Cycleave®PCR Legionella (5S rRNA) Detection Kit Ver.2.0
Contents
1. Cycleave TaKaRa Ex Taq Thermal Cycler Dice http www takara bio co jp 29 CY210 http wwwr takara bio coJp 30 CY210 http wwwr takara bio coJp 31 CY210 IafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio co jp 22 2 GC ARRS v201406Da2. 5S rRNA PCR Hot Start PCR 7aKaRa Ex Tag HS RNA DNA RNase H
3. FAM ROX 2 DNA DNA http www takara bio co jp 28 CY210 X Fields B S Benson R F Besser R E 2002 Legionella and Legionnaires disease 25 years of investigation C in Microbiol Rev 15 506 526 Benin A l Benson R F Besser R E 2002 Trends in legionnaires disease 1980 1988 declining mortality and new patterns of diagnosis Clin Infect Dis 35 1039 1046 3 Schraft H et a 1995 Applied and Enviromental Microbiology 61 98 102 4
4. 55 rRNA dH20 5S Positive Control 2 5S rRNA EASY Dilution for Real Time PCR DNA DNA TE tRNA rRNA http www takara bio co jp 3 CY210 II 20 C
5. RNase H RNA 1 Ver 2 5S Positive Control 2 5S rRNA FAM ROX 5S rRNA
6. 1 2 3 PCR PCR Auto PCR Manual http www takara bio co jp 7 CY210 VII 5S Positive Control 2 5S Positive Control 2
7. FAM ROX DNA PCR PCR DNA
8. Serratia marcescens Alcaligenes faecalis Klebsiella pneumonia subsp pneumoniae Staphylococcus epidermidis Achromobacter xylosoxidans subsp denitrificans Streptcoccus pyogenes Pseudomonas fluorescens Pseudomonas aeruginosa Pseudomonas stutzeri Acinetobacter baumannii Acinetobacter lwoffii Myroides odoratus Escherichia coli O157 H7 Salmonella Typhimurium Salmonella Agona Salmonella Enteritidis Salmonella Dublin Salmonella Infantis Salmonella Arizonae Citrobacter freundlii Morganella morganii Staphylococcus aureus enterotoxin A B C D E F G H human genome Vl 1 2 PCR 3
9. Omit http www takara bio cojp 13 CY210 27727 BBRD Negative control N p 5 Control DNA 5 00E 000 5 5 00E 002 Control DNA 500 5 00E 003 Control DNA 5 0000 5 00E 004 Comp sp sono i i NEN 5 Primary Curve FAM gt FE G K ROX NIC 5 00E 001 TC STD STD STD STD STD N FAM lt ROX Co NEMO el RAMOS Cow ee 1723242313811 To SUEDE M MORS YU 34 050 22 00 08 4041 424344 48 http www takara bio coJp 14 CY210 9 FAM
10. http www takara bio co jp 4 CY210 1 Dew see ee e Demen occ FRE Nords rag t eusE ws noo eana F waeg 322 3 wenns r baad E veneta 752 Ca wiosr Lbmwwghamess c worm 4m HO OC G GE DEN NUT RN Fs vos Lorem NIIB0114 L brunensis 441 1 43878 t pu D emm s EEEE O EE mi woe L donaso c oae lmweml Pu wewoe Lamm mm zol _ neos eamm r ws 322 Li woo re r r ss pass s eorde reeeas msE sese ie nevo Lem F nao 7092 Neoewslzeeef r r we 32 ie wiseae 3 c gmwm 36 e 9 niso koena F sm 04 c x wo komn r mm 3292 a neona koen bwmeeon sns 22 winar oe E eeswnp ross O wanwa s s ry P pl rn eran aseo 25 NIIBOO53 L sraelensis Bercovier 4 43119 dmm D C CHERHEE mo sa 29 gole4 names 3 ee 2 39 Mt255 onde 2 t MuhoweB less 3 Weoeg2 ezgpeeiee c ogg 3942 32 eoeg ones 3
11. Excel sample1 sample1 B5 sample2 BB sample2 E C5 sample3 UN C6 sample3 UN D5 sample4 D6 sample4 EE sample5 EB sample5 UNKN OK OK Nega Nega Nega Nega Nega Nega Nega Nega Nega Nega Posi Posi Posi Posi Posi Posi Posi Posi Pos Posi OK OK_ n http www takara bio cojp 26 CY210 3 1 FAM 5S rRNA 1 5S rRNA 1 5S rRNA 5S rRNA ab k3 2 5S Positive Control 2 RoX x omen I _ P 5S 5S 55 rRNA 5 4 3 3 evene C 5SrRNA
12. Thermal Cycler Dice Real Time System http www takara bio co jp 12 CY210 2 PCR FAM ROX FAM ROX G lt Collect Data PCR 3 Step PCR Hold 95 C 10 3 Step PCR 95 5 _55 10 _72 20 X 45 cycles Hold Cycle 1 95 10 3step PCR Cycle 45 95 5 55C 10 72 20 3 L 4
13. 19 3 2 5 s XI Cycleave PCR Legionella 16S rRNA Detection Kit CY240 CY2405S Viable Legionella Selection Kit for LC EMA qPCR 7730 77305 Cycleave RT PCR Cryptosporidium 18S rRNA Detection Kit CY230 Cycleave RT PCR Giardia 18S rRNA Detection Kit CY231 NucleoSpin Tissue XS 740901 10 50 250 Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 0 2 ml 8 strip tube individual Flat Caps NJ600 96well Hi Plate for Real Time NJ400 Sealing Film for Real Time NJ500 Plate Sealing Pads 9090 0 2 ml Hi 8 Tube NJ300 0 2 ml Hi 8 Flat Cap NJ302
14. Ct CP CP CP BeBIsEBDDBEEH p e c Lg o Fn Hw n n nw a 9 9 i LU nm 2 e or K Wy NUN nme n 60 EEEEEEEEEEEEEEEEEEEEEEEEEE samplel i AB sample B5 sample2 B6 sample2 c5 sample3 C6 sample3 D5 sample4 D6 sample4 ES sample5 EB sample5 UNKN http www takara bio co jp 16 CY210 Dip aky ewe A NM ND c 3b DNA 7 cfu cfu 17 cfu E sarmplk UNKN 5 FAM 223 4112E 003 4 D465E 003 E i5enpl
15. ROX c c B ad 5 PC ROX http www takara bio coJjp 23 CY210 U FAM Filter lt ROX 5S FAM ROX Alk PCR Multi Target 45
16. IV 1 2 3 121 20 2 5
17. DNA y 4 41 2 37 1 4 1 cfu 5S DNA 17 x 2 http www takara bio co jp 8 CY210 VIII lt 5S Positive Control 2 gt 1 2 PCR 3 5S Positive Control 2 4 CN 4 ERI git cfu http www takara bio co jp 9 CY210 VII 1
18. ROX U FAM Filter lt ROX 38 55 FAM ROX PCR http www takara bio coJjp 15 CY210 CP 5 55 Positive Control 2 Omit ets Im ut 3904 uen ters E
19. 5S Positive Control 2 PCR i 4 1 1 1 a 20 ul 1 5 ul N 2 1 2 X Cycleave Reaction Mixture 12 5 ul 1x 5S Primer Probe Mix 5 X conc 5 ul 1x 5S Positive Control 2 amp 5 ul Sca ERZK dH20 O 25 ul Total 25 ul 3 2 3 5S Positive Control 2
20. lt gt PCR Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 Thermal Cycler Dice Real Time System Software Applied Biosystems 7500 Fast Real Time PCR System Life Tecnologies 200uk 20uk 10ul DNA gt 47 mm 47 mm 0 22 um Millipore Code GTTP04700 lsopore RA 50m 2 0 ml NucleoSpin Tissue XS 740901 10 50 250 gt 99 56 70
21. Positive Control lt P gt altay F E OK OUT Unknown U Posi Nega ND Internal Contro PCR Error Replicate http www takara bio co jp 25 CY210 CP t CP CP CP DI mm 23 32921 2151332822 22 32 928 bibitur Oo EEE A ppp E BE a h CECEEEEECEEEEEEEEEEEEEEE5 NE 6
22. 2 3 PCR PCR 1 47 mm 47 mm 022 um 0 45 um 2 500 ml 3 50 ml 4 5 ml
23. 5S rRNA DNA ODzeo 1 X 10 copies EASY Dilution for Real Time PCR 5S Positive Control 2 10 EARRA BEI 19 P17 P29 1 P23 34 y 13979Ln x 41 173 R 0 9964 Ct lt Gar 14151Ln x 37 079 R 0 9995 7 7 7 7 7 7 7 o EN k o A e cfu tube 2 PCR DNA PCR DNA M
24. PCR Ct http www takara bio coJjp 24 CY210 170 8 10 cfu 5S Positive Control 2 1 x 104 copies ul 300 5 ul Positive Control Ct Manual 5S Positive Control 2 1 x 104 copies ul 300 5 ul Positive Control Ct 34 FAM Manual 34 RI Negative Control N
25. 1 0 2 ml lt PCR 2 PCR 3 PCR Thermal Cycler Dice Real Time System CycleavePCR Kit http www takara bio co jp 20 CY210 CycleavePCR Kit 1 CycleavePCR Kit OK CycleavePCR Ki v F e Cycle ave PC
26. 1 5S rRNA 1 5S Positive Control 2 3 EASY Dilution OTUFO 1 5 1 5 N 2 1 10 copies ul 5S Positive Control 2 2 10 copies ul 5S Positive Control 2 5 ul EASY Dilution 45 ul 3 10Zcopies u 2 O 103 copies u l ul EASY Dilution 45 ul 4 10 copies ul 3 10 copies u lA 5 ul EASY Dilution 45 ul 5 1copy ul 4 10 copies ul ul EASY Dilution 45 ul X 5 Xx 10 5 X 104 copies 4 2 1 g 20 ul
27. 1 5 2 ml 2ml 6 13 000 15 000 rpm 4 5 950 u x 2 100 ul DNA NucleoSpin Tissue XS 1 60 ul BufferT1 160 ul 2 Proteinase K 16 ll 5 X2 3 56 10 4 Buffer B3 160 5 X2 5 70 5 6 96 10096 160 ul 5 X2 7 NucleoSpin Tissue XS Column Collection Tube 2 m 8 6
28. 11 000 Xg 9 Collection Tube 2 ml 10 Buffer B5 2 50 11 000 X g 1 Collection Tube 11 Buffer B5 2 50 11 000 x g 2 12 1 5 ml 13 Buffer BE 20 11 000 x g 1 DNA 14 5 ul X PCR 1 Proteinase K 740901 50 50 Proteinase K 20 mg 1 vial Proteinase Buffer 1 ml Proteinase K 20 2 BufferB5 Wash Buffer B5 concentrate 2 ml 8 ml http www takara bio co jp 10 CY210 VIII 2
29. 1 5 N 2 1 2 X Cycleave Reaction Mixture 12 5 ul 1X 5S Primer Probe Mix 5 X conc 5 ul 1x 5 ul dH20 O 25 ul 25 ul 3 3 3 2 0 2 ml PCR http www takara bio co jp 11 CY210 VIII 3 PCR
30. Primary Curve FAM ROX Primary Curve PCR 5 copies Primary Curve ROX FAM 5S Primer Probe Mix 5S Positive Control 2 FAM ROX Primary Curve PCR
31. 3T oroa 7 EAM erue B 1 http www takara bio co jp 2 CY210 I 25 ul 50 1 2 x Cycleave Reaction Mixture 2 X conc 625 ul 2 5S Primer Probe Mix FAM ROX 5 X conc 250 ul O 3 dH20 1ml 4 5S Positive Control 2 2 1 x 10 copies ul 100 ul Q 5 EASY Dilution for Real Time PCR 1mlx2 1 2 PCR 1 3 PCR 1 3 2 X Cycleave Reaction Mixture PCR Buffer dNTP Mixture 5S Primer Probe Mix
32. cfu 3 PCR Z Cl amp Thermal Cycler Dice Real Time System amp Lite Applied Biosystems 7500 Fast Real Time PCR System Life Tecnologies 18 Thermal Cycler Dice Real Time System 1 0 V OK Y VERB lt gt M X Thermal Cycler Dice Real Time System Software lt Absolute Quantification gt
33. 5S 3 1 5S rRNA 2 Ac s 3 PCR 4 55 55 5S 5 http www takara bio co jp 27 CY210 IX FAM
34. R Kit SNPs CycleavePCR Kit Thermal CyclerDice Real Time System Software PM M Plus Minus Assay gt Thermal CyclerDice Real Time System 2 FAM ROX PCR Hold Cycle 1 95 10 3step PCR Cycle 45 95 5 55C 10 72 20 3 http www takara bio cojp 21 CY210 4 E 005 X
35. a il ila ila L pneumophila subs NIIBO304 5 E i L pneumophila subs NIB0050 P ohila j la ila la LC870 BAA 538 LLAP 6 700991 N NIIBO196 L quateirensis N E WA 270A C2 35304 1 L quinlivanii 2 L rowbothamii L sainthelensi L santicrucis L shakespearei L spiritensis 2 L spiritensis NIIBO305 Legionella genomospecies 1 NIIBOO39 L sainthelensi 5 5 5 5 5 6 6 6 6 6 6 C 7 C C C E E C E C E C C C C C C E E E E E E E E C E E E E E E E E E 4 5 6 57 8 9 1 2 3 4 5 7 70 71 72 3 74 NIIBO451 L pneumophila untypable E Hi3 192 http wwwr takara bio coJp 6 CY210 2 10cfu tube DNA 10 cfu tube DNA 100 cfu tube DNA 10000cfu tube DNA 19
36. a h oj p w L xD S E XG p p lp ER HU Internal Control ROX Omit 2 5 http www takara bio co jp 22 CY210 FAM ROX Elk Auto N FAM lt ROX gt TSE NC ROX P FAM
37. c ei 3 mmus r macan t Pee 3 w wmooe Lmetde c mno sms 5 mmonelrmoma e 363 4382 De weows Lmumm t 24 9 7 Neooe r oads t omo 3w8 ET t pc 3529 i HH Hf http wwwr takara bio coJp 5 CY210 A L pneumophila subsp i NIIB0001 22uyoo P Philadelphia 1 33152 L pneumophila subs L pneumophila subs NIIB0003 7euyoo P Bloomington 2 33155 NIIB0004 L pneumophila subsp fraseri NIIBOOO5 L pneumophila subsp fraseri Los Angeles 1 33156 L pneumophila subs NIIBO15 ETE IN 23 G1 C2 35289 797 PA H 4 2 4 4 4 4 L pneumophila subs 45 NIIBOO06 pleumophi p 46 NIIB0033 L pneumophila 7 4 i 4 4 i 0 L pneumophila subs NIIBOO34 OO P 0 1 L pneumophila subsp NIIB0051 L pneumop L pneumophila subsp NIB0060 Fed P L pneumophila subsp NIIB0061 pem pa SUDSP 1 570 CO H 82A3105 ne 4 39 0 1 2 3 4 5 6 7 8 9 50 5 52 1 CO w NINJ gt CO O w O o o FEE EE pP EE EE 9 1 1 L pneumophila subs NIB0062 460moo P NIIB0063 L pneumophila subsp fraseri 1 2 3 4 5 LU Un N Un anms 3525 untypable 3703 IB0453 L pneumophila Hi32300 NIIBO462 L pneumophila H329 1335 9507 248 02 untypable il
38. e5 UNKN 5 FAM 3002 3898 E 003 4 O45E 003 sample 5 5S Positive Contorol 2 2 4 046 x 10 2 4 x 102cfu H5 rarmpleG UNKN 8 FAM 18 68 4283Er007 4 208E 007 H samples UNKN 8 FAM 18 05 4333E 007 4 28BE4007 sample 8 5S Positive Contorol 2 2 4298 x 107 2 5 x 106cfu http www takara bio coJp 17 CY210 Applied Biosystems 7500 Fast Real Time PCR System Life Technologies 1 Advanced Setup New Experiment 2 Experiment Properties C Quantification Standard Curve TaqMan Re agents Other Other Include Melt Curve Plate Setup Define Target Target Name 5S Reporter FAM Quencher none 4 Plate Setup Define Target Target Name IC Reporter ROX Quenche
39. l RO 10 Standard Curve 11 View Well Table Seed aie vam edton c poor wm StepOnePlus Real Time PCR System Life Technologies ROX target ROX IC FAM ROX target http www takara bio co jp 19 CY210 lt gt 1 10 2 PCR 3 l
40. r none 5 Define Samples CC NC PC 6 3 A 5 Plate Layout Passive Reference none 3 7 Instrument Reaction Vokas Par Wel uL psdMode Hold hioldng Staga CInd Stags Cycle 1 te 95 C 10 sto ox E73 3 step PCR Cycle 45 KT NN 95 5 55 10 72 25 Step t Ste 1 Sue 2 Sun 3 8 Start 9 Analysis Amplification Plot Target Threshold Baseline Manual Amplification Plot on 200 1 08 000 ce ceo w teo zs 000 oe 006 ee oco WOe amp samo 4m roo x goo SO xx Doo 204 000 LI 160 006 ss 000 tameo gt RN same 1 5S rRNA FAM http www takara bio co jp 18 CY210 Amplification Plot amp 9 3 4 4 S 4 53 5 B 8 W 4 4 Cn Internal Contro
41. usz r CY210 Tahaha Cycleave PCR Legionella 5S rRNA Detection Kit Ver 2 0 v201406Da eg o7e 8 pneumophila 55 e gionella pneumophila 21 4 3 PCR PCR PCR
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