Page 1 PF0888A PathoProof Software Norden Lab Mastitis Studio.pdf · Page 1 PF0888A PathoProof...

PF0888A PathoProof Software

Norden Lab Mastitis Studio

Instructions for use

Analyze the results using Norden Lab Mastitis Studio software

Calibrate Norden Lab Mastitis Studio software:

1. Perform real-time PCR calibration setup

2. Perform real-time PCR calibration run

3. Calibrate the real-time PCR instrument using Instrument calibration wizard

Getting started with Norden Lab Mastitis Studio

When using the system for the first time, add real-time PCR instrument to the software. See Section A

See Sections B1 and B2

See Section B3

See Section B4

See Sections 2–7

See PathoProof instructions

for use

Now the instrument and software are ready for samples

• Follow the instructions in your PathoProof assay’s instructions for use

• Calibrate when using the PathoProof Mastitis PCR assays for the first time

• Calibrate, if change in qPCR plastic type or instrument maintenance activities affect threshold values (see Section B).

1.2 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 instrument 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 the selected run(s)

Report templates – Opens report templates for editing

Legend – Lists the status 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 http://diagnostics.finnzymes.fi/pathoproof/ and http://www.nordenlogic.com/

Database settings – Allows you to change the location of the Mastitis Studio database

About – Shows general information about the application

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 Thermo Scientific PathoProof Mastitis PCR assays. The software is applicable for use with:

• PathoProof Mastitis Complete-12 kits together with 7500 Fast Real-Time PCR System (Applied Biosystems™), Stratagene Mx3005P™ or Mx3000P™ QPCR System (Agilent Technologies) and Chromo4™ Real-Time PCR Detection System (Bio-Rad Laboratories).

• PathoProof Mastitis Complete-16 kits together with Stratagene Mx3005P QPCR System.

• PathoProof Mastitis Major kits together with Stratagene Mx3005P or Mx3000P (for Major-3) QPCR System.

Norden Lab Mastitis Studio is highly recommended as an integral part of the working procedure for the PathoProof Mastitis PCR assays. Note: before you use PathoProof Mastitis 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 qPCR 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, 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

Describe plate samples window allows you to inspect and edit sample names 6, determine possible dilutions 7 and specify Extraction negative control (if included; you can also use Extraction negative from another run) 8. 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 9. Note: If you want to see a sample’s position on the plate, click the Show plate icon AT. A window opens, indicating the location of the currently selected sample on the plate.

2. Importing a run to the application

See “Real-Time PCR instrument settings and run” in your PathoProof assay’s instructions for use in order to create a data file.

Click Browse 2 to select the run file. Then click Next 3.

Only when using Applied Biosystems 7500 Fast Real-Time PCR System or Bio-Rad Chromo4 Real-Time PCR Detection System:

In the Select samples name file window, click Browse 4 to select the plate setup file (the file containing the names of the samples).

Note: This step is optional. If you do not specify a plate setup file, you can enter sample names in the next window. When you have finished, click Next 5.

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,

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Selected sample

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In the next window, enter a name for the run (or use the default value) AK, and then click Finish AL to save the run and close the wizard.

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Note: PathoProof assays require separate software licenses. For example, you cannot import a run obtained with Major kit if you have a license for Complete kit only. In this case, “Error importing data” message appears.

The default location of latest 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 as long as there are other runs referring to it.

3. Viewing the imported runs

Before printing a report of an imported run, you must inspect every sample visually and 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 results 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% prevalence of the target in question.

• Turquoise background color in sample name refers to dilution.

• 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 detected in low quantity.

Run viewer for PathoProof Mastitis Complete-12 kit.

Run viewer for PathoProof Mastitis Major-3 kit.

Further information 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.

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.

4. Interpretation of the results 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 inspecting results

For each sample, check the following parameters:

• Internal Amplification Controls (IAC)

• Amplification curves of the bacterial targets

• Negative Controls and their IACs

• For detailed instructions, see Sections 4.1–4.6.

4.1 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 recommended Ct cutoff value of the PathoProof Mastitis PCR assay is 37. It 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 involved 40 cycles, Ct 37 was the latest Ct value where the above requirement would be possible to meet. However, Ct values above 37 do not mean that the result is a false positive.

4.2 Internal Amplification Control

An Internal Amplification Control (IAC) is included in the Primer Mixes of all PathoProof Mastitis kits. IAC consists of a control DNA template, a primer pair and a probe that will 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 IACs in that sample. If the Ct values of the Internal Amplification Controls are not within the acceptable

range, Norden Lab Mastitis 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 messages appear, refer to the “Troubleshooting” Section in your PathoProof assay’s instructions for use for recommended action.

4.3 Real-time PCR amplification curves

The amplification curves of true positive targets are exponential. Figure 1 shows an example of an ideal and an abnormal amplification curve. 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 value of the selected target is discarded and no longer shown as positive in the report. Note: The Ct value of the target is permanently removed from the run.

Figure 1. A. Example of ideal amplification curves. B. Example of abnormal curves that do not represent true amplification.

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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 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 your PathoProof assay’s Instruction for user for recommended action.

4.5 Interpretation of Staphylococcus aureus and coagulase-negative staphylococci

PathoProof Mastitis PCR assays detect the presence of Staphylococcus aureus and coagulase-negative staphylococci. Interpret the results for S. aureus and Staphylococcus spp. as follows: If a sample gives positive results for the S. aureus reaction as well as for the Staphylococcus spp. reaction, S. aureus is present in the sample. If the Ct value of Staphylococcus spp. target is more than three cycles lower than that of S. aureus target, both S. aureus and Staphylococcus spp. are most likely present in the sample. 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 bovis and Mycoplasma spp.

PathoProof Mastitis PCR assays detect the presence of M. bovis and Mycoplasma spp. Interpret the results for M. bovis and Mycoplasma spp. as follows: If a sample gives positive results for the M. bovis reaction as well as for the Mycoplasma spp. reaction, M. bovis is present in the sample. If the Ct value of Mycoplasma spp. target is more than 4.6 cycles lower than that of M. bovis target, both M. bovis and Mycoplasma spp. are most likely present in the sample. If, however, the M. bovis result is negative and Mycoplasma spp. result positive, the sample is interpreted as containing Mycoplasma spp (other than M. bovis).

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 Mastitis 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. A positive blaZ result from the Major-4.1 assay can originate from Staphylococcus spp., which the assay does not differentiate. To check if the sample contains Staphylococcus spp, other than S. aureus, please use the PathoProof Mastitis Complete assays.

5. Creating reports

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 window of the Reports wizard, you can select the runs and samples 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 template for the report (see Section 7 on report templates). Once the template is selected, click Next to proceed to the next window.

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In the next window, you can adjust the following parameters of the report: Choose which data to include in Include in report 2. For detailed instructions on inspecting the results, see Section 4.

• Negative targets – include negative targets

• Negative samples – include negative 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

• Always include Beta-lactamase result sample – β-lactamase result reported also for Staphylococcus spp. 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. In case this option is left unchecked, Staphylococcus spp. target is not shown for S. aureus results unless the Ct value of Staphylococcus spp. target is more than three cycles lower than that of S. aureus target. In these cases, both S. aureus and Staphylococcus spp. targets are present in the reports. Note: This data applies only for PathoProof Mastitis Complete-12 and Complete-16 kits.

• Always include Mycoplasma spp. result for M. bovis – for each M. bovis result, Mycoplasma spp. result is reported. In case this option is left unchecked, Mycoplasma spp. target is not shown for M. bovis results unless the Ct value of Mycoplasma spp. target is more than 4.6 cycles lower than that of M. bovis target. In these cases, both M. bovis and Mycoplasma spp. targets are present in the reports. Note: This data applies only for PathoProof Mastitis Complete-16 kits.

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

Specify the type of report to be generated in Report type 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

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).

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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 below the assay’s cutoff 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.

Click the Report Templates 1 icon in the main window.

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 5.

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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.

Click the Instruments 1 icon in the main window to open the instrument browser window.

Click Add instrument 2 in the instrument browser window to open an “Edit instrument” dialog.

Select the PathoProof application 3 you are using and click Next 4.

Select the instrument model (5; only for PathoProof Mastitis Complete-12 kit) and type a name for the new instrument 6. Then click Save 7 to save the newly added instrument in the database. To see more details on the instrument model, click Show model information.

Click Close 8 to close the instrument browser.

Note: If you use more than one kit configuration, repeat the procedure described in Section A in order to add the new instrument for all kits.

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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 Mastitis PCR assays for the first time. Calibration may also need to be performed when qPCR plastic type 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, then the instrument needs to be re-calibrated. If the calibration has already been done, proceed to Section 2.

Calibration PCR protocol for PathoProof Mastitis Complete assays is described in Section B1, and for Major assays, in Section B2. Instructions for instrument-specific real-time settings, performing a run and handling files are provided in Section B3. Instructions for calibrating the Norden Lab Mastitis Studio are provided in Section B4. 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 Mastitis 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:

• 35μL PathoProof Master Mix

• 17.5μL PathoProof Primer Mix 1

• 17.5μL PathoProof Universal Amplification Standard












• 17.5μL PathoProof Universal 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

3. Prepare a 96-well PCR plate by dispensing 20μL of each Calibration PCR solution into its respective wells as follows.

• Calibration PCR solution 1 into 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 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 B3.

B2. Calibration PCR setup for PathoProof Mastitis 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 Mastitis Major Primer Mix and PathoProof Universal Amplification Standard in a microcentrifuge tube.

Calibration PCR solution:


• 17.5μL PathoProof Mastitis Major Primer Mix


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 20μL 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 B3.

B3. Instrument-specific settings and instructions for run and file handling

The PathoProof Mastitis Complete-12 kit is compatible with the following real-time PCR instruments: 7500 Fast Real-Time PCR System (Applied Biosystems), Stratagene Mx3005P or Mx3000P qPCR System (Agilent Technologies) and Chromo4 Real-Time PCR Detection System (Bio-Rad Laboratories). The PathoProof Mastitis Complete-16 and Major assays are compatible with the following real-time PCR instruments: Stratagene Mx3005P or Mx3000P (for Major-3) QPCR System (Agilent Technologies). Download a ready-made, instrument-specific calibration template file from http//diagnostics.finnzymes.fi/. Save the template on the computer connected to the real-time PCR instrument. Below are separate instructions for each real-time PCR instrument.

B3.1 Applied Biosystems 7500 Fast Real-Time PCR System

Instructions for Sequence Detection Software v 1.4

In the real-time PCR instrument software, open the calibration template file (Calibration_ template.sdt).

Verify that the settings are now as follows:

• Thermal Cycler Protocol:

1. Stage 1 (Reps 1)

a. 10 min. at 95°C

2. Stage 2 (Reps 40)

a. 5 sec. at 95°C

b. 1 min. at 60°C

3. Stage 3 (Reps 1)

a. 5 sec. at 25°C

• Sample Volume: 20μL

• Run Mode: Fast 7500

• Data Collection: During Stage 2, Step 2b (60.0°C @ 1 min.)

Save the file in .sds format. The instrument should now be connected to the software. Start the run by clicking the Start button.

After the calibration real-time PCR run, save the calibration data file as follows:

• Open the Results menu.

• Choose Amplification plot and click Analyze.

• Go to the File menu, choose Export and then Delta Rn.

• Save the file in .csv format.

• Copy this calibration data file to the computer on which Norden Lab Mastitis Studio is installed.

Proceed as instructed in Section B4.

B3.2 Stratagene Mx3OO5P or Mx3000 QPCR System* *The following Stratagene Mx3005P or Mx3000P QPCR System instrument filters are compatible with the PathoProof Mastitis PCR assays: FAM/SYBR Green, HEX/JOE/VIC, ROX/Texas Red, CY5 and ATTO (for Mx3005P). If your Stratagene instrument does not have these filters, please contact Thermo Fisher Scientific techsupport: [email protected].

In the real-time PCR instrument software, open the calibration template file (Calibration_ template.mxp). Switch the instrument’s lamp on by clicking the lamp icon.


• Gain settings: 1x for CY5, 1x for ROX, 2x for HEX-JOE, 4x for FAM and 4x for ATTO (Instrument -> Filter Set Gain Settings)

• Thermal Profile:

1. Segment 1 (1 cycle)

a. 10 min. at 95°C

2. Segment 2 (40 cycles)

a. 5 sec. at 95°C

b. 60 sec. at 60°C, Endpoint read (End symbol on Step 2b)

Verify that the lamp status field in the lower right corner of the window indicates “Lamp Ready” (green) or “Warm-Up”. Save the run file in .mxp format. Insert the PCR plate and press the Run button to start the run.

After the calibration run, save the calibration data file as follows.

• Click the Analysis button, then the Results tab.

• Go to the File menu and select Export Chart Data to Text file and Format 1 - Vertically Grouped by Plot....

• Save the file in .txt format.


Proceed as instructed in Section B4

B3.3 Bio-Rad Chromo4 Real-Time PCR Detection System

Instructions for Opticon Monitor 3.00

In the real-time PCR instrument software, open the calibration template file (Calibration_ template.mxp).


Plate Setup

• Plate type: clear

• For all wells that are in use:

– FAM, VIC, TXR and CY5 dye filters are defined

– All reactions are defined as sample (red)

– Data is collected from all four channels

Protocol Setup

• Temperature Control: Sample Calculation

• Lid Settings: Constant 100°C, Shutoff <30°C

• Reaction Volume: 20μL

• PCR protocol:

1. 10 min. at 95°C

2. 5 sec. at 95°C

3. 60 sec. at 60°C

4. Plate read

5. Go to line 2 for 39 more times

6. 5 sec. at 25°C

After completing the Plate Setup and Protocol Setup, save the data as a .tad file, insert the PCR plate and press the Run button to start the run.

After the calibration run, save the calibration data file as follows:

• Go to the Quantitation menu and choose Export.

• Clear the Interleaf Multiple Dyes box and activate all wells by clicking with the left mouse button.

• Save the file in .csv format.


Proceed as instructed in Section B4.

B4. Software calibration

Click the Calibrate icon 1 to start the instrument calibration wizard. 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 B3).

When you have selected the data file, click Next 3. Note: The Next button is disabled until you have selected a valid data file.

In the Select calibration Ct file window, click Browse 4 to select the calibration Ct file. Preferably use the Ct values file you have received from Thermo Fisher Scientific during instrument installation. If needed, Ct values file can be downloaded from http://diagnostics.finnzymes.fi. Then click Next 5.

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When you have finished downloading, click Next 6.

In the last screen of the Instrument calibration wizard, you can inspect the calculated thresholds for targets by clicking the View calibration run link. If the calibration yielded abnormally low or high threshold values, refer to section B4.1 to adjust the threshold values. The information you type in the comments field 7 will be visible as notes attached to this calibration. Click Finish 8 to save the calibration data in the database. The wizard closes, and the main application window reappears.

When you have completed the calibration, you can begin processing samples. Start with DNA extraction as instructed in your PathoProof instruction manual.

Note: Calibration may fail if there is too much variation between the replicates, for example. If calibration fails, a warning message appears. In such case, redo the calibration.

B4.1 Setting threshold values

After the calibration, each target should have a threshold line which locates approximately within the marked range of the amplification curve (see Figure 2).

Figure 2. Amplification curve with marked range for an acceptable threshold line.

Acceptable range for a threshold line

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If a threshold value is outside this range, you can adjust the value manually in Edit calibration Ct’s window by clicking the target and typing a new Ct value. Then click Next 1.

In the next window, click View calibration run link 2.

In the calibration run screen, choose a sample 3 for viewing the adjusted threshold values. If the threshold values are within the acceptable range, close the calibration run screen and click Finish 4. Otherwise, return to Edit calibration Ct’s window and readjust the Ct values as shown on top of the page, until the threshold values are within the acceptable range as illustrated in Figure 2.

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10/2011/v1

Contact Information: +44 (0) 1256 841144 [email protected] www.thermoscientific.com/finnzymes

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 enumeration 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). Not for use in veterinary diagnostic procedures.

Trademark, copyright and license information

Norden Lab Mastitis Studio General Edition is copyright of Norden Logic Oy. PathoProof is either a trademark or registered trademark of Thermo Fisher Scientific. Applied Biosystems is a trademark or registered trademark of Life Technologies Corporation. Mx3005P and Mx3000P are either trademarks or registered trademarks of Stratagene, Inc. Chromo4 and Opticon Monitor are either trademarks or registered trademarks of Bio-Rad Laboratories. Microsoft, Windows and Vista are either trademarks or registered trademarks of the Microsoft Corporation.

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