Software manual. Thermo Scientific PathoProof Norden Lab Mastitis Studio Software Instructions for use

Transcription

1 Software manual Thermo Scientific PathoProof Norden Lab Mastitis Studio Software Instructions for use PF0888A Revision Date: January 22, 2015

2 Contents Getting started with Norden Lab Mastitis Studio Overview of Norden Lab Mastitis Studio Main functions Importing a run to the application Viewing the imported runs Interpretation of the results with Norden Lab Mastitis Studio Cycle threshold values Internal Amplification Control Real-time PCR amplification curves Negative (no template) control Interpretation of Staphylococcus results Interpretation of Mycoplasma results Interpretation of β-lactamase results Creating reports Result categories in the reports Report templates A. Adding a new real-time PCR instrument to Norden Lab Mastitis Studio B. Calibrating Norden Lab Mastitis Studio B1. Calibration PCR setup for PathoProof Complete assays B2. Calibration PCR setup for PathoProof Major assays B3. Calibration PCR setup for PathoProof Mycoplasma-8 assay B4. Instrument-specific settings and instructions for run and file handling B4.1 Applied Biosystems 7500 and 7500 Fast Real-Time PCR System (7500 Software v2.0.6, Mycoplasma-8 only) B4.2 Agilent Mx3005P or Mx3000P QPCR System (MxPro - Mx3005P v4.10 software) B5. Software calibration B5.1 View Calibration Run B5.2 The Calibration Warning Messages Norden Lab Mastitis Studio Software - Instructions for use

3 PathoProof Software - Instructions for use Getting started with Norden Lab Mastitis Studio Creating instruments When using the system for the first time, add real-time PCR instrument to the software. See section A Calibrate when using the Thermo Scientific PathoProof PCR assays for the first time Calibrate, if change in real-time PCR plastic type or instrument maintenance activities affect threshold values (see Section 8). 1. Perform real-time PCR calibration setup See section B1 to B3 2. Perform real-time PCR calibration run See section B4 3. Calibrate the real-time PCR instrument using Instrument calibration wizard See section B5 Performing analyses Now the instrument and software are ready for samples Follow the instructions in the PathoProof assay's instructions for use See PathoProof instructions for use Result interpretation Analyze the results using Norden Lab Mastitis Studio software See Sections of 23

4 PathoProof Software - Instructions for use 1. Overview of Norden Lab Mastitis Studio Norden Lab Mastitis Studio General Edition is a software application designed for viewing, reporting and storing the results obtained using PathoProof PCR assays. The software is applicable for use with: PathoProof Mastitis Complete - 12 kit together with o Agilent Mx3005P and Mx3000P QPCR Systems with MxPro TM - Mx3005P v4.10 software PathoProof Mastitis Complete - 16 kit together with o Mx3005P QPCR System with MxPro - Mx3005P v4.10 software PathoProof Mastitis Major - 3 kit together with o Mx3005P and Mx3000P QPCR System with MxPro - Mx3005P v4.10 software PathoProof Mastitis Major - 4 kits together with o Mx3005P QPCR System with MxPro - Mx3005P v4.10 software PathoProof Mycoplasma - 8 kit together with o Applied Biosystems 7500 and 7500 Fast Real-Time PCR Systems with 7500 Software v2.06 o Agilent Mx3005P QPCR System with MxPro - Mx3005P v4.10 software Norden Lab Mastitis Studio is highly recommended as an integral part of the working procedure for the PathoProof PCR assays. Before you use PathoProof PCR assays for the first time, you must add the new instrument to the software and calibrate the Norden Lab Mastitis Studio. Calibration may also need to be performed when changing real-time PCR plastic type or when the real-time PCR instrument has undergone maintenance. See Section A for instructions on adding a new instrument to the software. Instructions for performing calibration can be found in Section B. Norden Lab Mastitis Studio has the following hardware and software requirements: Operating System: Microsoft Windows 2000, 2003, XP, XP 64-Bit, Windows 7 and 8, Vista or Vista 64 Bit, including product variants Processor: Intel or AMD DualCore or QuadCore Processor recommended Memory (RAM): 1 GB or more recommended Storage: 60 GB hard disk or larger recommended; DVD/CD-ROM drive Display: At least 1024x768 resolution; at least 16-bit color (65,000 colors), 32-bit color recommended 2 of 23

5 PathoProof Software - Instructions for use 1.1 Main functions The following icons located at the top of the application's main window give access to the main functions of Norden Lab Mastitis Studio: Import Run - Loads the run data to the database Instruments - Opens an instrumentt editor for adding a new real-time PCR instrument or to manage existing instruments Calibrate - Performs calibration for the selected real-time PCR instrument Reports - Creates reports on theselected run(s) Report templates - Opens report emplates for editing Legend - Listss the statuss icons used in the application windows with explanations In addition, the following functions are available in the application's main menu: License manager - Shows the current state of the software license Software updates - Accesses the software update pages on Archive database and Open Archive - Allows accesss to archive databases and open archived databases About - Shows general information about the application 2. Importing a run to the application See "Real-Time PCR instrument settingss and run" in the PathoProof assay's instructions for use in order to create a data file. 1 To start the Import run wizard, click the import run icon 1. If more than one kit or instrument has been defined, select the appropriate kit or instrument from the list. 3 of 23

6 PathoProof Software - Instructions for use Click Browse 2 to select the run file. Then click Next Selected sample 8 4 PCR Negative control Describe plate samples windoww allows you to inspect and edit sample names 4, to determine possible dilutions 5 and specify Extraction negative control (if included; you can also use the Extraction negativee control from another run) 6. If you did not specify a plate setup file, the samples are named according to the wells they occupy. For example, the first sample is named "A1-A4". When you have finished, click Next 7. Note: If you want to see a sample's position on the plate, click the Show plate icon 8. A windoww opens, indicating the location of the currently selected sample on the plate and the PCR Negative control. 9 In the next window, enter a name for the run (or use the default value) 9, and then click Finish 10 to save the run and close the wizard of 23

7 PathoProof Software - Instructions for use Note: PathoProof assays require separate software licenses. For example, you cannot import a run obtained with PathoProof Major kits or PathoProof Mycoplasma-8 kit if you have a license for Complete assays only. In this case, "Error importing data" message appears. To acquire license keys, please contact your local sales representative for more information. The default location of latestt run is at the top of the database. The database can be sorted by clicking the column name. Several runs can be simultaneously deleted from the database by holding the Shift key down on your keyboard and selecting the runs that you want to delete using left mouse click. Note: You cannot delete a run containing the Extraction negative control if there are other runs referring to it. 5 of 23

8 PathoProof Software - Instructions for use 3. Viewing the imported runs Beforee printing a report of an imported run, it is highly recommended thatt every sample is visually inspected in order to verify the key parameters described in Section 4. This Section contains technical instructions for inspecting the samples in Norden Lab Mastitis Studio. Open the run viewer either by double-clicking the run in the application's main screen or by clicking View run in the Import run wizard. The run viewer shows the resultss for each sample in the run. Grey background color of the bacterial target refers to over 90% prevalence of that target. Black background color refers to over 99% prevalencee of the target. Turquoise background color in sample name refers to dilution. Red dot under the + symbol refers to possible contamination issues noticed in the negative control that affect the result interpretation of this sample. Orange dot under the + symbol refers to possible contamination issues noticed in the negative control but it does not affect the result of this sample. Blue dot under the minus symbol refers to very low quantity of target present in the sample (see section 4.1 for more information). Blue minus refers to negative result The color of the + symbols represent the abundance of the detected bacterial targets: Red = bacterial DNA detected in high quantity. Orange = bacterial DNA detected in intermediate quantity. Green = bacterial DNA detectedd in low quantity. Run viewer for PathoProof Mastitis Complete-16 kit. 6 of 23

9 PathoProof Software - Instructions for use Run viewer for PathoProof Mastitis Major-3 kit. Further informationn on the results can be obtained by holding the mouse pointer over the target. To access the detailed information on each sample, you can open the sample viewer by double-clicking a sample in the run viewer. The sample viewer shows the real-time PCR amplification curves and Internal Amplification Controls (IACs) for each sample and for each real-time PCR reaction. In the sample viewer, you can switch between samples of the current run by clicking and or by choosing a sample in the dropdown menu between these arrows. You can zoom in on a part of the amplification curve by holding the left mouse button and dragging to the right. Zoom out by holding the left mouse button and dragging to the left. 7 of 23

10 PathoProof Software - Instructions for use The Show control above the table allows you to filter the table display to simplify the visual analysis. You can select Show positive targets to show only the targets for which the assay result was positive. Clicking the Report icon starts the Report wizard with the current sample preselected. When the sample viewer displays the Negative control, certain functions are disabled. For example, you cannot create report from only negative control. 4. Interpretation of the resultss with Norden Lab Mastitis Studio This Section gives instructions on the important parameters that should be inspected for each sample using the sample viewer of Norden Lab Mastitis Studio. Checklist for nspecting results For each sample, check the following parameters: Internal Amplification Controls (IAC) Amplification curves of the bacterial targets Negative Controls and their IAC For detailed instructions, see Sections Cycle threshold values The results of the PathoProof Mastitis PCR assays are based on cycle threshold (Ct) values obtained from the amplification curves of the bacterial targets. The Ct value represents the number of cycles required to reach a particular threshold fluorescence signal level. The fewer cycles it takes to obtain a detectable fluorescence level, the greater the amount of bacterial DNA in the milk sample. The specified Ct cut-off value of the PathoProof PCR assay is 37. This was chosen because obtaining a 3-cycle difference in target Ct, in comparison to the negative control Ct, is the generally accepted norm for reliable separation of a true positive signal from a contamination (Bustin, 2004). As the assay's thermal cycling protocol involves 40 cycles, therefore Ct 37 is the appropriate 3 cycle different Ct value. Ct values above 37, but below 40 may represent casess where target is present at very low levels. As it is it difficult to separate thesee results from possible minor contamination, it is recommended to consider Ct values higher than 37 as negativee with quarter milk samples. If however PathoProof assays are being used for screening purposes, it is recommended to use the whole 40 (1-40) cycle range. 8 of 23

11 PathoProof Software - Instructions for use 4.2 Internal Amplification Control An Internal Amplification Control (IAC) is included in the Primerr Mixes of all PathoProof kits. IAC consists of a control DNA template, a primer pair and a probe that willl result in an amplification reaction if the PCR was correctly performed. The purpose of the IAC is to confirm, for each sample and PCR reaction, that the reaction conditions were acceptable for the identification of the bacterial targets and that the plate setup (pipetting) was correctly performed. The graph on the lower left of the sample viewer shows the IAC in that sample. If the Ct values of the Internal Amplification Controls are not within the acceptable range, Norden Lab Mastitiss Studio displays the warning icon beside the sample name in the run viewer and in the upper left corner of the sample viewer. Additionally, the word "Failed" appears after the IAC Ct values in the sample viewer. If these warning messagess appear, refer to the "Troubleshooting" Section in the PathoProof assay'ss instructions for use for recommended action. 4.3 Real-time PCR amplification curves The amplification curves of true positive targets are exponential. Figure A shows an example of an ideal and figure B showss an abnormal amplification curve. Note: To remove the Ct of an abnormal curve from the analysis, open the sample viewer and select the corresponding target, then press the letter 'F' key on the keyboard. The Ct report. value of the selected target is discarded and no longer shown as positive in the Note: The Ct value of the target is permanently removed from the run. Figure A. Example of ideal amplification curves. Figure B. Example of abnormal curves that do not represent true amplification. 4.4 Negative (no template) control If DNA amplification occurs in negative control reactions (either in PCR Negative control or in Extraction negative control) ), the application shows a small red or orange exclamation mark as a warning sign for that target in all samples (in the sample viewer, Ct value for Extraction negative sample is in italic font and for PCR negative control in roman font). If the Ct value of the target in a negative control is less than 3 cycles apart from the Ct of the same target in a sample, that target in 9 of 23

12 PathoProof Software - Instructions for use that sample fails to qualify as positive and must be disregarded in the analysis. In the report, a warning is printed for such targets if the box Quality controls is checked (see Section 5 on creating reports). If the Ct values of the IACs in the negative controls are not within the acceptable range, the warning icon appears beside the name of the run in the application's main window and in the upper left corner of the run viewer. Additionally, the word "Failed" appears after the IAC Ct values in the sample viewer. If these warning messages appear, refer to the "Troubleshooting" Section in the PathoProof assay's instruction for use for recommended action. 4.5 Interpretation of Staphylococcus results PathoProof Complete assays detect the presence of Staphylococcus aureus and coagulase-negative staphylococci. The software will automatically determine if Staphylococcus spp. result is from S. aureus target and will exclude the Staphylococcus spp. result from the final report if this option is left unchecked when creating reports. If, on the other hand, the S. aureus results are negative and Staphylococcus spp. results positive, the sample is interpreted as containing Staphylococcus spp. (other than S. aureus). 4.6 Interpretation of Mycoplasma results PathoProof Mycoplasma-8 and Complete-16 assays detect the presence of M. bovis and Mycoplasma spp. In addition the Mycoplasma-8 detects M. alkalescens, M. bovigenitalium, M. californicum and M. canadense. The software will automatically determine if Mycoplasma spp. result is due to M.bovis, M.alkalescens, M.bovigenitalium, M. californicum or M. canadense target and will exclude the Mycoplasma spp. result from the final report if this option is left unchecked when creating reports. The sample is interpreted as containing Mycoplasma spp. (other than M. bovis, M. alkalescens, M. bovigenitalium, M. californicum, M. canadense) if the Mycoplasma targets are negative or the target is present in a very low quantity below the assay s detection limit. 4.7 Interpretation of β-lactamase results The presence of the β-lactamase gene (blaz, responsible for penicillin resistance) is clinically relevant only with S. aureus and Staphylococcus spp. While the blaz gene can be found also in some other species (for example, enterococci), there is no compelling scientific evidence to demonstrate its relevance in any other bacteria than staphylococci. Consequently, the PathoProof PCR assays should be used to designate a sample as penicillin resistant only if the sample is β-lactamase positive AND positive for S. aureus or Staphylococcus spp. 10 of 23

13 PathoProof Software - Instructions for use 5. Creating reports 1 Reports can be produced in four different formats: html,.xls,.txt or.csv. To start the Reports wizard, click the Reports 1 icon in the application's main window. Note: When the Reports wizard is launched from the run viewer or the sample viewer, the current run and sample are preselected for the report. In the first windoww of the Reports wizard, you can select the runs and sampless to be included in the report. To add a run or a sample to a report, select it in the Available runs/samples list and click. To remove a run or a sample from the selection, select it in Selected runs/samples and click. Once you have selected the runs or samples, proceed to the next window by clicking Next. In the Select report template window you can select a templatee for the report (see Section 7 on report templates) ). Once the template is selected, click Next to proceed to the next window In the next window, you can adjust the following parameters of the report: Choose whichh data to include in Include in report 2. For detailed instructions on nspecting the results, see Section 4. Negative targets - include negative targets Negative samples - include negativee samples +/- targets - include targets with Ct values between 37.1 and 40 Ct values - for each included target, show Ct value Quality controls - include quality controls Negative control - Include results from the Negative controls and Negative extraction controls in the report 11 of 23

14 PathoProof Software - Instructions for use Always include Beta-lactamase result for sample - β-lactamase result reported also for Staphylococcus spp. and Staphylococcus aureus negative samples. Note: This data applies only for PathoProof Mastitis Complete-12 and Complete-16 kits. Always include Staphylococcus spp. result for S. aureus - for each S. aureus result, Staphylococcus spp. result is reported. The software will automatically determine if Staphylococcus spp. result is from S. aureus target and will exclude the Staphylococcus spp. result from the final report if this option is left unchecked. Note: This data applies only for PathoProof Mastitis Complete-12 and Complete-16 kits. Always include Mycoplasma spp. result for Mycoplasma targets - for each M. bovis, M. alkalescens, M. bovigenitalium, M. californicum and M. canadense result, Mycoplasma spp. result is reported. The software will automatically determine if M. sp result is due to M.bovis, M.alkalescens, M.bovigenitalium, M. californicum or M. canadense target and will exclude the Mycoplasma spp. result from the final report if this option is left unchecked. Note: This data applies only for PathoProof Mastitis Complete-16 and Mycoplasma-8 kit. Sorting options 3 : Sort samples by Name - sample names are listed in alphabetical or numerical order, depending on how the samples have been named Sort samples by Sample location - samples are listed according to the sample order on the plate Sort samples by Number of positive targets - samples that have the greatest number of positive targets are listed first Sort targets by Name - targets are listed in alphabetical order Sort targets by Quantity - within a sample, the target having the greatest amount of DNA is listed first Report type - Specify the type of report to be generated in 4 : HTML file - file formatted for display in a web browser xls file - Microsoft Excel spreadsheet file txt file - text file, no formatting csv file - comma-separated value file 6 Specify a location for the report file 6. Open report in application ( 5, previous picture) - Once the report has been generated, start the application to display the report (Web browser, Microsoft Excel, text editor or csv, depending on the selected report type). 12 of 23

15 PathoProof Software - Instructions for use 6. Result categories in the reports In the reports, each bacterial target is assigned to one of five result categories depending on the Ct value. The table below describes these categories. Results category in the report Interpretation - Bacterial DNA not detected. +/- Bacterial DNA detected in quantity above the assay's cut-off value. + Bacterial DNA detected in low quantity. ++ Bacterial DNA detected in intermediate quantity. +++ Bacterial DNA detected in high quantity. In addition, when multiple bacterial targets are detected in a sample, the software reports the percentage of the most abundant bacterial species, if its proportion is over 90%. 7. Report templates Norden Lab Mastitis Studio contains several different report templates. You can also modify these to create your own report template. 1 Click the Report Templates main window. 1 icon in the 2 To edit an existing template, double-click the template name. To add a new template, click Add template 2. In the next dialog, you can change the template parameters and the name. For a description of the parameters, see Section of 23

16 PathoProof Software - Instructions for use A. Adding a new real-time PCR instrument to Norden Lab Mastitis Studio When you first start to use Norden Lab Mastitis Studio, you must add the real-time PCR instrument to the software. If this has already been done, proceed to Section B. 1 Click the Instruments 1 icon in the main window to open the instrument browser window. 2 Click Add instrument 2 in the instrument browser window to open an "Edit instrument" dialog. 3 Select the PathoProof application are using and click Next. 4 3 you 4 5 Select the instrument model and type a name for the new instrument 5. Then click Save 6 to save the newly added instrument in the database. To seee more details on the instrument model, click Show model information. 6 Click Close browser. 7 to close the instrument Note: If you use more than one kit configuration, repeat the procedure e described in Section A in order to add the new instrument for all kits of 23

17 PathoProof Software - Instructions for use B. Calibrating Norden Lab Mastitis Studio In order to obtain consistent performance with Norden Lab Mastitis Studio, it is necessary to calibrate the software with each real-time PCR instrument and reagent kit type when using PathoProof PCR assays for the first time. Calibration may also need to be performed when real-time PCR plastic type changes, real-time PCR reagent lot changes or when the real-time PCR instrument has undergone maintenance. After such activities, examine to confirm that the threshold values of the target are within the acceptable range (see Section B4.1). The need for calibration can be checked by re-running samples that have been run on the same instrument before plastic change or instrument maintenance. If the Ct-values differ more than 1 cycle, the instrument needs to be re-calibrated. If the calibration has already been done, proceed to Section 2. Calibration PCR protocol for PathoProof Complete assays is described in Section B1, for PathoProof Major assays in Section B2 and for PathoProof Mycoplasma-8 assays in Section B3. Instructions for instrument-specific real-time settings, performing a run and handling files are provided in Section B4. Instructions for calibrating the Norden Lab Mastitis Studio are provided in Section B5. Please complete the calibration before you start processing samples, as described in the DNA extraction protocol in your PathoProof assay's instructions for use. Note: the calibration runs and the experiment runs must be performed using the same real-time PCR instrument, the same type of vessels and the same sealing method (sealers or caps). B1. Calibration PCR setup for PathoProof Complete assays Make sure that all the reagents are thoroughly thawed. Vortex the PathoProof Master Mix and PathoProof Primer Mixes 1-4 briefly and spin down. 1. Prepare four separate Calibration PCR solutions by combining PathoProof Master Mix, PathoProof Complete Primer Mixes 1-4 and Universal amplification Standard in four separate microcentrifuge tubes. Calibration PCR solution 1: Calibration PCR solution 2: 40 µl PathoProof Master Mix 40 µl PathoProof Master Mix 20 µl PathoProof Primer Mix 1 20 µl PathoProof Primer Mix 2 20 µl PathoProof Universal 20 µl PathoProof Universal Amplification Standard Amplification Standard Calibration PCR solution 3: Calibration PCR solution 4: 40 µl PathoProof Master Mix 40 µl PathoProof Master Mix 20 µl PathoProof Primer Mix 3 20 µl PathoProof Primer Mix 4 20 µl PathoProof Universal 20 µl PathoProof Universal Amplification Standard Amplification Standard The formulas provide excess volume to compensate for volume loss due to reagent pipetting. 2. Vortex the PCR solutions briefly and spin down 15 of 23

18 PathoProof Software - Instructions for use 3. Prepare a 96-well PCR plate by dispensing 20 µl of each Calibration PCR solution into its respective wells as follows. Calibration PCR solution 1 into wells A1, B1 and C1 Calibration PCR solution 2 into wells A2, B2 and C2 Calibration PCR solution 3 into wells A3, B3 and C3 Calibration PCR solution 4 into wells A4, B4 and C4 4. Close the 96-well PCR plate with a compatible optically clear sealer or optically clear caps and spin the plate down with a plate centrifuge (3000rpm, 5 sec). Note that the experiment runs must be performed using the same real- time PCR instrument, the same type of vessels and the same sealing method. 5. Place the 96-well plate in a real-time PCR instrument and start the PCR program using the instrument-specific settings given in Section B4. B2. Calibration PCR setup for PathoProof Major assays Make sure that all the reagents are thoroughly thawed. Vortex the PathoProof Master Mix and PathoProof Primer Mix briefly and spin down. 1. Prepare a Calibration PCR solution by combining PathoProof Master Mix, PathoProof Major Primer Mix and PathoProof Universal Amplification Standard in a microcentrifuge tube. Calibration PCR solution: 40 µl PathoProof Master Mix 20 µl PathoProof Major Primer Mix 20 µl PathoProof Universal Amplification Standard The formula provides excess volume to compensate for volume loss due to reagent pipetting. 2. Vortex the PCR solution briefly and spin down. 3. Prepare a 96-well PCR plate by dispensing 20 µl of Calibration PCR solution into its respective wells A1, B1 and C1. 4. Close the 96-well PCR plate with a compatible optically clear sealer or optically clear caps and spin the plate down with a plate centrifuge (3000rpm, 5 sec). Note: the experiment runs must be performed using the same real-time PCR instrument, the same type of vessels and the same sealing method. 5. Place the 96-well plate in a real-time PCR instrument and start the PCR program using the instrument- specific settings given in Section B4. B3. Calibration PCR setup for PathoProof Mycoplasma-8 assay Make sure that all the reagents are thoroughly thawed. Vortex the PathoProof Master Mix and PathoProof Primer Mixes briefly and spin down. 1. Prepare two separate Calibration PCR solutions by combining PathoProof Master Mix, PathoProof Mycoplasma Primer Mixes 1-2 and Universal amplification Standard in two separate microcentrifuge tubes. 16 of 23

19 PathoProof Software - Instructions for use Calibration PCR solution 1: 40 µl PathoProof Master Mix 20 µl PathoProof Primer Mix 1 20 µl PathoProof Universal Amplification Standard Calibration PCR solution 2: 40 µl PathoProof Master Mix 20 µl PathoProof Primer Mix 2 20 µl PathoProof Universal Amplification Standard The formula provides excess volume to compensate for volume loss due to reagent pipetting. 2. Move to under table Vortex the PCR solution briefly and spin down. 3. Prepare a 96-well PCR plate by dispensing 20 µl of each Calibration PCR solution into its respective wells as follows. Calibration PCR solution 1 into wells A1, B1 and C1 Calibration PCR solution 2 into wells A2, B2 and C2 4. Close the 96-well PCR plate with a compatible optically clear sealer or optically clear caps and spin the plate down with a plate centrifuge (3000rpm, 5 sec). Note: the experiment runs must be performed using the same real-time PCR instrument, the same type of vessels and the same sealing method. 5. Place the 96-well plate in a real-time PCR instrument and start the PCR program using the instrument- specific settings given in Section B4. B4. Instrument-specific settings and instructions for run and file handling PathoProof assays are compatible with the following real-time PCR instruments. Complete-12 assay Agilent Mx3005P and Mx3000P QPCR Systems with MxPro - Mx3005P v4.10 software Complete-16 and all Major-assays Agilent Mx3005P QPCR System with MxPro - Mx3005P v4.10 software Mycoplasma-8 assay Agilent Mx3005P QPCR System with MxPro - Mx3005P v4.10 software Applied Biosystems 7500 and 7500 Fast Real-Time PCR System with 7500 Software v2.06 (Thermo Fisher Scientific) Please contact Thermo Fisher Scientific technical support: oxoid.techsupport@thermofisher.com for instrument specific template files. Save the template on the computer connected to the real-time PCR instrument. Below are separate instructions for each real-time PCR instrument. 17 of 23

20 PathoProof Software - Instructions for use B4.1 Applied Biosystems 7500 and 7500 Fast Real-Time PCR System (7500 Software v2.0.6, Mycoplasma-8 only) Please contact Thermo Fisher Scientific technical support: oxoid.techsupport@thermofisher.com for instrument specific template files. Save the template on the computer connected to the real-time PCR instrument. 1. Open template file In the real-time PCR instrument software, open the calibration template file (PathoProof_Mycoplasma-8_Calibration_template.eds). Wait until instrument has initialized itself. 2. Load the plate 3. Verify correct settings in the template file Verify that the settings for plate setup are as follows: S.aur, S.aga, My.sp, M.bov and IAC are in wells A1, B1 and C1 (these wells are marked in blue) M.bg, M.can, M.alk, M. can and IAC are in wells A2, B2 and C2 (these wells are marked in red). Verify that the settings for Run Method are as follows: Number of cycles: 40 Holding stage: Step1 95 C 10 min Cycling stage Step1 95 C 5 seconds Step2 60 C 60 seconds, Endpoint read (Collect data-symbol on step 2). 4. Save the run Save the calibration run file in separate location and with different file name for easier identification and to ensure, that the empty calibration template file is not written over. 5. Start the run Start the run by clicking the Start run -button in the Setup Screen. 6. Export raw data for the PathoProof software After the real-time PCR run, choose Analysis option from the left side of the screen if this has not yet been selected automatically. Select Export. Make sure that the following check boxes have been selected: Sample Setup Amplification Data Open file(s) when export is Save current settings as the default 18 of 23

21 PathoProof Software - Instructions for use Make sure that the following check boxes have been unselected: Raw Data Results Multicomponent Data Make sure that the file format is.xls and that the selection is One File. Select Start Export. Save the generated excel file to the computer on which Norden Lab Mastitis Studio is installed. Proceed as instructed in Section B5. B4.2 Agilent Mx3005P or Mx3000P QPCR System (MxPro - Mx3005P v4.10 software) *The following Mx3005P or Mx3000P QPCR System instrument filters are compatible with the PathoProof Mastitis PCR assays: FAM TM, HEX TM /JOE TM, ROX TM, CY5 TM and ATTO (for Mx3005P). If your instrument does not have these filters, please contact Thermo Fisher Scientific technical support: oxoid.techsupport@thermofisher.com. 1. Open template file In the real-time PCR instrument software, open the calibration template file (PathoProof_Mycoplasma-8_Calibration_template.mxp). Switch the instrument's lamp on by clicking the lamp icon. 2. Load the plate 3. Verify correct settings in the template file Before first run and after instrument maintenance, verify that the Filter Gain Settings are as follows (from the Instrument selection -> Filter Set Gain Settings). 1x for CY5, 1x for ROX, 2x for HEX/JOE 4x for FAM 4x for ATTO Also before first run and after instrument maintenance, verify from optics configuration that Dyes CY5, ATTO, ROX, JOE and FAM have been selected. Verify that the plate setup has all the eight targets and IAC active, and that filter sets CY5, ROX, HEX/JOE, FAM and ATTO have mark in the check boxes at right side of the screen (under the Collect fluorescence data). Verify that the settings for Thermal Profile Setup are as follows: Segment 1 10 min. at 95 C Segment 2 (40 cycles) 95 C 5 seconds 60 C 60 seconds, Endpoint read (End symbol on Step 2) 4. Save the run Save the calibration run file in separate location and with different file name for easier identification and to ensure, that the empty calibration template file is not written over. 19 of 23

22 PathoProof Software - Instructions for use 5. Start the run Start the run by clicking the Start run -button in the Thermal Profile Setup. 6. Export raw data for the PathoProof software After the real-timee PCR run: Click the Analysis button, then the Results tab. Select File menu from the top left Select Export Chart Export Chart Data to Text file Format 1 -- Vertically Grouped by Plot... Copy the resulting files to the computer on which Norden Lab Mastitiss Studio is installed. Proceed as instructed in Section B5. B5. Software calibration 1 Click the Calibrate icon calibration wizard. 1 to start the instrument Note: The instrument selection window does not appear if there is only one instrument in the application. In that case, the wizard starts with the Select calibration data file window. Click Browse 2 to navigate to the calibration data file generated with your real-time PCR instrument (for creating a calibration data file, see Section B4). 2 3 When you have selected the data file, click Next 3. Note: The Next button is disabled until you have selected a valid data file. 4 In the last screen of the Instrumentt calibration wizard, you can inspect the calculated thresholds for targets by clicking the View calibration run link. Refer to section B4.1 for more information. The information you type in the comments field 4 will be visible as notes attached to this calibration. Click Finish 5 to save the calibration data in the database. The wizard closes, and the main application window reappears of 23

23 PathoProof Software - Instructions for use When you have completed the calibration, you can begin processing samples. Start with DNA extraction as instructed in your PathoProof assay s instruction for use. Note: Calibration may fail if there is too much variation between the replicates or if the amplification control amplifies later than expected. If the calibration fails, a warning message appears. In such cases, redo the calibration. Refer to section B5.2 regarding different warning messages. B5.1 View Calibration Run After you have selected any of the replicates, amplification screen shows up. You can view the amplification of each target by checking the boxes next to the target. Acceptable range for a threshold line Each target should have a threshold line which locates approximately within the marked range of the amplification curve. If the threshold-level is clearly set elsewhere, something has gone wrong with the calibration. In case of threshold-level being elsewhere than within the acceptable range, please contact technical support. 21 of 23

24 PathoProof Software - Instructions for use B5.2 The Calibration Warning Messages There are three kinds of warning messagess that can appear due to failed calibration. 1. The calibration has failed due to remarkable late Ct values of the amplification control. Please repeat the calibration run or contact oxoid.techsupport@thermofisher.com for technical help. This warning appears when the amplification control has failed to amplify within the first 30 cycles. In such a case it is recommended to repeat the calibration run. Please contact Thermo Fisher Scientific technical support if the problem reoccurs. 2. The first calibration sample, target M. bovis has failed. This warning occurs when one of the replicates, in this examplee the M.bovis, does not amplify at all. In such a case it is recommended to repeat the calibration run. 3. The first calibration sample, target E.coli has large deviation (3.3) from averaged Ct (23.8). This warning occurs when one replicate, in this example the Escherichia coli, amplifies earlier or later (3.33 cycles) than rest of the replicates. It is recommended to repeat the calibration run. However, the calibration may be adequate if the threshold-levethreshold-level. is set on correct level. Refer to section B5.1 for the correct range of the 22 of 23

25 PathoProof Software - Instructions for use Permissible use This product is intended to be used for the purpose of detection and/or analysis of microorganisms in milk for quality assurance and quality control purposes (Food Testing Applications), as well as for identification or semi-quantification of microorganisms in raw material samples, process control samples or finished product samples of an industrial process for the purpose of detecting the presence, absence or amount either of a contaminant or of an intended component (Industrial Microbiology Applications). Trademark, copyright and license information 2015 Thermo Fisher Scientific Inc. All rights reserved. NordenLab Mastitis Studio General Edition is copyright of Norden Logic Oy. Mx3005P, MxPro and Agilent are registered trademarks of Agilent Technologies. Cy5 is a registered trademark of GE Healthcare. Microsoft, Windows and Vista are trademarks, or registered trademarks of Microsoft Corporation in the United States and/or other countries. Intel is a trademark of Intel Corporation in the U.S. and/or other countries. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. 1/2015/v3 Contact Information: +44 (0) oxoid.techsupport@thermofisher.com 23 of 23