DEVICE MANUAL Thermo Fisher Scientific

QuantStudioTM 3 / QuantStudioTM 5
Real-Time PCR System

Designed for GeneProof diagnostic kits

See www.geneproof.com for the current kits list

GeneProof a.s.
Vídeňská 101/119, 619 00 Brno – Dolní Heršpice, Czech Republic · info@geneproof.com
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CONTENTS

1. PURPOSE ................................................................................................................................................. 3

1.1. PCR REACTION PREPARATION .................................................................................................................3

1.2. DEVICE PROGRAMMING ............................................................................................................................3
1.3. PCR AMPLIFICATION START.....................................................................................................................4
1.4. QUALITATIVE ANALYSIS OF THE RESULT AND EVALUATION OF DETECTION ................................................8
1.5. RESULT QUANTITATIVE ANALYSIS AND DETECTION EVALUATION .............................................................14

2. GENETICAL DIAGNOSTICS .................................................................................................................. 16

2.1. DEVICE PROGRAMMING ..........................................................................................................................16

2.2. STARTING THE SOFTWARE ......................................................................................................................17
2.3. ANALYSIS OF THE RESULT AND EVALUATION OF DETECTION ...................................................................21

3. CUSTOMER SERVICE ........................................................................................................................... 29

4. CONTACT INFORMATION .................................................................................................................... 29

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1. Purpose
This device manual describes in detail the process of using GeneProof PCR kits for microbiological
diagnostics the following devices: QuantStudio 3 Real-Time PCR System and QuantStudio 5 Real-Time
PCR System.

1.1. PCR Reaction Preparation

Prepare PCR reaction according to the Instruction for use of the used GeneProof PCR kit.

1.2. Device Programming

In case the software does not include predefined templates, it is necessary, before the first use with
GeneProof PCR kits, to programme them according to the Instruction for use of the used GeneProof kits ,
or download them from the product site of the used GeneProof PCR kits from the website of the company
www.geneproof.com. Save the downloaded templates on your local disc and open them in the
QuantStudio™ Design & Analysis Software.

Fig. 1.1 Save template

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For easy searching is recommended to create a GeneProof file on the desktop.

1.3. PCR Amplification Start.

1.3.1 Starting the Software

1. Start the QuantStudio™ Design & Analysis Software.

2. Click the arrow next to the Create New Experiment button and choose Template.
3. Open file according to used GeneProof PCR kit.

Fig. 1.2 Open template

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1.3.2 PCR plate editing

1. In Properties tab, enter experiment name into the Name row.

2. In Plate tab, switch to Advanced Setup and use Add in Targets section to define targets according to
the kits used in the experiment.

E.g. for HSV detection (3 channels) set Target Name: HSV 1, Reporter: FAM, Quencher: None; Target
Name: HSV IC, Reporter: VIC, Quencher: None and Target Name: HSV 2, Reporter: Cy5, Quencher:
None. For MT detection (2 channels) set Target Name: MT, Reporter: FAM, Quencher: None and Target
Name: MT IC, Reporter: VIC, Quencher: None. Use Action -> Save to Library and Action -> Import
from Library to save and reuse targets.

3. Assign the appropriate targets for used wells by checking the boxes.
4. For Negative Controls set N in the Task column of targets.
5. For calibrators (in the case of quantitative detection) set S in the Task column of pathogen target and
enter the corresponding quantity in the Quantity column – according to the Package Insert of the used
GeneProof PCR kit, e.g.: 10 000, 1 000, 100 a 10.

Fig. 1.3 Assign targets

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6. Use Add in Samples section to define the samples used in the experiment.
7. In case of qualitative detection define positive control as the sample, e.g. Positive Control MT.
8. Assign the appropriate samples and controls for used wells by checking the boxes.

Fig. 1.4 Assign samples

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1.3.3 Starting the experiment

Save the experiment before starting the device.

1. Select File in the main menu, click Save and save the created experiment as the Test Document
Single files (*.eds) file type. To make search easier it is recommended to create the Experiments folder.

Fig. 1.5 Save experiment

2. In Run tab click button to start the experiment.

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1.4. Qualitative analysis of the result and evaluation of detection

PCR detection result evaluation must be always performed qualitatively first; if you use the PCR kit for
quantitative assessment, continue to quantify positive samples in the second step.

When the experiment is finished, Amplification Plot is displayed.

Fig.1.6 Amplification curves

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1.4.4 Analysis settings

1. Open Analysis Settings.

2. Uncheck Default Settings for all targets.
3. Uncheck Automatic Threshold and leave original value.
4. Uncheck Automatic Baseline and leave Start Cycle 3 and End Cycle 15.
5. Click Apply to confirm.

Fig.1.7 Analysis settings

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1.4.5 Detection analysis of the studied microorganism

1. In Show Plot Settings select the target microorganism (e.g. HBV) and select Graph Type Log.
2. Move the Threshold line just above the reaction basal noise.

Fig.1.8 Target and threshold set up

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3. In Show Plot Settings select Graph Type Linear.

Fig. 1.9 Amplification curves of the studied microorganism in linear scale

In the case of multiplex kit, follow the instructions for all the studied microorganisms.

Use button to switch to the Results table for Ct values.

Perform evaluation according to the Instruction for use of the used GeneProof PCR kit.

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1.4.6 Internal Standard detection analysis

1. In Show Plot Settings select the internal standard (e.g. HBV IC) and select Graph Type Log.
2. Move the Threshold line just above the reaction basal noise.

Fig. 1.10 Internal standard and threshold setting

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3. In Show Plot Settings select Graph Type Linear.

Fig. 1.11 Amplification curves of the internal standard in linear scale

Use button to switch to the Results table for Ct values.

Perform evaluation according to the Instruction for use of the used GeneProof PCR kit.

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1.5. Result quantitative analysis and detection evaluation

1. In Standard Curve, evaluate the calibration quality. The R2 parameter in a well-performed calibration
achieves a minimum value of 0.98 or higher. If the R2 parameter is lower than 0.98, move the
Threshold and repeat the analysis.

Fig. 1.12 Calibration curve

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2. Use button to switch to the Results table. Concentrations of positive samples are displayed in the
Quantity column of the table.

Fig. 1.13 Results table

Perform evaluation, including the virus concentration calculation according to the Instruction for use of the
used GeneProof PCR kit.

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2. Genetical diagnostics
This chapter describes in detail the process of using GeneProof PCR kits for genetic diagnostics using the
the following devices: QuantStudio 3 Real-Time PCR System a QuantStudio 5 Real-Time PCR System.

2.1. Device Programming

In case the software does not include predefined templates, it is necessary, before the first use with
GeneProof PCR kits, to programme them according to the Instruction for use of the used GeneProof kits,
or download them from the product site of the used GeneProof PCR kits from the website of the company
www.geneproof.com.
Save the downloaded templates on your local disc and open them in the QuantStudio™ Design & Analysis
Software.

Fig. 2.1 Save template

After saving, the template can be opened from the file GeneProof. With each next usage of GeneProof
PCR kits continue from the chapter 2.2 Starting the software.

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2.2. Starting the software

2.2.1 Opening of the saved template

1. Start the QuantStudio™ Design & Analysis Software.

2. Click the arrow next to the Create New Experiment button and choose Template.
3. Open file according to used GeneProof PCR kit.

Fig. 2.2 Open template

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2.2.2 PCR plate editing

1. In Properties tab, enter experiment name into the Name row.

2. In Plate tab, switch to Advanced Setup and use Add in Targets section to define targets according to
the kits used in the experiment.

E.g. for FII detection set Target Name: FII WT, Reporter: FAM, Quencher: None and Target Name: FII
MUT, Reporter: VIC, Quencher: None. For FV detection set Target Name: FV WT, Reporter: FAM,
Quencher: None and Target Name: FV MUT, Reporter: VIC, Quencher: None. Use Action -> Save to
Library and Action -> Import from Library to save and reuse targets.

3. Assign the appropriate targets for used wells by checking the boxes.
4. For Negative Controls set N in the Task column of targets.

Fig. 2.3 Assign targets

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5. Use Add in Samples section to define the samples used in the experiment.
6. Define positive controls as the samples, e.g. Positive Control WT, Positive Control MUT and Positive
Control HET.
7. Assign the appropriate samples and controls for used wells by checking the boxes.

Fig. 2.4 Assign samples

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2.2.3 Starting the experiment

Save the experiment before starting the device.

1. Select File in the main menu, click Save and save the created experiment as the Test Document
Single files (*.eds) file type. To make search easier it is recommended to create the Experiments
folder.

Fig. 2.5 Save experiment

2. In Run tab click button to start the experiment.

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2.3. Analysis of the result and evaluation of detection

When the experiment is finished, Amplification Plot is displayed.

Fig. 2.6 Amplification curves

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2.3.4 Analysis settings

1. Open Analysis Settings.

2. Uncheck Default Settings for all targets.
3. Uncheck Automatic Threshold and leave original value.
4. Uncheck Automatic Baseline and leave Start Cycle 3 and End Cycle 15.
5. Click Apply to confirm.

Fig. 2.7 Analysis settings

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6. In the plate, select only positive controls WT, MUT and HET of one evaluated kit (e.g. FXIII).
7. In Show Plot Settings select WT target of evaluated kit (e.g. FXIII WT).

Fig. 2.8 Select WT target

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8. Move the Threshold line so that only controls WT and HET are positive (the Threshold line intersects
only the Positive Control WT and Positive Control HET curves).

Fig. 2.9 WT Threshold settings

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9. In Show Plot Settings select MUT target of evaluated kit (e.g. FXIII MUT).

Fig. 2.10 Select MUT target

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10. Move the Threshold line so that only controls MUT and HET are positive (the Threshold line intersects
only the Positive Control MUT and Positive Control HET curves).

Fig. 2 MUT Threshold settings

11. Repeat steps 6. – 10. for the other kits in the experiment.

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2.3.5 Evaluation

1. Use button to switch to the Results table.

2. If there is a numerical value only for the WT target in the Ct column – this is a standard genotype;
numerical value only for the MUT target – this is a mutant genotype; numerical values for both targets
WT and MUT – this is a heterozygote genotype.

Fig. 2.12 Evaluation

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2.3.6 Examples of typical curves

Typical WT curve

Typical MUT curve

Typical HET curve

Fig. 2.13 Typical curves

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3. Customer Service
We appreciate all our customers and besides high-quality products we provide, in cooperation with
our partners, above-standard customer service including the following:

▪ Demonstration PCR kits

▪ Express deliveries
▪ Quick solution of issues related to the supplied products – service guaranteed within 24 hours from
the time of report
▪ Consultations concerning technological and clinical interpretations

To assure the quickest possible solution of any issue we always require the GeneProof PCR Kit
users to provide the following information:

▪ Kit name
▪ Issue definition
▪ Kit lot - specified on the kit package
▪ Used device
▪ File with the examination log from the used device, if available

4. Contact Information

Support and customer care Orders

Phone: +420 730 176 222 Phone: +420 543 211 679
e-mail: support@geneproof.com e-mail: sales@geneproof.com

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