Software manual Thermo Scientific PathoProof

Norden Lab Mastitis Studio Software

Instructions for use

PF0888A

Revision Date: January 22, 2015

Norden Lab Mastitis Studio Software - Instructions for use

Contents

Getting started with Norden Lab Mastitis Studio ......................................................... 1 1. Overview of Norden Lab Mastitis Studio ................................................................. 2

1.1 Main functions ................................................................................................... 3 2. Importing a run to the application ............................................................................ 3 3. Viewing the imported runs ....................................................................................... 6 4. Interpretation of the results with Norden Lab Mastitis 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 .......................................................................... 9 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 A. Adding a new real-time PCR instrument to Norden Lab Mastitis Studio .............. 14 B. Calibrating Norden Lab Mastitis Studio ................................................................ 15

B1. Calibration PCR setup for PathoProof Complete assays ................................ 15 B2. Calibration PCR setup for PathoProof Major assays ...................................... 16 B3. Calibration PCR setup for PathoProof Mycoplasma-8 assay .......................... 16 B4. Instrument-specific settings and instructions for run and file handling ............ 17

B4.1 Applied Biosystems 7500 and 7500 Fast Real-Time PCR System (7500 Software v2.0.6, Mycoplasma-8 only) ................................................. 18 B4.2 Agi lent Mx3005P or Mx3000P QPCR System (MxPro - Mx3005P v4.10 software) ............................................................................................................ 19

B5. Software calibration ......................................................................................... 20 B5.1 View Calibration Run ............................................................................. 21 B5.2 The Calibration Warning Messages .................................................... 22

PathoProof Software - Instructions for use

1 of 23 www.thermoscientific.com/contactus

Getting started with Norden Lab Mastitis Studio

See section AWhen 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 change in real-time PCR plastic type or instrument maintenance activities affect threshold values (see Section 8).

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

See section B1 to B3

See section B4

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

Analyze the results using Norden Lab Mastitis Studio software

See PathoProof instructions

for use

Result interpretation

See Sections 2-7

PathoProof Software - Instructions for use

2 of 23 www.thermoscientific.com/contactus

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 MxProTM - 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

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PathoProof Software - Instructions for use

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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.

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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 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 : • 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 Mastitis 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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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 Amplification Standard

• 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 • 20 µL PathoProof Universal Amplification Standard

• 20 µL 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

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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 StandardThe 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.

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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.

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.

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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

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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 Agi lent 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: FAMTM, HEXTM/JOETM, ROXTM, CY5TM 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.

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PathoProof Software - Instructions for use

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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) 1256 841144 oxoid.techsupport@thermofisher.com