DNA増幅試薬 D
Contents
1. 1 ARDA AINE Rn TCD EA 2 PAILBAODEIRVHBSEDEUCERBU CROIRU MEST ANF FRA RHBUTCK ES 3 SCS RHE ICHIMRRA RECETAT SIS DYTKOWMAASNSRIMR GI B SSSCOC FINREBRBICANSCEABITCIES KERIKTIPO SYFERSVEMHSCSlA USHSAREBID DIEORRGHEHED TCHEUTCIESU 4 RZD CAO BRICNBLECHEI BHICABOKT HHICRURL M EBPwHNNSRMOFS CeBIT TCE ALOFT 1 RACK Ti CORDICKY LAMP RDAS LEL BERNA BENS O zs 2 ARRIAREROREORBMEAICIDE BEOMBAACREUTCKIEAUN 3 RMFA TPIARBBLOTWOT MIRUUCIBEBUT lt ES 4 RMFA PSASHICFAs EESDRVCCeBR CHRBULTCIER Rai Fa DicFAZ ERESMHSCECK lt KAECSRIDOD FAa DORIBICK ORBESLISTREMHOSS UPILITHSEAEREM ST YIN S ORMID vy DA CF 2 POMBIB LIES IA RUDTTROREAAN IW LL BR AARRESS HR OMMBORACROES 5 RMFA FOR CUPOAMA CRER RAN TIETOC ATA EDEREK ALTES KE BONMEBRF CHRSBWK SICERBUTCKIESU 6 ROERMFA PABSICHOPISNyYICRL SSACHBRMANTIISCE Steal PEORIA CREUT 7 Positive control dBP PC dBP RUMMHCKEDNSIRASIA home CHEL TR ELUTE 8 MRFRADMBWORRACCAIRE BABROHESRSSIEDHDOLIO T KRRRORAICHE o THERAN RRES OCRMBORBOEC CHA SRiBLT lt IERU 9 ARROMARICHRUAL GRVETDRERIIERSS CRIE KE SOAUECHR CHE VEBSEICMUT MHtlt WOBESBWNEKA 10 AReAEAARRAICHEALT lt IERU 11 ARROSH HERSSBUANSHOBNICRALBAUVC lt CIESU 2 72 12 RIMMFA D DS1V SyIARRBMBF a TIS IMRRH LIR2. PRBOICMIEIC P VEE inner primer DOOHERSKRMAEO RIT CUETLES TNICKYO LAMP AISA SHARD 1 BM CHSICEHBNSS Sik IBARRLIECHKCS fH ARM RE Ost MIC OLTI Eiken GENOME SITE CURL http loopamp eiken co jp amp CBIR lt ES 18 FAA 1 MSGR BM AS ARBICASENTKCAO CHBBRBUTCK ER 1 DSAV SyIARRAMEF A TF 0 5 mL Rid 1 5 mL 2 YT7OERYR 3 71I ENY KFYT DNase RNase 7 4 RMF I P AMA IVSRS yD 5 K DayvyaP 2 RUPTARYD 6 MBA ELH 7 8BV1 2720F 7AA RN 8 MILF YIZSI Y 9 Loopamp DY KO ILEY A DNA KIMHC RAAB E Primer mix dBP PM dBP Positive control dBP PC dBP Negative control NC A UPILAT Waa UPILST ABER LAMP 48A B tS Rah 1 Loopamp y Baawets ROC SURAA BITTE 2 UPILAT WSEAS LAMP SAS 2 LAT YF CREED 0 5 CIA hy KY Ry BT 3 E b7OY7 BRAS 4 SOMRIRMB GR 240 260nm 350 370nm 2 5 DIBORRNVAHES x1 ARRCMASDE CEAISIY KO RAAY KTI 2 MRE RIO LAE FRIMRERR ORC DU CId Eiken GENOME SITE CURL http loopamp eiken co jp 2CBIR lt IES 2 751 Nit LAMP KICK SISIBICIABWERDIS1 V BUOMBERAFCROES Et feo Tid
3. LAMP amp BAHOIS 1 V RstSIEY Db LAMP AIT Vast SRV I KIIP Primer Explorer Eiken GENOME SITECURL http loopamp eiken co jp KO CAIFRUVEIE lt K CEMCEEST KE F51V dHEREDSWECRMREIGR lt CO RMBAEBRELES 2 DI4V Sy IADR OKER A UPIL T LBB C TARDE ER SAIL VCR D RNAD SOS LEDS TT FS1TVY D RAD VISBHCUTEKI SREISHBAIL BRIL KFIYLE DS1V ERET SRRURERIA DECC FIP BIP CD TIR HPLC RJU RICKSSMEHBLEAT tt 3 TROBRDE 1 MBAR OYT IUH 2Y KOLO O Dried DNA Amplification Reagent R BLEF RMFI PaWMISRSlS SISSESSOOACTOIMUDS PREN RICGBSSABWUAKSDICIAAAS COMETS BO ORMF 1 FItBS5 ICHOP IVE Ny 7 CBHUCRELET BRR UCIS1Y SyIARRARMEF A DICB IS 1V aMBETAL BO FROS 1 FALHED CHRLET LAMP RibRCUTSEt 25 0uL CISSKD FOAV SYIRZCVY TIVEROSSEBE UTK IES a BISIDAIS1V By FEMS NE TEAST SRSA SIS1V BYbO PARECE T lt IESU O FYTILEIOR Cl lt at gt lt Ais gt Distilled Water DW xX ul Cas J5417 FIP 40 pmol BIP 40 pmol LF 3 20 pmol LB 3 20 pmol F3 5 pmol B3 5 pmol 6 at 15 0 uwlL FAb K3 KF USBMBHOECAD Loop primer FEANSCE TiBMBSGADH 1 3 CE anes O ARROIYF JLI CLoopamp DY FO Ly gt DNA lt at gt lt A
4. NA SA RMEOMAS A NUE RNADSOBIMEWRETS 172 6 isah S y FO L RIYO 07 06 05 5 o4 i 03 02 o1 o p 01 sR 23 LOIN BR NS Y om a so M1 DYF lt RIEICHIE TOIA T 6 LAMP RSJ RICREA RRF OSEM CERATS CROEBRECESTRACESHENDHIET COLISGIVISR Y3 VEDES SIEOIC RARWROMBBHIRFISAR SPAS SBECISEIOR BE CAIHI SD HSV ARAKWEDEUCHOLUP CHT9T lt IESU WBC MUTT PU YNYF ORARLER OP 1 UL YaVADYOSASCIVY SR YaVeMCBBECO CSS SRREROMIRICIA MAM ORDER DNase RNase DIYSSR Ya Veet CCHS SCRE OMIA RICAI DCSE BORDERS BEDIERISS4 DISHED TS SNBWCCMBHO ES ICA LIDAR CS 2 DAMENE LEU TES TUTFIVERERGER RIMTROREICVANHSCRHEORACRSEOT RE VIDYUTC AERMICROBRLYT CER RIMBOFA PORIARECHIIAUICC ESL HICRMBOF TERED SROWS CSICFI POBURMSAS BBICROWLC lt EAU SBE MICKSDYVISR Y 3 VIPSBHEORACESILDO TEX HERETOEO BBR SRETEICPRALEUMRO MEE UU ERD SNE lt SS TRE HOST BRAGS COMBE MOIR MAGI TESEN MERNE ER OER
5. UVC IES BRE OTS T o KIRRACKIEE BAS CR OCMRECICSIREMOHIES RMEBOF 1 FISBeRII SIC GANUERISB RAC SSEI ORE Cie Ui DICE UT CIES SIE MORAUBS LE OIE PMEBISTTIVIEVVC lt IERU D RROMCA rIL 2 RIHFA PAAKIFOEY PP RMFA PALU Td PET PIVSNYD AZPIL Fy KI Zd EESE UTERT 3 KeSARVEHEOAROSERUSSAlL FRWOULRV SCI SABO KES BIA SICHE SHOR ECHVCUBUTCKES itm BORA ORF RRI F Rm amp TEDA Se Geet RIF Loopamp AAS a a DNA ISIE D 1 30 C 14 fa 96 FAK LMP207 BVH 1 Notomi T et al Nucleic Acids Research 28 12 e63 2000 2 Nagamine K et al Clin Chem 47 9 1742 1743 2001 3 Mori Y et al Biochem Biophys Res Commun 289 1 150 154 2001 4 Tomita N et al Nat Protoc 3 5 877 882 2008 5 Nagamine K et al Molecular and Cellular Probes 16 3 223 229 2002 BER OAM Mii 54 3 667 715 1999 6 AMIBFA V1 ALTIT 4 WSN A x THEFRAAt PBS yard ER D J A4 VP T 0120 308 421 HAR ET BS oh SA A143 g ith For research use only 4 Loopamp LAMP Loop mediated Isothermal Amplification method DNA Amplification Reagent D This product is a reagent for research purpose Do not use this product for making or supporting a diagnosis Read this expl
6. anatory leaflet carefully before use Introduction The LAMP Loop mediated Isothermal Amplification method is a gene amplification technique characterized by 1 isothermal gene amplification reaction 2 high specificity due to the use of 4 primers recognizing 6 regions 3 high amplification efficiency resulting in amplification in a short time 4 a large amount of amplification product facilitating simple detection This product is designed for amplifying and detecting the target gene sequence with the LAMP method by combining with an originally designed LAMP primer or a separately provided primer set product This product is a dry formulated DNA amplification reagent and allows the amplification of the target gene only by dissolving the amplification reagent in a primer solution and a sample solution containing template DNA and keeping the reagent at a constant temperature Contents For 96 tests Dried DNAAmplification Reagent 48 tubes X2 Measurement Principle The LAMP method is an isothermal gene amplification technique using 4 primers and a DNA polymerase with strand displacement activity to cause a reaction Of the 4 primers 2 inner primers recognize 2 different regions in the target gene sequence on their 3 and 5 sides The sequence on the 5 side is set so that it will be annealed in the complementary strand region synthesized by the elongation reaction from the 3 side This amplification reac
7. asses or shield 1 Control set to be used with this product x2 For more information on compatible equipment deactivation or inactivation and ultraviolet irradiation conditions please visit the Eiken GENOME SITE CURL http loopamp eiken co jp e 2 Primer design Appropriate primer design is an important factor to the amplification by the LAMP method Primer design support software dedicated for the LAMP method LAMP Method Primer Design Support Software Primer Explorer is available for designing primers on the following website Eiken GENOME SITE CURL http loopamp eiken co jp e To select appropriate grade for purification of primer please note that the rate of reaction is increased and reaction becomes more stable as the degree of refining for a primer is increased Therefore simple column refining or higher grade is recommended for synthesizing primers for primary screening HPLC refining grade is recommended for deciding a primer or after decision for at least FIP and BIP 3 Reagent preparation method 1 Take a necessary number total number of samples and controls of the Dried DNA Amplification Reagent To cut a bridge of reaction tube use scissors to avoid any impact on the dried reagent do not tear the tube Restore the remaining reaction tube to the original aluminum pack and seal it for storage 2 Preparation of primer mix Coperation on ice Prepare a necessary number of each primer for the test
8. e of their components This product should be stored as specified while avoiding freezing or sudden change in temperature Carefully handle reaction tubes because they are fragile Visually check reaction tubes for flaws or chaps Flaws or chaps of reaction tubes if any may result in incorrect measurements or the contamination of measurement devices The damage of a tube in the real time turbidimeter or incubator reaction block may result in the leakage of the reaction solution causing unrecoverable contamination or breakdown Take care not to apply excessive impact to the cap of the reaction tube inside of the rib because the detection reagent is kept dry and retained in it Avoid the direct contact between the inside of the cap and hands Restore the remaining reaction tube to the original aluminum pack Confirm that it is surely sealed and store it as specified Keep the positive control dBP PC dBP and potentially positive specimens away from other reagents Perform gene test with this product only under the supervision of experts with the knowledge and experience of gene test because the lack of knowledge or experience may result in incorrect judgment of the test result Eiken Chemical Co Ltd does not bear any responsibility for false judgment or any consequential damage derived from the false judgment caused by non capability problems such as operation error Use this product within the expiration date Do not recycle the conta
9. hee O C Detection with commercially available fluorescent pigment 6 The target gene can also be detected using a commercially available fluorescent pigment and fluorescence measuring device Amplification from RNA The target gene can be amplified from RNA using the specimen after the cDNA synthesis reaction Amplification curve pattern Positive control Negative control 07 06 A 05 04 03 f Turbidity 02 5 0o pE J 01 Time min Figure 1 Control amplification curve pattern lt Precautions for measurement gt 1 6 Because the LAMP reaction is very sensitive any contamination with only a minute amount of the target gene or amplification product may result in an incorrect result To avoid such contamination the use of this product and specimen collection nucleic acid extraction procedure should be performed in separate rooms or in different areas by partitioning the laboratory area Take appropriate measures to prevent contamination including the use of clean benches gloves and isolation gowns as required Avoid the contamination by microorganisms or nucleic acid degrading enzymes such as DNase and RNase in handling this product Completely dissolve the dry reagent Incomplete dissolution may result in poor performance including low sensitivity Do not leave more than 2 minutes in state of inverting reaction tubes Air bubbles may appea
10. in a separately prepared sterilized tube for preparing primer mix according to the ratio in the following table per test Adjust the volume of the primer mix and sample solution so that the total volume of the LAMP reaction solution will be 25 0 u L If a separately provided primer set is combined follow the instruction manual of the primer set For sample reaction example lt Reagent gt lt Dose gt Distilled Water DW X uL Appropriate amount Primer FIP 40 pmol BIP 40 pmol LF 3 20 pmol Lg 20 pmol F3 5 pmol B3 5 pmol Total 15 0 uL test X3 The use of Loop primer which is not essential reduces amplification time to approximately one third O For control reaction of this product Loopamp control set DNA lt Reagent gt lt Dose gt Primer mix dBP PM dBP 15 0 wL test 3 Mix by tapping or inverting the tube or using a vortex mixer for 1 second 3 times and spin down before using as the primer mix Operation on ice 4 Operating procedure Operation on ice Dispense 15 0 u L of the primer mix to each reaction tube Dispense 15 0 u L of PM dBP for control reaction Add 10 0 u L of the sample solution or control 25 0 uL in total as the LAMP reaction solution Use negative control NC for the negative control and positive control dBP PC dBP for positive control After closing the cap invert the reaction tube to transfer the solution onto the cap and allow it to stand o
11. iners or accessories of this product or use them for other purposes 4 2 2 2 Do not expose reaction tubes and tubes for preparing primer mix to UV light A change in color or degeneration caused by ultraviolet lamp sometimes results in misjudgment Precautions for disposal 1 Appropriately dispose of tubes after reaction with the cap closed by putting them in double plastic bags that can be incinerated or sealed To prevent dispersion of amplification products do not autoclave tubes before disposal 2 Reaction tubes are mainly made of polypropylene PP The tray for reaction tubes is mainly made of PET The aluminum pack is mainly made of aluminum The case is mainly made of paper 3 Dispose of this product containers and materials before or after use on the responsibility of the laboratories in compliance with applicable laws on waste disposal and cleaning and water pollution prevention law Storage method shelf life packaging unit and product code Product name Storage Shelf life Package Product method unit code Loopamp For 96 1 30 C 1 LMP207 DNA Amplification Reagent D year tests References 1 Notomi T et al Nucleic Acids Research 28 12 e63 2000 2 Nagamine K et al Clin Chem 47 9 1742 1743 2001 3 Mori Y et al Biochem Biophys Res Commun 289 1 150 154 2001 4 Tomita N et al Nat Protoc 3 5 877 882 2008 5 Nagamine K et al Molecular a
12. n ice for 2 minutes Repeat the inversion 5 times and spin down the reaction mixture using an 8 microtube simple centrifuge lt LAMP reaction gt Set it in the real time turbidimeter or incubator reaction block to start reaction 30 to 60 minutes at 60 to 67 C 4 The control reaction of this product occurs at 66 C for 40 minutes Enzyme deactivation 80 C for 5 minutes or 95 C for 2 minutes Automatically processed in the real time turbidimeter 172 Turbidity measurement evaluation X4 The optimum condition for the designed primer varies and has to be individually examined 5 Detection A Real time turbidity detection The target gene can be detected in real time using the real time turbidimeter designed for the LAMP method Refer to the package insert or operation manual etc for the detailed operating procedure B Fluorescent visual detection The target gene can be visually evaluated using the separately provided Loopamp fluorescent visual detection reagent Refer to the instruction manual of the fluorescent visual detection reagent for details 380233 A
13. nd Cellular Probes 16 3 223 229 2002 6 The guideline for the bio safety and bio hazard by the Japanese Society for Bacteriology Japanese Journal of Bacteriology 54 3 667 715 1999 Manufacturer EIKEN CHEMICAL CO LTD Biken 143 Nogi Nogi machi Shimotsuga gun Tochigi 329 0114 Japan Date of Revision April 2015 Ver 1 380233 A RAB 2015 4 AYER 35 1h 4 Loopamp LAMP Loop mediated Isothermal Amplification it TEE DNA isi ats D ARRISHABAE CS BMNISCOMHS BHI CUTIAGEAUBLYC lt IERU CORBEES lt i CO SILAUTCKIESU id UIC LAMP Loop mediated Isothermal Amplification Aid O SIR CHBFISIBRM DETTS OQ 6 MRABAT S 4 ENTIT ERAT SEORSHD SU BBDD MPMICISIBDHE CHS EBEE HBA CWS SORWMAE ST SHBFISIBATS ARGS BSE LE LAMP AIST VY KEIO IS 1 V vy FRR CHA BENET LAMP ATRO RES FEIE RT SIEDOFY KCTS FRA BYCANUE DNA ISIE A D51V SRC DNA SEOUL TILIEM CBRL SEREICR OIE CRF ASBI DT TENTE RR AS FZ K Dried DNA Amplification Reagent 48 tubes X2 CAERE LAMP id 4 HOPS V Ci SIE HO DNA Polymerase FRUV TR SWS SRARTISIBACS 4 BOIS 1V OSD5 2 FO inner primer ld Z OIME SATRI PORD 2 Haw SIS 1V TC SOR UIS EO 3 AP SOMRRM CSM CERIN ICP LISKABELES Said TM inner primer ICKOFE MFI SAZTFAIL TPMWiEP SORBCARRM CC IV
14. r on the liquid level of the reaction solution after mixing the sample solution Remove them to prevent measurement errors by spinning down the reaction mixture Never open the tube cap after reaction Particularly carefully remove the tube from the equipment so as not to open the cap after reaction The contamination with amplification products not only results in an erroneous decision but also causes the contamination of the measurement environment Such contamination may persistently inhibit correct measurement unless it is completely eliminated Avoid handling amplification products using electrophoresis Precautions for handling hazard prevention 1 2 3 This product is not designed as an in vitro diagnostic IVD Carefully handle specimens as potentially infectious substances and take necessary biohazard prevention measures Take care not to directly gaze at the ultraviolet ray sterilizing ray from the lamp of the ultraviolet irradiation device for fluorescent visual evaluation because it might cause serious damage When it is necessary to gaze at the lamp that is on make sure to do that through a glass plate or using wide eyeglasses or shield If the reagent accidentally enters the eyes or mouth or attaches to skin immediately rinse it off with a large amount of water and seek medical treatment if needed Precautions 1 2 Some specimens may inhibit the LAMP reaction and produce incorrect results becaus
15. s gt Primer mix dBP PM dBP 15 0 uL FAP 3 FA PeB lt MUVCRSIS SVEVI PD NAER DSVMAMILT Y DASTU 1 WAX ORL GREER CYSDVEISTV SyIAZACUES 4 RUF OKER REF 1 ABENIS1V SYIA15 0uL EWF A JUBUMFBITIA PM dBP 15 0 uL 2DSE HYPER EEY KOENEN 10 0 uL HH J LAMP fii UCSat 25 0 uL BIIY KO LICI amp Negative control NC BIIY KO LICId Positive control dBP PC dBP 1 BAADH RF 1l Peih CAREEN BL afl LIAR OKE 2 DAKE CMET D a RUF A De 5 Opr 8sev17 F1i TP RBA CACYIIVIS LAMP Ri UPIVAT LSBWERE IIT YIN O RWwA7OyVICey KUTRMAZI b 60 67C 30 60 DA ARROIY FO I URMlt 66C 40 DS BRE 80C 5 DARC 22 UPILST DSEWESE CASOMBsnay BERT He X4 i Rat lIeF51 Tko CHBRADRESOC RHRAIDUBICBVIES UPILST WSR RE SEI AFOSR TBR BIED Loopamp 85 BWMRKAMRAA SCC CARHEDURETI iol St BRM ARO PARAS SCSIRCICAU LAMP ASAD 2AUSCOC UPIS TARET ORUNLEROWRHBSS amp CEIRCIERU iS E HARDENER CC HERE CORLEDHCS cD
16. tion proceeds by alternating the self elongation from the stem loop structure generated from the inner primer and strand displacement synthesis from the inner primer annealed to the loop part This process of the LAMP method allows isothermal amplification using only 1 enzyme For more information on the reaction principle please visit the Eiken GENOME SITE CURL http loopamp eiken co jp e Instructions for use 1 Essential apparatus equipment and reagents etc Not included in the product Prepare them separately 1 Sterilized tube for preparing primer mix 0 5 mL or 1 5 mL 2 Micropipette 3 Pipette tip with filter DNase RNase free 4 Aluminum rack for cooling reaction tubes 5 Ice crushed ice and ice box 6 Simple microvolume centrifuge 7 8 microtube simple centrifuge 8 Vortex mixer 9 Loopamp control set DNA separately provided by Eiken Chemical Primer mix dBP PM dBP Positive control dBP PC dBP Negative control NC A Real time turbidity detection Real time Turbidimeter designed for LAMP method B Fluorescent visual detection 1 Loopamp fluorescent visual detection reagent separately provided by Eiken Chemical 2 Real time Turbidimeter designed for LAMP method or incubator with a temperature precision of within 0 5 C with hot bonnet 3 Heat block for enzyme deactivation 4 Ultraviolet irradiating equipment wavelength 240 to 260 nm and 350 to 370 nm 2 5 Wide eyegl
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