RealArt™ M. tuberculosis TM PCR Kit
Contents
1. Browse Barcode 2 OK Cancel Fig 16 Pre settings for creating a new file New Document 2 25 RealArt M tuberculosis TM PCR Kit 100 20000 8 5 3 2 Creating Selecting the Detectors With the help of the submenu Detector Manager from the Tools menu alternatively Setup level Add Detector select function assign the corresponding detector dyes to the file For the detection of M tuberculosis complex DNA as well as the Internal Control by means of the RealArt M tuberculosis TM PCR Kit the reporters quenchers listed in the following table are to be defined Detection Reporter Quencher M tuberculosis complex DNA Non Fluorescent Internal Control JOE M tuberculosis TM IC To create these detectors select the option New bottom left of the Detector Manager Name artus M tub Name artus M tub IC Description RealArt M tub TM PCR Kit Description RealArt M tub TM PCR Kit 1 Reporter FAM Reporter JOE v Quencher Non Fluorescent Quencher TAMRA Color EM Color sd cancel Cancel Fig 17 Creating the WM tuberculosis Fig 18 Creating the IC specific detector complex specific detector Detector Manager Detector Manager For the detection of M tuberculosis complex DNA define the reporter quencher combination FAM Non Fluorescent in the now appearing window For the detection of the nternal Control select the combination JOE TAMRA as shown in Fig 17 a2. Sample 10 ul each PCR assay Pipet 15 ul of the Master Mix into each required reaction tube or well of the 96 well reaction plate Subsequently add 10 ul of the eluate from the DNA isolation Mix the solution well by repeated up and down pipetting Close the reaction tubes with the corresponding caps or alternatively when using a 96 well reaction plate with an optical adhesive cover optical adhesive covers Applied Biosystems Centrifuge the reaction tubes in a storage rack for PCR The volume increase caused by adding the IC is neglected when preparing the PCR assay The sensitivity of the detection system is not impaired RealArt M tuberculosis TM PCR Kit 000 00000 tubes or the 96 well reaction plate in a centrifuge with a microtitre plate rotor for 30 seconds at 1780 x g 4000 rpm in order to collect the prepared reaction volume in the bottom of the tubes or wells If such a centrifuge is not at your disposal please make sure that both the Master Mix and the sample volume are pipetted to the bottom of the tubes or wells Store the prepared reactions at 4 C until the ABIPRISM SDS Instrument is programmed see 8 5 Programming the ABI PRISM SDS and subsequently transfer them into the instrument Attention e When using optical reaction tubes in combination with optical caps always insert a retaining rack 96 Well Tray Retainer Set Applied Biosystems into the instrument ABI PRISM 7000 7700 and 7900HT SD
3. Auto Increment sample volume 25 uL Dissociation Protocol 60 0 C iw 3600 Emulation Fig 8 Creating the temperature profile 17 i e eZ RealArt M tuberculosis TM PCR Kit D 00000 8 5 1 5 Saving the PCR Run Save the settings Setup as a template in order to make use of them again later in a modified or unchanged form By saving the Template file as SDS Template sdt in the Template Directory Local Disk C lProgram Files ABI PRISM 7000 Templates created by Applied Biosystems this file may be selected directly in the New Document window from the Template drop down list Copies stored in other folders have to be opened via Browse Before starting the PCR run save it again as an SDS Document sds in order to guarantee the saving of the data that will be collected during the course of the PCR 8 5 1 6 Starting the PCR Run Start the PCR run by selecting the option Start from the menu item nstrument or the field Start on the nstrument level 18 RealArt M tuberculosis TM PCR Kit 000 00000 8 5 2 Programming the ABI PRISM 7700 SDS For the detection of M tuberculosis complex DNA create a profile on your ABI PRISM 7700 SDS according to the following seven steps 8 5 2 1 8 5 2 7 All specifications refer to the ABI PRISM 7700 SDS Software Version 1 9 1 For details of programming the ABI PRISM 7700 SDS refer to the ABI PRISM 7700 SDS Users Manual For a better overview the software se
4. Conditions menu contains the option Show Data Collection By selecting this option the window depicted in Fig 14 is opened Each ramp and each plateau temperature shows a Data Collection icon which illustrates the data being collected at this stage of the run Remove all symbols except the one for the Annealing Extension step StageZ Step2 in order to exclude unnecessary fluorescence measurements The total run time and the data quantity will thus be kept at a minimum 22 RealArt M tuberculosis TM PCR Kit 000 00000 Thermal Cycler Conditions Stage2 Repeat Auto Increment Sample Yolume Show Time And Temp 25 a Lok JlI 2 Fig 14 Data collection 8 5 2 5 Important Additional Settings For the setting of the exposure time excitation of the fluorescence dyes as well as for the selection of the Pure Spectra Background files switch from the Setup level to the Analysis level Select the activated sub item Advanced Options to be found in the nstrument menu under Diagnostics Adjust the settings according to Fig 15 By inactivating the optional function Spectra Components Analysis the actual calibration files stored in the file Spectra Components at the moment of data generation are automatically used when reevaluating already analysed runs For an analysis of previous runs by using newly entered Spectra Components activate these two fields Please note that for a PCR run with the Rea Art M tuberculosis
5. IC have been thawed and frozen too often gt Please mind the storage conditions given in 2 Storage Repeat the PCR using a new M tuberculosis TM Master e The M tuberculosis TM Master has been kept at 4 C for longer than five hours gt Please mind the storage conditions given in 2 Storage Repeat the PCR using a new M tuberculosis TM Master e The PCR was inhibited gt Make sure that you use a recommended isolation method see 8 1 DNA Isolation and stick closely to the manufacturer s instructions e DNA was lost during extraction gt Make sure that you use a recommended isolation method see 8 1 DNA Isolation and stick closely to the manufacturer s instructions 36 RealArt M tuberculosis TM PCR Kit 00 500000 If you have any further questions or if you encounter problems please contact our technical support at techsupport artus biotech com 11 Specifications 11 1 Sensitivity In order to determine the sensitivity of the RealArt M tuberculosis TM PCR Kit a dilution series has been set up from 10 to nominal 0 05 M tuberculosis copy equivalents ul It was subsequently analysed by means of the ABI PRISM 7000 7700 and 7900HT Sequence Detection System with the help of the Rea Art M tuberculosis TM PCR Kit Testing for all instruments was carried out on three different days on eight replicates The results were determined by a probit analysis A graphical illustration of the probit analysis ABI
6. Programming the ABI PRISM 7000 SDS For the detection of the M tuberculosis complex DNA create a profile on your ABI PRISM 7000 SDS according to the following six steps 8 5 1 1 8 5 1 6 All specifications refer to the ABI PRISM 7000 SDS Software Version 1 0 1 For programming details of the ABI PRISM 7000 SDS please refer to the ABI PRISM 7000 SDS User Guide For a better overview the software settings are framed in bold black 8 5 1 1 Pre Settings for Creating a New File Select the item New from the File menu on the ABI PRISM 7000 SDS and program the following initial settings for the new file Fig 3 A backup template SDS Template sdt is available from the Template list or by selection using the Browse function see 8 5 1 5 Saving the PCR Run Confirm your settings OK 13 RealArt M tuberculosis TM PCR Kit 000 00000 VT x Assay Absolute Quantitation Container 96 well Clear 1 Template Blank Document Browse cancel Fig 3 Pre settings for creating a new file New Document 8 5 1 2 Creating Selecting the Detectors With the help of the submenu Detector Manager from the Tools menu assign the corresponding dyes to the file For the detection of M tuberculosis complex DNA as well as the Internal Control by means of the RealArt M tuberculosis TM PCR Kit the reporters quenchers listed in the following table are to be defined Detection Reporter Quencher M tubercu
7. TM PCR kit ROX has to be set as a passive reference Heference The equal distribution of the ROX dye in all PCR preparations of a lot by mixing of the M tuberculosis TM Master guarantees the recognition and calculation of tube to tube variations fluorescence differences between several PCR preparations by means of the Sequence Detection Software normalization Attention When using 96 well reaction plates for optical measurements in combination with optical adhesive covers or optical reaction tubes with flat 23 RealArt M tuberculosis TM PCR Kit 000 00000 caps the exposure time is ten milliseconds If you use optical reaction tubes with domed caps you have to adjust this exposure time to 25 milliseconds Advanced Options Yiewer Display mse in Multicomponent View Display best fit in Raw Spectra View Analysis Spectra Components E se background in Spectra Components folder m se pure spectra in Spectra Components folder Miscellaneous Met 7700 Exposure Time for Plates 4 3 Mise Spectral Compensation for Real Time se Spectral Compensation for Endpoint MM Reference Qi 6 Fig 15 Important additional settings Advanced Options 8 5 2 6 Saving the PCR Run Save the settings Setup as a template in order to make use of them again later in a modified or unchanged form For this purpose store the file in the Stationary File Format Before starting a newly programmed PCR run please save
8. cell free body fluids and material low in DNA RNA content Produced by QIAGEN GmbH D 40724 Hilden for artus GmbH Legal manufacturer artus GmbH D 22767 Hamburg RealArt M tuberculosis TM PCR Kit 00 00000 e When using isolation protocols with ethanol containing washing buffers please carry out an additional centrifugation step before the elution to remove any remaining ethanol This prevents possible inhibition of PCR e The RealArt M tuberculosis TM PCR Kit should not be used with phenol based isolation methods Important The nternal Control of the RealArt M tuberculosis TM PCR Kit can be used directly in the isolation procedure see 8 2 Internal Control 8 2 Internal Control An Internal Control M tuberculosis TM IC is supplied This allows the user both to control the isolation procedure and to check for possible PCR inhibition For this application add the Internal Control to the isolation at a ratio of 0 1 ul per 1 ul elution volume For example using the QlAamp DNA Mini Kit QIAGEN the DNA is eluted in 100 ul AE buffer Hence 10 ul of the Internal Control should be added initially The quantity of IC used depends only on the elution volume Please note that the IC should be added to the mixture of lysis buffer and sample material Please do not add the IC to the lysis buffer or sample material directly The IC can optionally be used exclusively to check for possible PCR inhibition For this applica
9. in parallel To generate a standard curve use all supplied quantitation standards M tuberculosis TM QS 1 4 for each PCR run Before each use all reagents need to be thawed completely mixed by repeated up and down pipetting or by quick vortexing and centrifuged briefly If the two part retaining rack is used it is necessary to open the reaction tubes when inserting them into and removing them from the rack In order to avoid contamination due to this procedure please use the lower part of the retaining rack only RealArt M tuberculosis TM PCR Kit o0 ooooO If you want to use the nternal Control to monitor the isolation procedure and to check for possible PCR inhibition the IC has already been added to the isolation see 8 2 Internal Control In this case please use the following pipetting scheme for a schematic overview see Fig 1 Number of samples M tuberculosis TM Master 1 Preparation of y tuberculosis TM Mg Sol Master Mix M tuberculosis TM IC Total volume 2 Preparation of EU Le Een PCR assay Sample 10 ul each Total volume lf you want to use the IC exclusively to check for PCR inhibition the IC must then be added directly to the M tuberculosis TM Master In this case please use the following pipetting scheme for a schematic overview see Fig 2 Number of samples M tuberculosis TM Master Master Mix M tuberculosis MC ee 1 2 Preparation of Master Mx ls dia pleach
10. 1 x 1000 ul 1 x 1000 ul 1 x 1000 ul PCH grade Quantitation Standard Internal Control IC Applied Biosystems is a registered trademark and ABI PRISM is a trademark of Applera Corporation or its subsidiaries in the US and or certain other countries RealArt M tuberculosis TM PCR Kit a 00000 2 Storage The components of the HealArt M tuberculosis TM PCR Kit should be stored at 20 C and are stable until the expiry date stated on the label Repeated thawing and freezing gt 2 x should be avoided as this may reduce the sensitivity If the reagents are to be used only intermittently they should be frozen in aliquots Storage at 4 C should not exceed a period of five hours 3 Additionally Required Materials and Devices e Disposable powder free gloves e DNA isolation kit see 8 1 DNA Isolation e Lysozyme mix see 8 1 DNA Isolation e Pipettes adjustable e Sterile pipette tips with aerosol barrier e Vortex mixer e Desktop centrifuge with rotor for 2 ml reaction tubes e Centrifuge with rotor for microtitre plates optional e 96 well reaction plate reaction tubes for optical measurement with corresponding optical closing materials see 8 4 Preparing the PCR e 96 well two part retaining rack for use with optical reaction tubes 96 Well Tray Retainer Set Cat No 403 081 Applied Biosystems see 8 4 Preparing the PCR e Compression pad for use with optical adhesive covers Optical Cover Compression Pad
11. 36 PO GI ICAU ONS rara 37 11 1 Sensitivity cooocccocncocncconccnnncncncnononcnonnconannnnrnnnnnonanonannnnnnoncnnnanonans 37 Tie eed EE E ee ee ee 38 Qu MEN E UU T TTE 41 11 4 Robustness ocoocccoccccccocncocnnococononcnnonononnnnnnnnonononnnnnnnnnnnnannnnnnnninanins 43 11 5 LCcuituvyj 43 16 Diagnostic EVA ti d cupdter cm nasesuese decdcun ern 43 Product Use Limitations 43 Explanation of Symbols 44 RealArt M tuberculosis TM PCR Kit 100 20000 RealArt M tuberculosis TM PCR Kit for use with the ABI PRISM 7000 7700 and 7900HT Sequence Detection Systems Applied Biosystems Attention The RealArt M tuberculosis TM PCR Kit may neither be used in combination with the GeneAmp 5700 SDS nor with the 384 plate format of the ABI PRISM 7900HT SDS 1 Contents Labelling Art No 5511 01 Art No 5511 02 Art No 5511 03 and contents 24 reactions 48 reactions 96 reactions M tuberculosis M tuberculosis TM Mg Sol 1 x 400 yl 1 x 400 ul 1 x 400 yl M tuberculosis TM QS 1 1 x 200 yl 1 x 200 yl 1 x 200 ul 3 x 10 cop ul M tuberculosis TM QS 2 1 x 200 ul 1 x 200 ul 1 x 200 ul 3 x 10 cop ul M tuberculosis TM QS 3 1 x 200 ul 1 x 200 ul 1 x 200 ul 3 x 10 cop ul M tuberculosis TM QS 4 1 x 200 ul 1 x 200 ul 1 x 200 yl 3 x 10 cop ul M tuberculosis TM IC 1 x 1000 ul 1 x 1000 ul 1 x 1000 ul White er
12. 39 40 41 42 43 44 45 Cycle Number Fig 23 Detection of the quantitation standards M tuberculosis TM QS 1 4 by measurement of a FAM fluorescence signal ABI PRISM 7000 SDS NTC non template control WE INA n un la MEAE RARE LL m M Hegy ATT num EA illi iii 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 Fig 24 Detection of the nternal Control IC by measurement of a JOE fluorescence signal ABI PRISM 7000 SDS in parallel with a simultaneous amplification of the quantitation standards M tuberculosis TM QS 1 4 NTC non template control 33 RealArt M tuberculosis TM PCR Kit 000 ARn ARn 4 500 4 000 500 3 000 2 500 2 000 1 500 1 000 0 500 0 000 0 500 02 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 33 40 42 44 Cycle Fig 25 Detection of the quantitation standards M tuberculosis TM QS 1 4 by measurement of a FAM fluorescence signal ABI PRISM 7700 SDS NTC non template control 1 000 0 500 0 600 0 400 0 200 0 000 0 200 0 400 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Cycle Fig 26 Detection of the nternal Control IC by measurement of a JOE fluorescence signal ABI PRISM 7700 SDS in parallel with a simultaneous amplification of the quantitation standards M tuberculosis TM QS 1 4 NTC non template c
13. 8 8 5 1 6 Starting the PCR Run semana 18 8 5 2 Programming the ABI PRISM 7700 SDS 19 8 5 2 1 Pre settings for Creating a New File sssesessssss 19 8 5 2 2 Selecting the Fluorescence Dyes and Assigning the Sample nu 19 8 5 2 3 Assigning the Necessary Information to the Plate Positions 21 8 5 2 4 Creating the Temperature Profile oococoonccconnocconcnnoom 22 8 5 2 5 Important Additional SettingS ooocccconcncconcncconcnnconccnnnnnnnnnnns 23 RealArt M tuberculosis TM PCR Kit 00 10 11 12 13 0000 8 5 2 6 Saving the PCR RUN ccccccssecseseeecseeeeceseeceeseessensesueeesneees 24 8 527 Staring the POR Run nenne 24 8 5 3 Programming the ABI PRISM 7900HT SDS 25 8 5 3 1 Pre Settings for Creating a New File cccocooccccccconncncocooconos 25 8 5 3 2 Creating Selecting the DetectorS cooonnccccconncnccoconnnncnnonnnonnnnnnnos 26 8 5 3 3 Assigning the Necessary Information to the Plate Positions 27 8 5 3 4 Creating the Temperature Profile ooccooococonnccnnnconncnnos 28 8 5 3 5 Saving the PCR Run ccccccesecseseeecseeeceseeseeeeessenseseseesneees 30 8 5 3 6 Starting the POR RUN asocio obiter ad Roble Ute d etae Ee Laden ag dd 30 Data AINA SUS senil icaica s 31 Troubleshooting z 0 nie nern nassen
14. Add To Plate Document 2 Fig 6 Selecting the detectors Detector Manager 15 RealArt M tuberculosis TM PCR Kit 660 000090 8 5 1 3 Assigning the Necessary Information to the Plate Positions Open Well Inspector from the View menu to find the detectors selected under 8 5 1 2 see Fig 7 Well Inspector id x Well s B3 Sample Name Q351 _Use Detector Reporter Quencher Task Quantity V atus M tub FAM none Standar v 1 IV artus M tub IC JOE Omit ell Passive dd Detector Remove ROX 2 Fig 7 Assigning the necessary information to the plate positions Well Inspector Mark the plate positions reserved for the detection of M tuberculosis complex DNA Assign the selected detectors to these positions by activating the Use option of both detectors upon which a tick will appear For the denomination of each single reaction select the corresponding position on the plate and enter the name Sample Name Please note that reactions with an identical Sample Name and an identical detector assignment will be identified as replicates by the software and will be averaged with respect to the quantified pathogen load Then select the corresponding function Task for each sample type according to the following table Function Concentration WED Quantity Sample Type Reporter Quencher Non template NTC control Standard Standard see 1 Contents To generate a sta
15. Kit a 00000 The following results are possible 1 AFAM fluorescence signal is detected The result of the analysis is positive The sample contains DNA of one or more members of the M tuberculosis complex In this case the detection of a JOE fluorescence signal nternal Control is dispensable since high initial concentrations of M tuberculosis complex DNA positive FAM fluorescence signal can lead to a reduced or absent fluorescence signal of the nternal Control competition 2 No FAM fluorescence signal is detected At the same time a JOE fluorescence signal from the nternal Control appears In the sample no DNA of members of the M tuberculosis complex is detectable It can be considered negative In the case of a negative M tuberculosis complex PCR the detected signal of the IC rules out the possibility of PCR inhibition 3 Neither a FAM fluorescence signal nor a JOE fluorescence signal is detected No diagnosis can be concluded Information regarding error sources and their solution can be found in 10 Troubleshooting Examples of positive and negative PCR reactions are given in Fig 23 24 ABI PRISM 7000 SDS 25 26 ABI PRISM 7700 SDS and 27 28 ABI PRISM 7900HT SDS 32 RealArt M tuberculosis TM PCR Kit 000 Delta Rn Delta Rn 0 1 0 40 0 20 0 10 LUMI me 123 4 5 6 7 8 910111213 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
16. ND Standard UNKN Unknown 1 Sample Name UNKN No Template Control Replicate Sample Type Setup Quantity __Not In Use one ii 3 3 Fig 11 12 Assigning the necessary information to the plate positions To generate a standard curve use all supplied quantitation standards M tuberculosis TM QS 1 4 per PCR run and enter the corresponding concentrations see 1 Contents for each standard into the field Quantity see Fig 12 This is only possible however if the positions reserved for standards were defined as such before by means of the Sample Type menu 21 RealArt M tuberculosis TM PCR Kit 000 00000 8 5 2 4 Creating the Temperature Profile To create a temperature profile switch to the Thermal Cycler Conditions menu on the Setup level Enter the temperature profile special for the detection of M tuberculosis complex DNA according to Fig 13 Make sure that the reaction volume for the detection of M tuberculosis complex DNA by using the RealArt M tuberculosis TM PCR Kit is set to 25 ul The pre settings of the Ramp times and of the Auto Increment remain unchanged Ramp Time 0 00 Auto Increment 0 0 C 0 0 Seconds Thermal Cycler Conditions Stage2 Repeat 85 0 los 1 Add Cycle Auto Increment Sample Volume Show Data Collection ddHold 0 0 c 25 a 2 TS Rao Pim 0 0 Seconds Fig 13 Creating the temperature profile In addition the Thermal Cycler
17. PRISM 7000 SDS is shown in Fig 29 Detection limit p 0 05 l ABI PRISMP 7000 SDS 1 0 copy ul ABI PRISM 7700 SDS 1 0 copy ul ABI PRISMP 7900HT SDS 0 6 copies pl This means that there is a 95 96 probability that 1 copy ul ABI PRISM 7000 SDS 1 copy ul ABI PRISM 7700 SDS and 0 6 copies ul ABI PRISM 7900HT SDS will be detected The standard is a cloned PCR product the concentration of which has been determined by absorption and fluorescence spectroscopy 37 RealArt M tuberculosis TM PCR Kit 500 000090 Probit analysis M tuberculosis ABI PRISM 7000 SDS 1 0000 0 01995 1 copy pl 95 0 8000 0 7000 3 0 5000 Proportions 0 5000 0 4000 0 3000 0 2000 0 1000 3 2 1 0 1 3 log dose Fig 29 Sensitivity of the RealArt M tuberculosis TM PCR Kit ABI PRISM 7000 SDS 11 2 Specificity The specificity of the RealArt M tuberculosis TM PCR kit is first and foremost ensured by the selection of the primers and probes as well as the selection of stringent reaction conditions The primers and probes are checked for possible homologies to other known sequences by sequence comparison analysis The detectability of all members of the M tuberculosis has thus been ensured Moreover the specificity was validated with 90 different M tuberculosis complex negative samples 30 sputum 30 BAL and 30 bronchial secretion samples These did not generate any sig
18. RealArt M tuberculosis TM PCH Kit Quantitative in vitro Diagnostics for use with the AB PRISM Sequence Detection Systems Applied Biosystems User Manual February 2004 CE ENO40220 RealArt M tuberculosis TM PCR Kit mdi oN OO PF WwW LP Table of Contents COMENS noo 1 LOA CO APER TETTE 2 Additionally Required Materials and Devices 2 General Precautions es 3 Pathogen Information 3 Principle of Real Time PCR 4 Product Description 4 o 5 ol DNAISOIAON csi ias 5 8 2 Internal CONTFOl ocoocccoccccccccnconnconcconcononononononcnnanocanonnonaninnnnnnonono 6 83 QUINUA ION NER 7 0 J TODAOPEHCG the I Ricos alos 8 8 5 Programming the ABI PRISMP SDS ee 13 8 5 1 Programming the ABI PRISM 7000 SDS 13 8 5 1 1 Pre Settings for Creating a New File sessesseessssse 13 8 5 1 2 Creating Selecting the DetectorS c ooonncnccconnccccoconcnncnoonnnnnnonnnnos 14 8 5 1 3 Assigning the Necessary Information to the Plate Positions 16 8 5 1 4 Creating the Temperature Profile ooococoonccconnncconnnnnoo 17 8 5 1 5 Saving the PCR Run cccccseccseeeeeceeeeeceseeceeseecsenseseuseenneees 1
19. S If the two part retaining rack is used it is necessary to open the reaction tubes when inserting them into and removing them from the rack In order to avoid contamination due to this procedure please use the lower part of the retaining rack only e Using 96 well optical reaction plates in combination with optical adhesive covers requires covering by a compression pad Optical Cover Compression Pads Applied Biosystems 10 RealArt M tuberculosis TM PCR Kit 000 00000 Addition of the Internal Control to the Purification Procedure p oz n Fig 1 Schematic workflow for the control of both the purification procedure and PCR inhibition Please make sure that the solutions are thawed completely mixed well and centrifuged briefly RealArt M tuberculosis TM PCR Kit 000 00000 Addition of the Internal Control into the RealArt Master Fig 2 Schematic workflow for the control of PCR inhibition Please make sure that the solutions are thawed completely mixed well and centrifuged briefly 12 RealArt M tuberculosis TM PCR Kit a 00000 8 5 Programming the ABI PRISM SDS The software of the ABI PRISM 7000 7700 and 7900HT Sequence Detection Systems SDS requires some additional information before starting the PCR run The programming procedures of the instruments however differ considerably from each other which is why they are treated in separate chapters as follows 8 5 1
20. ariation Intra assay variability 2 17 4 72 7 22 M tuberculosis TM QS 4 Inter assay variability M tuberculosis TM QS 4 M tuberculosis 2 37 Inter lot variability M tuberculosis TM QS 4 Total variance M tuberculosis TM QS 4 42 RealArt M tuberculosis TM PCR Kit a 00000 11 4 Robustness The verification of the robustness allows the determination of the total failure rate of the RealArt M tuberculosis TM PCR Kit 30 M tuberculosis complex negative samples of sputum BAL and bronchial secretion were spiked with 3 copies ul elution volume of M tuberculosis control DNA threefold concentration of the analytical sensitivity limit After extraction using the QlAamp DNA Mini Kit QIAGEN see 8 1 DNA Isolation these samples were analysed with the RealArt M tuberculosis TM PCR Kit For all M tuberculosis samples the failure rate was O In addition the robustness of the nternal Control was assessed by purification and analysis of 30 M tuberculosis complex negative sputum BAL and bronchial secretion samples The total failure rate was O Inhibitions were not observed Thus the robustness of the Rea Art M tuberculosis TM PCR Kit is 2 99 96 11 5 Reproducibility Reproducibility data permit a regular performance assessment of the RealArt M tuberculosis TM PCR Kit as well as an efficiency comparison with other products These data are obtained by the participation in established proficienc
21. e corresponding position on the plate and enter the name Sample Name Please note that preparations with an identical Sample Name and an identical detector 2 RealArt M tuberculosis TM PCR Kit 00 500000 assignment will be identified as replicates by the software and will be averaged with respect to the quantified pathogen load Then select the corresponding function Task for each sample type according to the following table Function Concentration Task Quantity Sample Type Reporter Quencher Non Fluorescent PAM Non template NTC FAM Non Fluorescent control Standard Standard see 1 Contents Non Fluorescent To generate a standard curve use all supplied quantitation standards M tuberculosis TM QS 1 4 per PCR run and enter the corresponding concentrations see 1 Contents for each standard Quantity Please note that for a PCR run with the Rea Art M tuberculosis TM PCR kit ROX has to be set as a passive reference Passive Reference The equal distribution of the ROX dye in all PCR preparations of a lot by mixing of the M tuberculosis TM Master guarantees the recognition and calculation of tube to tube variations fluorescence differences between several PCR preparations by means of the Sequence Detection Software normalization 8 5 8 4 Creating the Temperature Profile To create a temperature profile switch from the Setup level to the nstrument level in the software Enter the temperature profil
22. e specific for the detection of M tuberculosis complex DNA according to Fig 21 Make sure that the reaction volume for the detection of M tuberculosis complex DNA by using the RealArt M tuberculosis TM PCR Kit is set to 25ul The option 9600 Emulation should be activated the pre settings of the Ramp time and the Auto Increment should remain unchanged Ramp Time 0 00 Auto Increment 0 0 C 0 0 Seconds 28 RealArt M tuberculosis TM PCR Kit 500 oo Setup Instrument Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Data Collection Stage 2 Repeats 45 05 0 Add Cycle Add Hold Add Step Delete Step sample Volume uL E Add Dissociation Stage Fig 21 Creating the temperature profile In addition the nstrument level contains the option Data Collection By selecting this option the window depicted in Fig 22 is opened Each ramp and each plateau temperature shows a Data Collection Icon which illustrates the data being collected at this stage of the run Remove all symbols except the one for the Annealing Extension step Stage2 Step2 by clicking on them in order to exclude unnecessary fluorescence measurements The total run time and the data quantity will thus be kept at a minimum 29 RealArt M tuberculosis TM PCR Kit Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Data Collection Stage 2 Repeats las 95 0 Sample Vol
23. ens M tuberculosis IC FAM JOE Mycobacterium ulcerans s o to Mycobacterium xenopi c0 o t _ Neisseria Ee C Nocardia brasiliensis Of Nocardia farcinia s o o o Nocardia otitidiscaviarum ooo o o Peptostreptococous productus t Porphyromonas gingivalis o Prevotella denticola o oo Propionibacterium acnes o t o Pseudomonas aeruginosa J ef t Salmonella enteritidis ef Salmonella typhi s o E Staphylococcus aureus 000 o o Staphylococcus epidermidis sf t Streptococcus agalactias o ooo Streptococcus pyogenes so o to Streptococcus mutans 0 0 Streptococcus pneumoniae o Streptomyces venezuela to Veillonella parvula co o 0 Xanthomonas maltophilia Control group 40 i e eZ RealArt M tuberculosis TM PCR Kit D 00000 11 3 Precision The precision data of the RealArt M tuberculosis TM PCR Kit allow the determination of the total variance of the assay The total variance consists of the intra assay variability variability of multiple results of samples of the same concentration within one experiment the inter assay variability variability of multiple results of the assay generated on different instruments of the same type and different operators and the inter lot variability variability of multiple results of the assay using various lots The data obtained were used to determine the standard deviation the variance and the coef
24. erferences appear during multicolour analyses between the emission spectra of the single fluorescence dyes The software of the ABI PRISM SDS compensates these interferences by calculations using the spectrum data of the individual dyes stored in the Pure Spectra Component File The software uses the same file for the assignment of the fluorescence data comprising the entire measurable spectrum collected in the course of the PCR to the programmed detectors The fluorescence data of the individual dyes are subsequently divided by the value obtained for the signal of the passive reference ROX in order to account for tube to tube variations fluorescence differences between several PCR preparations The thus normalized signals may then be evaluated by means of the Amplification Plot The calibration files used for the evaluation of a PCR run are automatically stored with the saving of the run If no calibration files are installed please create these files according to the instructions given in the AB PRISM SDS User Guide Manual If more than one RealArt TM PCR system is integrated in your PCR run note temperature profile these assays should be analysed separately samples with an identical Sample Name and an identical detector assignment will automatically be identified as replicates by the ABI PRISM 7000 and 7900HT SDS Software and will be averaged with respect to the quantified pathogen load 31 RealArt M tuberculosis TM PCR
25. ew infections and about three million deaths are reported each year Countries in the third world are largely affected but due to resistance development and the importation of infections tuberculosis incidents occur increasingly often in industrialized nations The homeless drug addicted and immunocompromised persons are particularly at risk of infection Transmission of the bacteria occurs via aerosols with an increased risk of infection in cases of active tuberculosis Tuberculosis is a chronic cyclic disease In the primary stage mainly parts of the lungs and the associated lymph nodes are affected Depending on the immune status of the patient however mycobacteria can colonize in the entire lung and other organs In cases of immunosuppression the infection may spread from these sites and even years later can be reactivated recrudescent Signs of such a RealArt M tuberculosis TM PCR Kit 00 00000 reactivation in the lungs include chronic fever weight loss night sweats and coughing Other symptoms depend on the affected organs 6 Principle of Real Time PCR Pathogen diagnosis by the polymerase chain reaction PCR is based on the amplification of specific regions of the pathogen genome In real time PCR the amplified product is detected via fluorescent dyes These are usually linked to oligonucleotide probes which bind specifically to the amplified product Monitoring the fluorescence intensities during the PCR run i e i
26. ficient of variation for the specific and the IC system Precision data of the RealArt M tuberculosis TM PCR Kit have been collected using the quantitation standard of the lowest concentration QS 4 30 copies ul Testing was performed with eight replicates The precision data were calculated on basis of the Ct values of the amplification curves Ct threshold cycle Table 2 In addition precision data for quantitative results in copies ul were determined using the corresponding Ct values Table 3 Based on these results the overall statistical spread of any given sample with the mentioned concentration is 1 41 Ct or 10 23 conc for the detection of the nternal Control 2 59 Ct These values are made up of the totality of all single values from the determined variabilities 41 RealArt M tuberculosis TM PCR Kit o0 Table 2 Precision data on basis of the Ct values Standard Deviation Coefficient of M tuberculosis Variation VEE U Intra assay variability M tuberculosis TM QS 4 Intra assay variability Internal Control Inter assay variability M tuberculosis TM QS 4 Inter assay variability Internal Control Inter lot variability M tuberculosis TM QS 4 Inter lot variability Internal Control Total variance M tuberculosis TM QS 4 Total variance Internal Control Table 3 Precision data for quantitative results in copies ul Standard Coefficient of deviation variance v
27. it again in the Normal File Format in order to guarantee the saving of data that will be collected during the course of the PCR 8 5 2 7 Starting the PCR Run Start the PCR run by selecting the option Run from the menu item nstrument or the field Run on the Analysis level 24 i e e RealArt M tuberculosis TM PCR Kit D 00000 8 5 3 Programming the ABI PRISM 7900HT SDS For the detection of M tuberculosis complex DNA create a profile on your ABI PRISM 7900HT SDS according to the following six steps 8 5 3 1 8 5 3 6 All specifications refer to the ABI PRISM 7900HT SDS Software Version 2 1 For details of programming the ABI PRISM 7900HT SDS refer to the ABI PRISM 7900HT SDS User Guide For a better overview the software settings are framed in bold black 8 5 3 1 Pre Settings for Creating a New File Select the item New from the File menu on the ABI PRISM 7900HT SDS and program the following initial settings for the new file Fig 16 A backup template ABI PRISM SDS Template Document sdf is available from the Template list or by selection using the Browse function see 8 5 3 5 Saving the PCR Run Confirm the pre settings OK Attention The HealArt M tuberculosis TM PCH Kit may not be used in combination with the 384 plate format of the ABI PRISM 7900HT SDS New Document XX Assay Absolute Quantification Standard Curve v Container o6 Wells Clear Plate z 1 Template Blank Template z
28. ively For the analysis of sputum we recommend a NALC NaOH decontamination stomach fluids should be neutralized with phosphate buffer After a final centrifugation the bacteria pellet can be used for the following DNA isolation for more details contact our technical support at techsupport artus biotech com Various manufacturers offer DNA isolation kits Sample volumes for the DNA isolation procedure depend on the protocol used Please carry out the DNA isolation according to the manufacturers instructions The following isolation kits are recommended Nucleic Acid Catalogue Carrier Isolation Kit Number Manufacturer DNA RNA Sputum BAL PureArt DNA bronchial secretion Mini Kit 50 90204 50 not included Sample Material CSF stomach ar DNA fluid peritoneal ul 51 304 QIAGEN not included Attention It is important to follow the instructions in Protocol D Protocols for bacteria described in the QIAGEN Kit User Manual We recommend a digestion with lysozyme lysozyme mix 20 mg ml lysozyme 20 mM Tris HCl 2 mM EDTA 1 2 Triton for at least one hour The final DNA elution should be performed once using 100 ul AE buffer e f the selected isolation kit does not contain carrier DNA RNA please note that the addition of carrier RNA RNA Homopolymer Poly A Amersham Biosciences at a concentration of 10 ug ml lysis buffer to the sample lysis buffer mixture is strongly recommended for extraction of nucleic acids from
29. losis complex DNA Internal Control JOE M tuberculosis TM IC To create these detectors select the option File bottom left of the Detector Manager and subsequently the option New 14 RealArt M tuberculosis TM PCR Kit 000 00000 New Detector EL x New Detector E x Name artus M tub Name artus M tub IC 1 Description Realan M tub TM PCR Kit Description Realart M tub TM PCR Kit 4 Reporter Dye JOE y Quencher Dye TAMRA y Reporter Dye FAM y Quencher Dye none Color m Color Notes Create Another Cancel Create Another Cancel Fig 4 Creating the M tuberculosis Fig 5 Creating the IC specific detector complex specific detector Detector Manager Detector Manager For the detection of M tuberculosis complex DNA define the reporter quencher combination FAM none in the now appearing window For the detection of the Internal Control select the combination JOE TAMRA as shown in Fig 4 and Fig 5 By confirming the input data OK return to the Detector Manager Mark the newly created detectors and transfer each selection to the Well Inspector by clicking on the option Add to Plate Document see Fig 6 Close the window Done Detector Manager yj X Detector List Find El El Detector Name Deseription Reporter Quencher Color Notes Last Modified artus M tub RealArt M tub TM PCR Kit FAM none 4 artus M tub IC RealArt M tub TM PCR Kit JOE TAMRA
30. n real time allows the detection and quantitation of the accumulating product without having to re open the reaction tubes after the PCR run T Product Description The RealArt M tuberculosis TM PCR Kit constitutes a ready to use system for the detection of DNA of all members of the M tuberculosis complex M tuberculosis Mafricanum Il Mbovis M bovis BCG M microti M canetti using polymerase chain reaction PCR in the ABI PRISM 7000 7700 and 7900HT Sequence Detection System Applied Biosystems The M tuberculosis TM Master contains reagents and enzymes for the specific amplification of a 163 bp region of the mycobacterial genome The amplicon is detected by measuring the FAM fluorescence in the ABI PRISM SDS In addition the RealArt M tuberculosis TM PCR Kit contains a second heterologous amplification system to identify possible PCR inhibition This is detected as an nternal Control IC by measuring the JOE fluorescence The detection limit of the analytical M tuberculosis complex PCR see 11 1 Sensitivity is not reduced External positive controls M tuberculosis TM QS 1 4 are supplied which allow the determination of the pathogen load For further information please refer to section 8 3 Quantitation RealArt M tuberculosis TM PCR Kit a 00000 8 Protocol 8 1 DNA Isolation Before the DNA isolation large sample volumes or strongly acidic samples must first be concentrated or neutralized respect
31. nals with the M tuberculosis complex specific primers and probes which are included in the RealArt M tuberculosis TM PCR Kit To determine the specificity of the RealArt M tuberculosis TM PCR Kit the control group listed in the following table Table 1 has been tested for cross reactivity None of the tested pathogens has been reactive 38 RealArt M tuberculosis TM PCR Kit O Table 1 part I Testing the specificity of potential cross reactive pathogens IC ae Control Group M tuberculosis Actinomyces israelii Aeromonas hydrophila Bordetella pertussis Candida albicans Chlamydia trachomatis Chlamydia pneumoniae Citrobacter freundii Corynebacterium diphtheriae Corynebacterium jeikeium Cryptococcus neoformans Eikenella corrodens Enterobacter aerogenes Enterobacter cloacae Enterococcus faecalis Enterococcus faecium Escherichia coli Fusobacterium nucleatum ssp polymorphum Haemophilus influenzae Haemophilus parainfluenzae Klebsiella pneumoniae Lactobacillus acidophilus Mycobacterium avium ssp avium Mycobacterium celatum Mycobacterium chelonae Mycobacterium fortuitum Mycobacterium gordonae Mycobacterium intracellulare Mycobacterium kansasii Mycobacterium lentiflavum Mycobacterium malmoense Mycobacterium marinum Mycobacterium scrofulaceum Mycobacterium szulgai 39 RealArt M tuberculosis TM PCR Kit o0 Table 1 part Il Testing the specificity of potential cross reactive pathog
32. nd Fig 18 By confirming the input data OK return to the Detector Manager Mark the newly created detectors and transfer each 26 RealArt M tuberculosis TM PCR Kit 000 000090 selection to the Setup level by clicking on the option Copy to Plate Document see Fig 19 Close the window Done Detector Manager ES Find aj v Nae Reporter Quencher Color Modification Date artus M tub FAM Non Fluorescent artus M tub IC JOE TAMRA m v New Open Save As Import Export Copy To Plate Document 2 Fig 19 Selecting the detectors Detector Manager 8 5 3 3 Assigning the Necessary Information to the Plate Positions After having closed the Detector Manager Done the detectors selected under 8 5 3 2 are found on the Setup level Well Inspector listed in a table see Fig 20 Setup Instrument YYell s C5 2 Sample Name J051 Detector Reporter Task Quantity Color artus M tub FAN Standard aii Et 1 E artus M tub IC Standard Add Detector Gleat Passive Reference ROX v 2 Omt Well s Fig 20 Assigning the necessary information to the plate positions Mark the plate positions reserved for the detection of M tuberculosis complex DNA Assign the selected detectors to these positions by activating the Use option of both detectors by clicking on them upon which a cross will appear For the denomination of each single reaction select th
33. ndard curve use all supplied quantitation standards M tuberculosis TM QS 1 4 per PCR run and enter the corresponding concentrations see 1 Contents for each standard Quantity Please note 16 RealArt M tuberculosis TM PCR Kit 500 00000 that for a PCR run with the Rea Art M tuberculosis TM PCR Kit ROX has to be set as a passive reference Passive The equal distribution of the ROX dye in all PCR preparations of a lot by mixing of the M tuberculosis TM Master guarantees the recognition and calculation of tube to tube variations fluorescence differences between several PCR preparations by means of the Sequence Detection Software normalization 8 5 1 4 Creating the Temperature Profile To create a temperature profile switch from the Setup level to the nstrument level in the software Enter the temperature profile specific for the detection of M tuberculosis complex DNA according to Fig 8 In order to remove the 50 C step stored in the pre settings mark it with the left mouse button simultaneously holding the shift key and subsequently delete it by using the backspace key Make sure that the reaction volume for the detection of M tuberculosis complex DNA using the RealArt M tuberculosis TM PCR Kit is set to 25 ul The option 9600 Emulation should be activated the pre settings of the Auto Increment should remain unchanged Auto Increment 0 0 C 0 0 Seconds Thermal Cycler Protocol Thermal Profile
34. ontrol 34 RealArt M tuberculosis TM PCR Kit 000 00000 Fig 27 Detection of the quantitation standards M tuberculosis TM QS 1 4 by measurement of a FAM fluorescence signal ABI PRISM 7900HT SDS NTC non template control 1 000 8 000 E 1 6 000 E 1 ARn Fig 28 Detection of the nternal Control IC by measurement of a JOE fluorescence signal ABI PRISM 7900HT SDS in parallel with a simultaneous amplification of the quantitation standards M tuberculosis TM QS 1 4 NTC non template control 35 RealArt M tuberculosis TM PCR Kit a 00000 10 Troubleshooting No FAM fluorescence signal with positive controls M tuberculosis TM QS 1 4 e Incorrect programming of the ABI PRISM Sequence Detection Systems gt Repeat the PCR with corrected settings Weak or no signal of the nternal Control JOE fluorescence signal and simultaneous absence of a FAM fluorescence signal for the specific M tuberculosis complex PCR e The PCR conditions do not comply with the protocol gt Repeat the PCR with corrected settings e The settings used for the data analysis under Options Extension Phase Data Extraction do not correspond to the settings of the Data Collection see ABI PRISM 7700 SDS 8 5 2 4 Creating the Temperature Profile gt Analyse the PCR run with corrected settings and repeat the data analysis Analysis e The M tuberculosis TM Master and or the M tuberculosis TM
35. s Cat No 4 312 639 Applied Biosystems see 8 4 Preparing the PCR e Applicator for sealing of the reaction plates using optical adhesive covers Adhesive Seal Applicator Kit Cat No 4 333 183 Applied Biosystems The use of reaction tubes for optical analyses with domed caps is only permitted with the ABI PRISMP 7700 SDS and requires an adjustment of the exposure time see 8 5 2 Programming the AB PRISMP 7700 SDS 8 5 2 5 Important Additional Settings RealArt M tuberculosis TM PCR Kit 000 00000 ABI PRISM 7000 7700 or 7900HT SDS Applied Biosystems Attention A valid calibration of the pure dyes Pure Spectra Component File and the background Background Component File is necessary when putting the instruments into operation 4 General Precautions The user should always pay attention to the following e Use pipette tips with filters e Extract and store positive material specimens controls and amplicons separately from all other reagents and add it to the reaction mix in a spatially separated facility e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Work quickly on ice or in the cooling block 5 Pathogen Information Mycobacteria are distributed worldwide It is estimated that one third of the world s population is infected with Mycobacterium tuberculosis the pathogen of tuberculosis About eight million n
36. s shown in Fig 10 Sample Type Setup Acronym Name Color Reporter icon JS Reference ROA v Quencher T APTE 4 cancel ok Remove a Fig 10 Selecting the fluorescence dyes and assigning the sample type Sample Type Setup Please assign the sample type to a corresponding function Acronym according to the following table Reporter Quencher Function Concentration Sample Type Acronym Quantity Sample UNKN control Standard STND see 1 Contents 20 RealArt M tuberculosis TM PCR Kit 500 00000 8 5 2 3 Assigning the Necessary Information to the Plate Positions For the assignment of the detectors and sample types to the individual plate positions select the corresponding fields Then open the dialogue window Dye Layer on the Setup level and assign the corresponding reporter Upon activation of the pop up menu Sample Type you will find the sample types assigned to the reporter in the Sample Type Setup in the appearing list see Fig 11 Select the adequate sample type see Table under 8 5 2 2 and assign the remaining plate positions by means of the Dye Layers and the Sample Type menu A name may be assigned to each sample in the field Sample Name Fields defined as Replicates entering of the name of the reference sample into the Replicate column are averaged by the software with respect to the quantified pathogen load and the standard deviation will be calculated e ST
37. tion add O 5yl of the IC and 2yl M tuberculosis TM Mg Sol per test mixture directly to 13 ul M tuberculosis TM Master For each PCR reaction use 15 ul of the Master Mix produced as described above then add 10 ul of the purified sample If you are preparing a PCR run for several samples please increase the volume of the M tuberculosis TM Master the M tuberculosis TM Mg Sol and the nternal Control according to the number of samples see 8 4 Preparing the PCR The volume increase caused by adding the IC is neglected when preparing the PCR assay The sensitivity of the detection system is not impaired RealArt M tuberculosis TM PCR Kit a 00000 8 3 Quantitation The enclosed quantitation standards M tuberculosis TM QS 1 4 are treated as previously purified samples and the same volume is used 10 ul To generate a standard curve in an ABI PRISM Sequence Detection System all four quantitation standards should be used and defined as standards with specification of the corresponding concentrations see 8 5 Programming the ABI PRISM SDS The import of standard curves from previous runs is not possible with the ABI PRISM 7000 7700 and 7900HT SDS software Attention The quantitation standards are defined as copies ul The following equation has to be applied to convert the values determined using the standard curve into copies ml of sample material Result copies ml Result copies jl x Elution Volume ul Sample Vol
38. ttings are framed in bold black 8 5 2 1 Pre settings for Creating a New File Select the item New Plate from the File menu on the ABI PRISM 7700 SDS and program the following initial settings for the new file Fig 9 Confirm the pre settings OK New Plate Plate Type Single Reporter T Data Acquisition 1 Plate Format Standard Plate Run Fig 9 Pre settings for creating a new file New Plate 8 5 2 2 Selecting the Fluorescence Dyes and Assigning the Sample Type With the help of the Sample Type Setup Setup level Sample Type Sample Type Setup assign the corresponding detector dyes and the corresponding sample type to the file For the detection of M tuberculosis complex DNA as well as the Internal Control by means of the RealArt M tuberculosis TM PCR Kit the reporters quenchers listed in the following table are to be defined 19 RealArt M tuberculosis TM PCR Kit a 00000 Detection Reporter Quencher M tuberculosis complex DNA TAMRA Internal Control JOE M tuberculosis TM IC For the analysis of M tuberculosis complex DNA by means of the RealArt M tuberculosis TM PCR Kit select the reporter dye FAM as given in the table This applies to standards STND and samples UNKN as well as to non template controls UNKN For the analysis of the nternal Control IPC define JOE as reporter As quencher set TAMRA The assignment of the dyes and sample types in the window Sample Type Setup i
39. ume ml If a concentration of the sample material was performed by e g centrifugation prior to nucleic acid extraction please enter the entire initial sample volume in the above equation RealArt M tuberculosis TM PCR Kit a 500000 8 4 Preparing the PCR Prepare the number of required reaction tubes or a 96 well reaction plate for the scheduled reactions Recommended materials are listed in the following table Article 96 well optical reaction plate Optical adhesive cover Optical reaction Catalogue Number 4 306 737 4 311 971 Description 96 Well Optical Reaction Plate Applied Biosystems Optical Adhesive Covers ABI PRISM Applied Biosystems Applied Manufacturer Retaining Compression Rack Pad Optical Tubes 8 Tubes Strip EIOS SIUS 4 316 567 N8010933 4 323 032 Attention The use of reaction tubes for optical analyses with domed caps is only permitted with the ABI PRISM 7700 SDS and requires an adjustment of the exposure time see 8 5 2 Programming the AB PRISM 7700 SDS 8 5 2 5 Important Additional Settings tubes Optical reaction tubes MicroAmp Optical Tubes Applied Biosystems ABI PRISM Optical Caps 8 Caps Strip Optical cap flat Applied Biosystems EE px EAE a When preparing the PCR reaction please make sure that at least one quantitation standard per PCR run as well as one negative control Water PCR grade are performed
40. ume uL 25 v 9600 Emulation Fig 22 Data collection 8 5 3 5 Saving the PCR Run Save the settings Setup as a template in order to make use of them again later in a modified or unchanged form By saving the Template file as an ABI PRISMPSDS Template Document sdt in the Template Directory D Program Files Applied Biosystems SDS 2 1 Templates created by Applied Biosystems this file may be selected directly in the New Document window from the Template list Copies stored in other folders have to be opened via Browse Before starting the PCR run save it again as an ABI PRISM SDS Document sds in order to guarantee the saving of the data that will be collected during the course of the PCR 8 5 3 6 Starting the PCR Run Start the PCR run by selecting the option Start from the menu item nstrument or the field Start on the Analysis level 30 i e e RealArt M tuberculosis TM PCR Kit D 00000 9 Data Analysis A valid calibration of the dyes Pure Spectra Component File and the background Background Component File is necessary when putting the instruments into operation These calibration files are required for the exact calculation of the results as follows All interfering signals generated by the instruments which influence the measurement are eliminated by the Sequence Detection Software of the ABI PRISM Sequence Detection Systems by means of the Background Component File Furthermore int
41. y programs 11 6 Diagnostic Evaluation Currently the RealArt M tuberculosis TM PCR kit is undergoing a series of evaluation studies 12 Product Use Limitations e All reagents may exclusively be used in in vitro diagnostics e The product is to be used by personnel specially instructed and trained in the in vitro diagnostics procedures EN375 only e Compliance with the user manual is required e Attention should be paid to expiration dates printed on the box and labels of all components Do not use expired components 43 RealArt M tuberculosis TM PCR Kit 00 00000 13 Explanation of Symbols gt Use by LOT Batch code Manufacturer Catalogue number Temperature limitation E Quantitation Standard IC Internal Control 44 artus GmbH K nigstr 4a 22767 Hamburg Germany ul Phone 49 40 41 36 47 00 Fax 49 40 41 36 47 10 E mail info artus biotech com Webpage www artus biotech com artus Biotech USA Inc China Basin Landing 185 Berry Street Suite 6400 oan Francisco CA 94107 USA Phone 1 415 512 7887 Fax 1 415 512 7884 E mail info USA artus biotech com artus Malaysia Sdn Bhd Level 13 Wisma E amp C No 2 Lorong Dungun Kiri Damansara Heights 50490 Kuala Lumpur Malaysia Phone 603 2094 5170 Fax 603 2094 5171 E mail info asia artus biotech com
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