Cycleave®PCR 呼吸器系感染症起因菌検出キット Ver.2
Contents
1. http www takara bio coJp 4 CY214 V V 1 DNA 2 EXTRAGEN II DNA EXTRAGEN II 1 0 5 ml 5 000 rpm 5 2 100 ul 8 ul 1 1 5 ml 10 12 000 rpm 3 2 2 500 10 4 12 000 rpm 3 3 2 200 ul 10 6 12 000 rpm 32. 7 RNase free dH2O 40 DNA RNA RNase free dH2O 3 5 V 2 1 1 6 DNA o 24 1 1 Il Probe Primer Mix 1 Probe Primer Mix 4 2 1 Probe Primer Mix 5 Probe Primer Mix 6 3
3. PCR hp Ver 2 Positive Control RNA DNA 1 DNA TTT 2 1 1 3 RNase H h 4 1 TT 5 i ee 1 http www takara bio coJp 2 CY214 25 MI X 10 X 6 1 2XCycleavePCR Reaction Mixture 750 60 2 Probe Primer Mix 1 2 20 ul 10 3 Probe Primer Mix 2 2 20 ul 10 4 Probe Primer Mix 3 2 20 10
4. Positive Control Probe Primer Mix 1 OECD LytA 319 bp 205 bp i Probe Primer Mix 2 OS 16S rRNA 167 bp 110 bp a Probe Primer Mix 3 CO 16S rRNA 225 bp 185 bp Probe Primer Mix 4 OO DO 16S rRNA 248 bp 165 bp Probe Primer Mix 5 Legionella pneumophila 16S rRNA 387 bp 274 bp Probe Primer Mix 6 A 16 TRNA 0 230 bp Streptococcus pyogenes LO 264 bp 187 bp http www takara bio co jp 11 CY214 VI Thermal Cycler Dice Real Time System 5 pyogenes B CZ7 SpyoB UNKN SpyoBLO ROX D7 Spye UNKN Spye168 FEAM D7 SpyoeC UNKN Spyo8LO ROX 2849 28 43 E77 SpyeG UNKN Smye168 EAM E7 BSpyoG UNKN SpyoBLO ROX 26 72 26 50 2 A B C G http www takara bio coJp 12 CY214 VII 1
5. http www takara bio coJp 8 CY214 VIII Morozumi M E Nakayama 5 Iwata Y Aoki K Hasegawa R Kobayashi N Chiba 1 Tajima and K Ubukata 2006 Simultaneous detection of pathogens in clinical samples from patients with community acduired pneumonia by realtime PCR with pathogen specific molecular beacon probes Clin Microbiol 44 1440 1446 IX Cycleave PCR Ver 2 CY216 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 X
6. 2 1 2 x CycleavePCR Reaction Mixture 12 5 1 Xx ProDbe Primer Mixture 2 DNA 2 Positive Control lt 1 ul 9 5 ul Total 25 ul 1 DNA 1 lt 5 uu 1 ul 25 ul 2 DNA Positive Control 2 http www takara bio coJp 5 CY214 2 DNA 3 DNA Positive Control 1 0 2 ml PCR Probe Primer Mix 5 6 2 Positive Control Positive Control FAM Positive Control
7. Sample Type UNKN Posi Nega Error Filter FAM LF ki hdre PG 4 35 Pesi Posi FU lg hg PU FE g A FE lg L1BB FM LT LI EL MG L164 HC A QR nK nR LIFM Td L 15 lr FC RU TE 7 t Pi t Fosi Fm FU L 1 klip FU UI I WT lll ll FC L 15 L15 FC ER TE 1 t UE t DE UF Positive Control 5 FAM Sample Name Mip PC Filter FAM Result OK Filter ROX Result OUT Positive Control 5 ROX Sample Name L 16S PC Filter FAM Result OUT Filter ROX Result OK Detected F Primary Curve Final Filter Detected CP Primary Curve Ct Filter Detected SDM 2nd Derivative Ct Filter P M Result F Primary Curve Final
8. 1 DNA 2 PCR Positive Control PCR 2 Thermal Cycler Dice Real Time System primary curve Ct SDM Primary curve Ct SDM 3 33 3 8 WW IHS 4 181W Wa Ciela
9. Positive Control 2 lt 8 9 16 20 Il EXTRAGEN II 1 200 20 10 2 PCR Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 1 0 2 ml 8 0 2 ml 8 strip tube individual Flat caps NJ600 Applied Biosystems StepOnePlus RealTime PCR System Applied Biosystems 7500 Fast Real Time PCR System Life Technologies Baseline Auto Threshold Manual StepOnePlus 7500 Fast Real T
10. NC Negative Control 2 Name Sample Name 3 Color 4 3 Target List Sample List Update http www takara bio coJp 7 CY214 Plate Image Filter FAM FAM Target Filter ROX ROX Target 1 Plate Image Plate Image Editor Target 2 Plate Image Plate Image Editor Sample Target amp Sampl Swng Pumaps mm OE eles x Pchmirmm r em FeehanFrier sy EE ebhFrirmmf Omit http www takara bio coJp 8 CY214 5 Result Analysis NC PC 5 1 NC
11. CY21 4 IdRaRa Cycleave PCR Ver 2 Ver 2 v201406Da 6 A PCR
12. Hot Start PCR TaKaRa Ex HS RNA DNA RNase H CC i RNase H RNA 1
13. 5 Probe Primer Mix 4 2 20 ul 10 6 Probe Primer Mix 5 2 20 ul 10 7 Probe Primer Mix 6 lt 20 ul 10 8 dH20 750 ul 9 Positive Control 1 FAM 10 ul 10 Positive Control 2 FAM 10 ul 11 Positive Control 3 FAM 10 ul 12 Positive Control 4 FAM 10 ul 13 Positive Control 5 FAM 10 14 Positive Control 5 ROX 10 ul 15 Positive Control 6 FAM 10 ul 16 Positive Control 6 ROX 10 ul 1 dNTP Mixture vortex 2 FAM ROX a lt ODN Streptococcus pneumoniae MM _ TN Haemophilusinfuenzae I Mycoplasma pneumoniae OM a Probe Primer Mix 4 Co HO 16S rRNA 2 2 Probe Primer Mix 5 i 16S rRNA 1A i gt 2 Probe Primer Mix 6 eB 16S rRNA 57O 1 Haemophilus influenzae
14. Cycleave TakKaRa Ex 729 Thermal Cycler Dice http www takara bio coJp 14 CY214 http www takara bio coJp 15 CY214 sm Iaf4R4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJjp v201406Da
15. Selector N gt Filter FAM Filter ROX Filter FAM Filter ROX 5 2 PC Selector P gt Positive Control FAM Filter FAM Filter ROX Filter FAM Filter ROX Positive Control ROX Filter FAM Filter ROX Filter ROX Filter FAM 5 3 Selector U gt http www takara bio coJp 9 CY214 6 Text Plate Format Scatter Plot 6 1 Analysis Data lt Text Report gt 6 2 Column Result Sample Type NC PC OK OUT
16. P M Result CP Primary Curve Ct P M Result SDM 2nd Derivative Ct http www takara bio coJp 10 CY214 V 3 40 PCR Thermal Cycler Dice Real Time System Result Analysis Analysis Data Text Report 2nd Derivative Ct Ct SDM Ct 33 PCR
17. 16S rRNA 2 2 L pneumophila Legionella 5 pyoge7ey EE pneumophila Legionella FAX ROX ROX 5 pyogenes 8 B FAX ROX ROX http www takara bio coJp 9 CY214 0 1 4
18. i a mp FTF Hold Cycle 1 k 95C 10sec 3 step PCR Hold Cycle 40 95C 5sec 55C 15sec 72 C 20 sec collect data els Te rs te kar del Hp Ti terri _ mm et http www takara bio coJp 6 CY214 3 Start Run 4 Plate Setup Target amp Sample Settind Target List Sample List 4 1 larget List Dye Dye of Internal Control none Target List Probe Primer Mix 1 Add Target List Delete 2 Dye 3 Name Target Name 4 Color 4 2 Sample List NC PC 1 Type Type UNKN Unknown PC Positive Control
19. ROX 2 1 3 PCR 3 Thermal Cycler Dice Real Time System Thermal Cycler Dice Real Time System Software Thermal Cycler Dice Real Time System 1 Run File New Experiment Options Experiment Type PM M Plus Minus Assay Multiplex gt gt Few Exherirmgnt ntinrms Exnerlrmgnt Thg alE Experiment gal Single 1 hultiplesx Aa a Gingle solute Luantitication aathd hdultiplesx Ra Single Rielatree Guahntitication Ra hdltiples Single Plus JImus ssay 3 hdltiplesx Fnntyhin kultinla 2 Thermal Profile Setup PCR Probe Primer Mix 5 Probe Primer Mix 6 2 FAM ROX Collect Data FAM ROX EL
20. ime PCR System Threshold IV 1 PCR 2 3 3 1 2 3
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