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Software manual

Thermo Scientific PathoProof

Norden Lab Studio

Instructions for use

PF0888A

Revision Date: January 11, 2016

PathoProof Norden Lab Studio Software - Instructions for use

Contents

Getting started with PathoProof Norden Lab Studio .................................................. 1

1. Overview of PathoProof Norden Lab Studio .......................................................... 2

1.1 Main functions.................................................................................................. 3

2. Importing a run to the application .......................................................................... 3

3. Viewing the imported runs ..................................................................................... 6

4. Interpretation of results with Norden Lab Studio .................................................... 8

4.1 Cycle threshold values ..................................................................................... 8

4.2 Internal Amplification Control ........................................................................... 9

4.3 Real-time PCR amplification curves ................................................................. 9

4.4 Negative (no template) control ....................................................................... 10

4.5 Interpretation of Staphylococcus results......................................................... 10

4.6 Interpretation of Mycoplasma results ............................................................. 10

4.7 Interpretation of β-lactamase results .............................................................. 10

5. Creating reports .................................................................................................. 11

6. Result categories in the reports ........................................................................... 13

7. Report templates ................................................................................................. 13

8. Adding a new real-time PCR instrument to Norden Lab Studio ........................... 14

9. Calibrating Norden Lab Studio............................................................................. 15

9.1 Calibration PCR setup for PathoProof Complete assays ................................ 15

9.2 Calibration PCR setup for PathoProof Major assays ...................................... 16

9.3 Calibration PCR setup for PathoProof Mycoplasma-8 assay .......................... 17

9.4 Instrument-specific settings and instructions for run and file handling ............ 17

9.4.1 Applied Biosystems 7500 and 7500 Fast Real-Time PCR System (7500 Software v2.0.6 or v2.3) ...................................................................... 18

9.4.2 Ag i lent Mx3005P or Mx3000P QPCR System (MxPro - Mx3005P v4.10 software) .......................................................................................................... 19

9.5 Software calibration ....................................................................................... 20

9.5.1 View Calibration Run ........................................................................... 21

9.5.2 Calibration Warning Messages .......................................................... 22

10. References ........................................................................................................ 22

PathoProof Software - Instructions for use

1 of 23 www.thermoscientific.com/contactus

Getting started with PathoProof Norden Lab Studio

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

Creating instruments

Calibrate when using the Thermo Scientific™ PathoProof™ PCR

assays for the first time

Calibrate, if there have been changes to the real-time PCR plastic

type

Calibrate if instrument has gone through maintenance

See section B1 to B3

1. Perform real-time PCR calibration setup

3. Perform real-time PCR

calibration setup

4. Perform real-time PCR

calibration run

5. Calibrate the real-time

PCR instrument using

Instrument calibration

wizard

Performing analyses

Instrument and software are

now ready for samples

Follow the instructions in the

PathoProof assay's instructions

for use

Analyze the results using

PathoProof Norden Lab

Studio software

See PathoProof instructions

for use

Result interpretation

See

Sections 2-7

See section 9.5

See section 9.4

See section 9.1 to 9.3

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Calibrating instruments



1. Overview of PathoProof Norden Lab Studio

Thermo ScientificTM PathoProofTM Norden Lab Studio is a software application designed for viewing, reporting and storing the results obtained using PathoProof PCR assays. The software is applicable for use with the following PathoProof kits:

PathoProof Complete - 12 kit

PathoProof Complete - 16 kit

PathoProof Major - 3 kit

PathoProof Major - 4.2 kit

PathoProof Mycoplasma - 8 kit

and with the following real-time PCR instruments:

Applied BiosystemsTM 7500 and Applied Biosystems™ 7500 Fast Real-Time PCR Systems with 7500 Software v2.0.6 and v2.3

AgilentTM Mx3000P and Mx3005P QPCR System with MxProTM - Mx3005P v4.10 software

Norden Lab 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 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 8 for instructions on adding a new instrument to the software. Instructions for performing calibration can be found in Section 9.

Norden Lab Studio has the following hardware and software requirements:

Operating System: Microsoft® Windows® 2000, 2003, XP, XP 64-Bit, Windows® 7, 8 and 10, 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



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 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 theselected 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 www.nordenlogic.com Archive database and Open Archive - Allows access 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 settings and run" in the PathoProof assay's instructions for use in order to create a data file.

To start the Import run wizard, click the import run icon . If more than one kit or instrument has been defined, select the appropriate kit or instrument from the list.

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

Describe plate samples window allows you to:

Inspect and edit sample name

Determine possible dilutions

Specify Extraction negative control (if included; you can also use the Extraction negative control from another run).

Inspect sample's position on the plate by selecting Show plate icon

Note: If you did not specify a plate setup file, the samples are named according to the wells they occupy. For example, the first sample will be named "A1-A4".

When you have finished, click Next .

Selected sample

PCR Negative control

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In the next window, enter a name for the run (or use the default value) , and then select Finish

to save the run and close the wizard.

Note: PathoProof assays require separate

software licenses. For example, you cannot import

a run obtained with PathoProof Major or

PathoProof Mycoplasma-8 kits 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 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 if there are

other runs referring to it.

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3. Viewing the imported runs

Before printing a report of an imported run, it is highly recommended that 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

PathoProof Norden Lab 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.

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 detected in low quantity.

Run viewer for PathoProof Complete-16 kit.



Run viewer for PathoProof 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. For

example, you cannot create report from only

negative control.

4. Interpretation of results with Norden Lab Studio

This Section gives instructions on the important parameters that should be inspected for each sample using the sample viewer of Norden Lab 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 IAC

• For detailed instructions, see Sections 4.1 - 4.7.

4.1 Cycle threshold values

The results of the PathoProof 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.

It is generally accepted that a 3-cycle difference in target Ct compared to the negative control Ct will reliably separate a true positive signal from a contamination result (Bustin, 2004). As the PathoProof assay's thermal cycling protocol involves 40 cycles, Ct 37 is the appropriate 3 cycle different Ct value, and with the exception of Staphylococcus spp. and beta-lactamase (see note below), the specified Ct cut-off value of the PathoProof PCR assays has been set at 37. Ct values above 37, but below 40 may represent cases where target is present at very low levels. As it is it difficult to separate these results from possible low level contamination results, it is recommended that Ct values higher than 37 are considered as negative with quarter milk samples. If however PathoProof assays are being



used for screening purposes, it is recommended that the whole 40 (1 - 40) cycle range is used.

Important Note: The Ct cut-off for coagulase-negative staphylococci has recently been reduced to 34.0 instead of 37.0 due to the fact that late Staph. species Cq values may not have clinical relevance to mastitis (Hiitiö et al 2015). Similarly the Ct cut-off for β-lactamase gene is now reduced to 36.0 instead of 37.0.

4.2 Internal Amplification Control

An Internal Amplification Control (IAC) is included in the Primer Mixes of all PathoProof 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 IAC in that sample. If the Ct values of the Internal Amplification Controls are not within the acceptable range, Norden Lab 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 the 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 A shows an example of an ideal and figure B shows 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 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 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 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.



5. Creating reports

Reports can be produced in four different formats: html, .xls, .txt or .csv. To start the Reports wizard, click the Reports 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.

In the next window, you can adjust the following parameters of the report: Choose which data to include in Include in report . 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

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• 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 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 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 Complete-16 and Mycoplasma-8 kit.

Sorting options : • 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 : • 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 .

Open report in application ( , 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 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 Studio contains several different report templates. You can also modify these to create your own report template.

Click the Report Templates icon in the main window.

To edit an existing template, double-click the

template name. To add a new template, click Add template .

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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8. Adding a new real-time PCR instrument to Norden Lab Studio

When you first start to use Norden Lab Studio, you must add the real-time PCR instrument to the software. If this has already been done, proceed to Section 9.

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

Click Add instrument in the instrument browser window to open an "Edit instrument" dialog.

Select the PathoProof application you are using and click Next .

Select the instrument model and type a name for the new instrument . Then click Save to save the newly added instrument in the database. To see more details on the instrument model, click Show model information.

Click Close to close the instrument browser.

Note: If you use more than one kit configuration, repeat the procedure described in Section 8 in order to add the new instrument for all kits.

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9. Calibrating Norden Lab Studio

In order to obtain consistent performance with Norden Lab 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 9.5.1). The need for calibration can be checked by re-running samples that have been run on the same instrument before the plastic change or instrument maintenance. If the Ct-values differ more than 2 cycles, the instrument needs to be re-calibrated. If the calibration has already been done, proceed to Section 2.

The calibration runs and the experiment runs must be performed using the same real-time PCR instrument, the same type of vessels and the wells needs to be sealed with optical clear caps.

Calibration PCR protocols for:

PathoProof Complete assays are provided in Section 9.1,

PathoProof Major assays are provided in Section 9.2,

PathoProof Mycoplasma-8 assays are provided in Section 9.3.

Instructions for:

instrument-specific real-time settings, performing a run and handling files are provided in Section 9.4.

calibrating the Norden Lab Studio are provided in Section 9.5.

9.1 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 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



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 20 µL 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 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 type of caps.

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 9.4.

9.2 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


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 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 9.4.



9.3 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.

2. 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.



4. Close the 96-well PCR plate with a compatible 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 9.4.

9.4 Instrument-specific settings and instructions for run and file handling

All PathoProof kits are compatible with the following real-time PCR instruments.

Applied Biosystems 7500 and 7500 Fast Real-Time PCR System with 7500 Software v2.06 or 2.3 (Thermo Fisher Scientific)

Agilent Mx3005P QPCR System with MxPro - Mx3005P v4.10 software

In addition, PathoProof Complete-12 and Major-3 kits are also compatible with Agilent Mx3000P instrument. Instrument specific template file needed for PathoProof calibration and sample runs will be installed into PathoProof Norden Lab Studio installation directory and link to these files will be generated into desktop during installation process.

Calibration PCR solution 1:


• 20 µL PathoProof Primer Mix 1


Calibration PCR solution 2:


• 20 µL PathoProof Primer Mix 2


The formula provides excess volume to compensate for volume loss due to reagent pipetting.



9.4.1 Applied Biosystems 7500 and 7500 Fast Real-Time PCR System (7500 Software v2.0.6 or v2.3) 1. Open template file

Select correct PathoProof kit folder to be used from your desktop and open the PathoProof

calibration template file. Wait until instrument has initialized itself.

2. Load the plate

3. Verify correct settings in the template file Verify that the correct PathoProof kit targets are selected in the Assign Targets and

Samples tab.

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

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 Studio is installed. Proceed as instructed in Section 9.5.



9.4.2 Agi lent Mx3005P or Mx3000P QPCR System (MxPro - Mx3005P v4.10 software)

*The following Mx3005P and Mx3000P QPCR System instrument filters are compatible with the PathoProof PCR assays: FAMTM, HEXTM/JOETM, ROXTM, CY5TM and ATTO (for Mx3005P). If your instrument does not have these filters, please contact Thermo Fisher Scientific technical support: [email protected].

1. Open template file

Select correct PathoProof kit folder to be used from your desktop and open the PathoProof

calibration template file. Wait until instrument has initialized itself. 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, 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.

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-time 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 Studio is installed. Proceed as instructed in Section 9.5.

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9.5 Software calibration

Click the Calibrate icon 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 to navigate to the calibration data file generated with your real-time PCR instrument (for creating a calibration data file, see Section 9.4).

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

In the last screen of the Instrument calibration wizard, you can inspect the calculated thresholds for targets by clicking the View calibration run link. Refer to section 9.4.1 for more information. The information you type in the comments field will be visible as notes attached to this calibration. Click Finish 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 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 9.5.2 regarding different warning messages.

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9.5.1 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.

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.

Acceptable range for a threshold line



9.5.2 Calibration Warning Messages

There are three kinds of warning messages that can appear due to failed calibration.

1. “Calibration has failed due to remarkable late Ct values of the amplification control. Please repeat the calibration run or contact [email protected] for technical support.”

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: [email protected].

2. “The first calibration sample, target M.bovis has failed.”

This warning occurs when one of the replicates, in this example 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 (3.3 cycles) than rest of the replicates. It is recommended to repeat the calibration run. However, the calibration may be adequate if the threshold-level is set on correct level. Refer to section 9.5.1 for the correct range of the threshold-level.

10. References

Bustin, S. A. 2004. Quantification of nucleic acids by PCR. IUL Press, La Jolla, CA.

Heidi Hiitiö, Rauna Riva, Tiina Autio, Tarja Pohjanvirta, Jani Holopainen, Satu Pyörälä and Sinikka Pelkonen 2015. Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture. Journal of Dairy Research 82 200 - 208



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. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. NordenLab 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. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Contact Information: +44 (0) 1256 841144 [email protected]

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mailto:microbiology.techsupport.uk@thermofish

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