PCR Kit
Contents
1. 0 8 0 6 0 4 Fluorescency N 2 8 14 20 26 32 38 44 50 Cycle number 0 6 0 2 Fluorescency A o e 2 8 14 20 26 32 38 44 50 Cycle number Fluorescency Sees oN A A OF 2 8 14 20 26 32 38 44 50 Cycle number Fluorescency Fluorescency Fluorescency Fluorescency Fluorescency Fluorescency Detector CY 5 4 3 2 1 0 2 8 14 20 26 32 38 44 50 Cycle number 4 3 2 1 0 2 8 14 20 26 32 38 44 50 Cycle number 1 0 8 0 6 0 4 0 2 0 2 8 14 20 26 3238 44 50 Cycle number 4 3 2 1 0 EE HH RR RN RG BG RG ERTE OG GN 2 8 14 20 26 32 38 44 50 Cycle number 1 0 8 0 6 0 4 0 2 0 2 8 14 20 26 32 38 44 50 Cycle number 1 0 8 0 6 0 4 0 2 2 8 14 20 26 32 38 44 50 Cycle number Fluorescency e N Q RA Fluorescency Fluorescency e N o 4A 1 1 1 1 1 Fluorescency cO e N UC n Fluorescency e N oC A Fluorescency Detector JOE 2 8 14 20 26 32 38 44 50 Cycle number 0 8 0 6 0 4 0 2 2 8 14 20 26 32 38 44 50 Cycle number 2 8 14 20 26 32 38 44 50 Cycle number 2 8 14 20 26 32 38 44 50 Cycle number 2 8 14 20 26 32 38 44 50 Cycle number 0 8 0 6 0 4 0 2 2 8 14 20 26 32 38 44 50 Cycle number Result Positive Positive Positive Positive Negative Non valid result Use the CY 5 detector only with the GeneProof Herpes Simplex Virus HSV PCR Kit mm See Detec2. 02 10 Z GURU HBV Test 03 12 2009 M SDS Documents sds Stage 3 Step 2 60 0 0 40 12 Qualitative analysis of the detection results PCR detection result evaluation must be always performed qualitatively first if you use the PCR kit for quantitative assessment continue to quantify positive samples in the second step 1 When the program is finished switch to the Results tab and select the Amplification Plot tab Dats Delta Rn vs Cycle Detector 41 Line Color Detector Color 102 002 i Trneshokt 0 0021516 C Auto Baseline Manual Bateline Start cycle f Erd cycte fis Anelyer Help Delta Rn 102 005 1 0e 006 1 0e 007 123 4 5 6 7 8 8 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number 13 Detection analysis of the studied microorganism 1 Select the Delta Rn vs Cycle display in the Data field 2 Enter FAM GeneProof or CY 5 GeneProof in the Detector field CY 5 detector is used only with tbe GeneProof Herpes Simplex Virus HSV PCR Kit where the fluorophor FAM signal presence indicates HSV 1 positivity and tbe fluorophor CY 5 signal presence indicates an HSV 2 positive sample 3 Enter Manual Ct and Manual Baseline in the Analysis Settings section The straight line depicting the baseline alignment will be
3. E IURE SDS Templates sdt 5 __Stomno 10 PCR Amplification Start When using the GeneProof PCR kits for the first time it is necessary to program the detectors and the amplification profile and save the program as a template See chapter Device Programming The software will remember the saved settings for subsequent GeneProof PCR kit uses It is not necessary to program the detectors and the amplification profile again Click the 7500 System Software icon to start the program Open the New Document Wizard tab in the main window Enter Absolute Quantification Standard Curve in the Assay field Click Browse and select GeneProof PCR sdt SDS Templates Click Finish to continue 7500 System SDS Software So 9E New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Absolute Quantification Standard Curve 96 well Clear GeneProof PCR sdt 6 Open Well Inspector in the Setup Plate window and enter the description of the inserted samples a 7500 System SDS Software Plate Absolute Quantification Unknown Unknown none Unknown 11 7 Select File in the main menu click Save As and save the created protocol under your own name as an SDS Documents sds file Pecha 3 d Dokumenty E BORRELIA 07 12 2009 _ E BORRELIA 08 12 2009 BORRELIA 03
4. GeneProof in the Name row Enter Positive signal in the Description row Enter CY 5 in the Reporter Dye row Enter None in the Quencher Dye row Press OK in the New Detector window to save these settings Oo RON Creation of a detector for reading the Internal Standard JOE fluoropbor signal 1 Press the New Detector button in the New Document Wizard Select detectors window 2 Enter JOE GeneProof in the Name row Enter Internal standard in the Description row 4 Enter JOE into the Reporter Dye row and then enter None into the Quencher Dye row Press OK in the New Detector window to save these settings p New Document Wizard Select Detectors Select the detectors you TET BOT tte tra JOE GeneProof I Detecdor Mame Internal Standard CY 5 GeneProof FAM GeneProof Inserting detectors into a protocol 1 Highlight the FAM GeneProof CY 5 GeneProof and JOE GeneProof detectors and then click Add to add them into the Detectors in Document table 2 Check the ROX selection in the Passive Reference field 3 Press Next to continue programming Use the CY 5 detector only with the GeneProof Herpes Simplex Virus HSV PCR Kit New Document Wizard Select Detectors Select the detectors you will be using in the document CY 5 GeneProof Positive signal CY 5 none FAM GeneProof FAM GeneProot Positive signal JOE GeneProof JOE GeneProot Internal Standa CY 5 GeneProof Filling out basic sample par
5. Problem resolution 1 Check whether MasterMix storage is in harmony with manufacturet s recommendations 2 Submultiple the MasterMix and do not freeze and thaw it 20 If you have any questions please contact our Product Support Department at support geneproof cz Notes Manufacturer GeneProof a s Vini n 235 615 00 Brno Czech Republic Phone Fax 420 543 211 679 e mail office geneproof cz www geneproof cz Issue date 17 2 2010 21
6. seneProof PCR Kit This manual is designed for the following kits Mycobacterium tuberculosis PCR Kit Mycoplasma pneumoniae PCR Kit Legionella pneumophila PCR Kit Chlamydia pneumoniae PCR Kit Chlamydia trachomatis PCR Kit Neisseria gonorrhoeae PCR Kit Borrelia burgdorferi PCR Kit Cytomegalovirus CMV PCR Kit Epstein Barr Virus EBV PCR Kit Herpes Simplex Virus HSV PCR Kit Herpes Simplex Virus 1 HISV 1 PCR Kit Herpes SimplexV virus 2 HSV Z PCR Kit V aricella Zoster Virus VZV PCR Kit Hepatitis B Virus HBV PCR Kit C in vitro Diagnostics User manual for use with the following device Applied Biosystems ABI 7500 Real Time PCR system VERSION K1 02 EN Contents GENEPROOF PCR IKDU iiiiretceetadeas ei eeeueeo eodeni Re be Ee E HEY SEO E ath taken TEE gelbe ea eon Eo cE e ead aa EE EET EE zv ede Eee Rueda dada 3 ISIN AND ISEX GENEPROOF PCR KIT VERSIONS ccccccccccccccccccccccccccccccccccccccccccccccccccccccccees 4 PCR REACTION PREPARATION uec pea ecsenzetesee voteuexedueUs sos Fes fU va eoa coUa gura eu ee Du eiua ees Peck eoe ecu oe viue ore eet eo pape sca 5 DEVICE PROGRAMMINQG 5 i eIieiszcsitcee es eos eee cene sue eos estas cons Gg e oS eot eee eue sue aus eove se voa deo osa eoe Cue Uos eco e ces esu ege Doe e oou con oae uU 5 SEM LMG SO ALC 02521900 ehi ia tdcctaa ioo aestatis a n osea oO esae azote da ctos Ai 5 BIS Se Ke ai eo as Ni NNT ey NITE mum 6 Amplification Profile Prog
7. ameters 1 Select the number of the inserted samples in the Set Up Sample Plate window 2 For selected samples select the FAM GeneProof CY 5 GeneProof and JOE GeneProof detector checkboxes 3 Click Finish to continue Select the CY 5 detector checkbox only with tbe GeneProof Herpes Simplex Virus HSV PCR Kit New Document Wizard Ed Set Up Sample Plate Setup the sample plate with tasks quantities and detectors T FAM GeneProot M JOE GeneProot JOE none Unknown T e 5 GeneProof zp cos _Dokon it Stono Amplification Profile Programming 1 2 2 4 Open the Thermal Profile tab in the Thermal Cycler Protocol screen Program the individual steps as follows Stage 1 Reps 1 Step 37 C 2 00 min Stage 2 Reps 1 Step 95 C 10 00 min Stage 3 Reps 45 Step 1 95 C 0 05 sec Step 2 60 C 0 40 sec Step 3 72 C 0 20 sec In the Settings section of the protocol enter Sample Volume uL 40 Select the 9600 Emulation checkbox In the Data Collection row enter Stage 3 Step 2 60 0 0 40 Ri 7500 System SDS Software Platel Absolute Quantification Stage 3 Step 2 60 0 0 40 gt saving the GeneProof PCR template i Select File in the main menu click Save As and save the created protocol under the name GeneProof PCR as an SDS Templates sdt file 500 System SDS Software Platel Absolute Quantification GeneProof PCR
8. andard detection is marked by the JOE fluorophor detected by the B filter 550 nm GeneProof PCR kits gt Use the hot start technology minimizing non specific reactions and assuring maximum sensitivity gt Contain uracil DNA glycosylase UDG eliminating possible contamination of the PCR reaction by amplification products gt All PCR kits for pathogen DNA detection can be amplified by means of a universal amplification program gt Easy to use the kits always contain one tube with MasterMix and one tube with Positive Control or with an Internal Standard or a set of Calibration Controls Designed for zz vitro diagnostics CE IVD certification ISIN and ISEX GeneProof PCR Kit Versions All GeneProof PCR kits include an Internal Standard providing for an effective monitoring of eventual inhibition of the PCR amplification and also of the isolation process efficiency The Internal Standard is a precisely defined and quantified construct of a plasmid and insert prepared by genetic engineering methods GeneProof develops and sells two basic variants of PCR kits which differ in the Internal Standard composition PCR kit ISIN Cat No PCR kit ISIN In this version of the PCR kit the Internal Standard is included in the MasterMix tube This kit version can be used to monitor the PCR reaction inhibition PCR kit ISEX Cat No PCR kit ISEX In this PCR kit version the Internal Standard is included in a separat
9. e tube within the package This PCR kit version enables pathogen amplification and detection with optional PCR inhibition control and with parallel DNA isolation process efficiency control When using PCR kits containing the Internal Standard as an independent package item the Internal Standard should be added at the beginning of the isolation process as follows 0 1 ul of the Internal Standard per 1 ul of the elution volume Elution Volume 25 ul 50 ul 100 ul 200 ul PCR kit ISIN PCR kit ISEX Y Y Jt Internal Standard 0 1ul IS per Tul of elution volume C purified DNA gt 30 ul MasterMix 4n INNTA PCR amplification i JOE JOE PCR inhibition control PCR inhibition and 4 DNA isolation efficiency control PCR Reaction Preparation 1 Add 30 pl of the MasterMix and 10 ul of the DNA isolate or 10 ul of the Positive Control into a PCR tube The final reaction mix volume should be 40 ul 2 Close the tubes centrifuge shortly insert into the device and start the PCR test see chapter PCR Amplification Start Device Programming When using the GeneProof PCR kits for the first time it is necessary to program the detectors and the amplification profile and save the program as a template During subsequent uses of the GeneProof PCR kits start from the PCR Amplification Start chapter The software remembers the saved settings It is not necessary to program the detectors and the amplification profile again Starting
10. l Negative control 1 00e 004 1003 005 1 00e4002 Unde Undet Undet Unet i uncos Ln Select the Plate tab in the Results tab and fill in parameters for Negative Control Enter Negative Control in the Sample Name row and enter NTC in the Task field T e SS Sa A samples samples samples samples amples 813e 003 5 66e 003 pe S3e003 247e 003 251e 003 S ge 003 Unde 1j Undet 8 Undet Undet Unda ij unde Wells F5 B samples samples sages Samples samples samples 6 73er002 HE 5 amp 0e 002 13e 002 2 4461002 3020 002 2 8 e002 Sample Name Negative cortrol Unda B Uncet Undel Undet Undat Bj Unde samples samples samples ganples samples samples M i 26 002 1 358002 1 2e 002 4 amp Se 001 2 38e 001 M 2802 001 M unea Undet Undet Undet Undet luce D angles samples Samples 345e 001 3 07e 001 3 206 001 Under Undet Undet i i i E somples samples samples samples samples semples samples samples 9378 003 7228 002 9 16001 8 59e 000 1 02e 004 1 0032 003 8 766 00 8 44e 000 Uncdet Undet Undet Undet Under aM unde Undet Undet T Ki x2 K3 1 00e 004 1003 003 1 006 002 Uncles Undet Undet 16 For quantitative evaluation of the positive signal presence in the detected microorganism channel FAM or CY 5 select the Delta Rn vs Cycle display in the Data field enter FAM GeneProof or CY 5 GeneProof i
11. n harmony with manufacturer s recommendations 2 Submultiple the Positive control and do not freeze and thaw it Invalid result of a Negative Control analvsis Fluorescency oF N Q RA e FAM CY 5 JOE Result 1 0 8 0 6 0 4 0 2 0 Fluorescency e N 55 A Fluorescency 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 Cycle number Cycle number Cycle number Problem PCR reaction contamination Problem resolution 1 Check the process of preparation and pipetting of the PCR mix into tubes 2 Check the handling of sterile plastics and filtered tips 3 Clean the PCR box 4 Ad uracil DNA glycosylase UDG into the reaction 19 Invalid result of an Unknown Sample analvsis Fluorescency FAM CY 5 JOE Result nN Fluorescency nN U Fluorescency o cR 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 Be Te A oit Mon Cycle number Cycle number Cycle number Problem PCR reaction inhibition PCR kit ISIN and ISEX Problem resolution 1 Repeat DNA isolation PA Check the process of preparation and pipetting of the PCR mix into tubes Problem Invalid process of DNA zsolation PCR kit ISEX Problem resolution 3 Repeat DNA isolation 4 Check the process of preparation and pipetting of the Internal Standard at the beginning of the isolation process Problem Incorrect storage of the MasterMix see Storage and transportation conditions
12. n the Detector field and enter Manual Ct and Manual Baseline in the Analysis Settings section The straight line depicting the baseline alignment will be red Xx 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number Move the line immediately above the basal noise of the reaction Press the Analysis button in the Analysis Settings tab to start the evaluation The straight line representing the baseline will turn green in case of the successful analysis performance 123 4 5 6 7 8 9 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number 17 5 Evaluate the calibration quality Display Standard Curve in the Result tab The R2 parameter in a well performed calibration achieves a minimum value of 0 98 or higher If the R2 parameter 1s lower then 0 98 move the baseline and repeat the analysis Only concentrations in the range specified by the calibration cutve may be measured for a quantitative evaluation of the results Quantification of samples where concentration exceeds the uppe
13. r measuring threshold determined by the calibration curve range a calibrator with the highest concentration is of reference value only You can dilute these samples and repeat the assessment to achieve a precise quantification samples with lower concentrations then the lowest concentrated calibrator can be quantified approximately only The following formula can be used to convert sample concentrations to units ml taking into account the isolation procedure Concentration ml cVZx EO sample concentration in units ul selected elution volume in ul material volume used for isolation in ml 6 Select the Report tab in the Results window to display the analysis results Numerical analysis results for the inserted samples will be displayed in this tab 18 Troubleshooting Invalid result of a Positive Control analvsis Fluorescency 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 2 8 14 20 26 32 38 44 50 FAM CY 5 JOE Result 1 0 8 0 6 0 4 0 2 0 Fluorescency Fluorescency N W U Cycle number Cycle number Cycle number Problem Incorrect programming of the PCR amplification Problem resolution T Check device programming according to the manual 2 Check correct temperature settings in the individual program blocks Problem Positive control incorrectly held in storage see Storage and transportation conditions Problem resolution 1 Check whether kit component storage is i
14. ramming sessssseeeeeeeeeeeeeeeeeee eene nene nnn nnn hn nnne esee sss s sns nn enn EEE TANTE 9 Saving the GeneProoE PCR template ee eerte ferie e ipe ut essa ortae tete qve REEE 10 PCRAAMPEIFICATION STAR d iiiiceeesces ovhbeiseceesctue ev edetoe cevauc deve bekaacoezesV eee Sese ae vev eos aeubelbecvexesveoveiebeaeevoee secos 11 QUALITATIVE ANALYSIS OF THE DETECTION RESULTS eee ee e eere ee eee e o eee een eee e ene e eee onoo 13 Detection analysis of the studied microorganism eesssssssssesesseeee nennen eene en esse nnne nnn nnn n nennen ens 14 Internal Standard CeteculOm Analysts 4 i oem ds dsoo a is asda inet ipid NDS id Lone testi iude ai MAL Do eS dod 14 Qualitative GeteChion CV alba OM ene a a E a 15 QUANTITATIVE ANALYSIS OF THE DETECTION RESULTS eesseeccseccccecccsscecsccccsccecoscecsscecsseecsseecscee 16 TROUBLES HOG DUNG Cem 19 MN HB ONERE LE LUCES 21 GeneProof PCR kit GeneProof PCR kits designed for the detection and quantification of pathogen DNA are based on the principle of amplifying specific target sequences of microorganisms and measuring the amplification product concentration growth in the course of the polymerase chain reaction by means of fluorescence marked probes the probe designated for pathogen detection is marked by the FAM detected by the E filter 650 nm or Cy5 detected by the E filter 650 nm fluorophor and the probe designated for the internal st
15. red 4 Move the line immediately above the basal noise of the reaction oF Press the Analysis button in the Analysis Settings tab to start the evaluation The straight line representing the baseline will turn green in case of the successful analysis performance ication Plat X Standard Curve Y Cissccietion y Report X 12345267 8 9 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number Internal Standard detection analysis Select the Delta Rn vs Cycle display in the Data field ds 2 Enter JOE GeneProof in the Detector field 3 Enter Manual Ct and Manual Baseline in the Analysis Settings section The straight line depicting the baseline alignment will be red Move the line immediately above the basal noise of the reaction oF Press the Analysis button in the Analysis Settings tab to start the evaluation The straight line representing the baseline will turn green in case of the successful analysis performance 0 02 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number 14 Qualitative detection evaluation Detector FAM Fluorescency m N Ow A 2 8 14 20 26 32 38 44 50 Cycle number Fluorescency N W 2 8 14 20 26 32 38 44 50 Cycle number Fluorescency e N oO A 2 8 14 20 26 32 38 44 50 Cycle number
16. the Software Click the 7500 System Software icon to start the program Open the New Document Wizard tab in the main window Enter Absolute Quantification Standard Curve in the Assay field Click Next to continue x Eom Hew Document Wizard Define Document Select the assay container and template Far the document and enter the operator name and comments Absolute Quantification Standard Curve 96 Well Clear Blank Document Detector Programming Creation of a detector for reading the detected microorganism FAM uoropbor positive sienal Press the New Detector button in the New Document Wizard Select detectors window Enter FAM GeneProof in the Name row Enter Positive signal in the Description row Enter FAM in the Reporter Dye row Enter None in the Quencher Dye row Press OK in the New Detector window to save these settings Ar RON New Document Wizard x Select Detectors Name Fam GeneProof Find Description Positive signal Detector Name De Reporter Dye MM o Quencher Dye fehl Color m Notes 4 New Detector Create Another Cancel lt Zp t Dal gt Dokon it Stormo Creation of a detector for reading the detected microorganism CY 5 fluorophor positive signal Use only with tbe GeneProof Herpes Simplex Virus HSV PCR Kit Press the New Detector button in the New Document Wizard Select detectors window Enter CY 5
17. tion Troubleshooting page 19 Select the Report tab in the Results window to display the analysis results Numerical values Ct of the analysis results for the inserted samples will be displayed in this tab 15 Quantitative analysis of the detection results Quantitative analysis sbould be performed for samples evaluated as positive in the course of tbe qualitative analysis procedure E Select the Plate tab in the Results tab and fill in parameters for Calibration Controls Always select Standard in the Task field and enter the corresponding calibrator concentration in the Quantity field psam Y rete Yresuns Y A samples samples samples samples samples 9130003 5658 003 3 961003 2 474003 2516 003 ed 003 Undet Undet Undet Undet Unda M unde Wells F4 B samples samples samples samples Saenples samples 6 73e 002 6 60e 002 13e 002 Il 24401002 302e 002 285e 002 Semple Name e Unda Undet Undet E Urdei Urdal MI Unox samples Sart nes samples sampilas ples I i 22 002 f 1 56 002 123e 002 i 4 65e 001 9 238e 001 M 2202 00 M unce I uncet Undet goose gos M uno D samples samples samoes 3 45e 001 T 3 07e 00 3 20e 001 Unet Jj Uncet Undet E sempies sam samples samples semples samples samples samples 9378 003 7 22e 002 3 188001 3 59e 000 1 02e 004 1 03e 003 8 764 00 8 44e 000 Unciet Undet Undet Undet Undet Uncle Undet Undet K1 Negative contro
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