use by professional users such as technicians and physicians experienced and trained in molecular biological techniques including experience with serum and plasma samples and viral nucleic acid isolation For safety instructions please refer to the respective chapter in the user manual NucleoSpin Dx Virus kit shall exclusively be used in an adequate test environment i e a suitable laboratory setting The respective user is liable for any and all damages resulting from application of the NucleoSpin Dx Virus kit for use deviating from the intended use as specified in the user manual
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