Thermo Fisher Scientific MycoSEQ User Manual (2024)

Contents

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Prepare the sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Prepare the kit reagents and premix solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Prepare the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Setup, run, and anlayze samples with AccuSEQ™ Software v3.1 on the

QuantStudio™ 5 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Sign in to the AccuSEQ™ Real Time PCR Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Create a MycoSEQ™ experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Start the run (QuantStudio™ 5 Real Time PCR Instrument) . . . . . . . . . . . . . . . . . . . . . . 16 Monitor the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Guidance for unknown samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Guidance for controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Example results with AccuSEQ™ Software v3.1 or later . . . . . . . . . . . . . . . . . . . . . . . . . 28

Cycle threshold value (Ct) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Melting temperature (Tm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Derivative value (DV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Results interpretation using the acceptance criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Other acceptance criteria considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Start the run (7500 Fast Real Time PCR Instrument) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Set the baseline and threshold values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Review the Results Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Guidance for unknown samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Guidance for controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Guidance for inconclusive results with AccuSEQ™ software v2.0 . . . . . . . . . . . . . . . . 52 Example results with AccuSEQ™ Software v. 2.1.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Positive control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Negative control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Blank extraction control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Positive extraction control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Inhibition control and positive control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Unknown sample: Negative result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Unknown sample: Positive result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Unknown sample: Positive result with decreased detection of DPC . . . . . . . . . . . . . . 60 PCR inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Multicomponent plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Set the baseline and threshold values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Guidance for unknown samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Guidance for controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Example positive results with SDS v1.4 software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix in this document.

Product description

The MycoSEQ™ Mycoplasma Real-Time PCR Detection Kit detects Mycoplasma species simply, reliably, and rapidly. To detect the presence of these microorganisms, the assay uses the polymerase chain reaction (PCR) to amplify a target unique to a wide variety of Mycoplasma species.

Contents and storage

Table 1 MycoSEQ™ Mycoplasma Real-Time PCR Detection Kit (Cat. No. 4460623)

[1]To purchase the MycoSEQ™ Mycoplasma Real-Time PCR Detection Kit that includes the PrepSEQ™ Mycoplasma Sample Preparation Kit, use Catalog Number 4460626.

[2]The kit contains reagents for 100 reactions.

Workflow

Prepare the sample (page 9)

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Prepare the kit reagents and premix solution (page 9)

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Prepare the PCR reactions (page 63)

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Create a MycoSEQ™ experiment (page 12)

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Start the run (QuantStudio™ 5 Real Time PCR Instrument) (page 16)

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Monitor the run (page 17)

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Analyze the results (page 18)

IMPORTANT! This chapter describes how to prepare and run PCR samples using AccuSEQ™ Software v3.1 or later on the QuantStudio™ 5 Instrument (0.1 mL block). If you are using AccuSEQ™ software v2.x on the 7500 Fast, see Appendix C, “Use the kit with 7500 Fast System AccuSEQ™ Real-Time PCR Detection Software v2.x”. If you are using SDS software v1.4 or later, see Appendix D, “Use the kit with 7500 System SDS Software v1.4 or later”.

IMPORTANT! For information on how to avoid PCR contamination, see Appendix G, “Good PCR practices”.

Prepare the sample

Prepare the DNA template for the PCR reactions using the PrepSEQ™ Mycoplasma

Nucleic Acid Extraction Kit.

For more information, see:

•The PrepSEQ™ Sample Preparation Kits for Mycoplasma, MMV, and Vesivirus User Guide (Pub. No. 4465957)

•The PrepSEQ™ Express Nucleic Acid Extraction Kit for Mycoplasma, MMV, and Vesivirus Detection User Guide (Pub. No. MAN0016799)

Prepare the kit reagents and premix solution

1.Thaw all kit reagents completely.

2.Vortex briefly, then spin down the reagents.

Prepare the PCR reactions

1.Dispense the following into each well to be used, gently pipetting at the bottom of the well.

Note: The MycoSEQ™ Mycoplasma Discriminatory Positive/Extraction Control can be used as a spike control that is added to the unknown sample or lysate before sample preparation.

2. Mix each sample by gently pipetting up and down.

3.Seal the plate with MicroAmp™ Optical Adhesive Film. See “Seal the plates” on page 65.

4.Briefly centrifuge the reaction plate.

Setup, run, and anlayze samples with AccuSEQ™ Software v3.1 on the QuantStudio™ 5 Instrument

Sign in to the AccuSEQ™ Real Time PCR Software

Thermo Fisher Scientific recommends configuring the Windows™ 10 screen save feature to require sign in when the screensaver is activated. This prevents other users from accessing the AccuSEQ™ Real Time PCR Software and making changes.

1.Launch the AccuSEQ™ Real Time PCR Software by double-clicking the AccuSEQ icon .

2.Enter the Username, then Password.

(First login only) The default username is Administrator and the default password is Administrator.

3.Click Sign in.

The following restrictions may be seen in the software:

•Access to functions in the software is based on the permissions associated with the individual user account.

•If a user account does not have permission to perform a function, the function is grayed out in the software.

•If the system is configured for password expiration, you will be periodically prompted to change your password. If the system is configured to monitor failed log in attempts, you will be locked out of the software if you incorrectly enter your user name or password more than a specified number of times.

Create a MycoSEQ™ experiment

1.In the Home screen, click the Factory default/Admin Defined Template tab, then select MycoSEQ.

Note: To create a copy of an existing MycoSEQ™ experiment, see chapter 6 of the AccuSEQ™ Real Time PCR Software v3.1 User Guide (Pub. No. 100094287).

Custom MycoSEQ™ experiments must be set up as Quantitation-Standard Curve type experiments with SYBR™ chemistry.

2

1Factory default/Admin Defined Template tab

2MycoSEQ template

2.In the Experiment Properties pane of the Setup tab:

a.(Optional) Change the system generated name of the experiment.

b.(Optional) Enter the plate Barcode, then add Comments. Note: Comments are not editable post analysis.

Default MycoSEQ™ settings (These settings cannot be changed in the default template. See the AccuSEQ™ Real Time PCR Software v3.1 User Guide (Pub. No. 100094287) for creating custom templates).

•Experiment Type—Quantitation-Standard Curve

•Chemistry—SYBR™ Green Reagents

•Ramp Speed—Standard - 2hrs

c.Click Next.

3.In the qPCR Method pane of the Setup tab, view the default volume and cycling conditions (These settings cannot be changed in the default template. See the AccuSEQ™ Real Time PCR Software v3.1 User Guide (Pub. No. 100094287) for creating custom templates).

Figure 1 MycoSEQ™ template default cycling conditions

4.Click Next.

5.In the Samples pane of the Setup tab, enter the sample Name. Add additional Samples if needed.

Note: Only the sample Name is necessary for experiments run from the factory default MycoSEQ template.

IMPORTANT! Do not change the Targets.

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1

1Samples pane

2Add—adds additional samples

7

Figure 2 MycoSEQ™ template default sample plate layout

1Toolbar (in order: Undo, Redo, Copy, Paste, Delete, View)

25 default Samples with and without an inhibition control (IC)

3Positive control (P)

4Selected samples. Click-drag to add additional samples.

5No template control (N)

6.(Optional) Double-click wells to add Comments. Comments can also be added post-analysis.

7.Click Next.

The Run tab is displayed.

8.Experiments are auto saved in the software. To save additional changes made to the experiment, exit the experiment. The software prompts you to save changes. Click Yes.

Note: Clicking Save As will create a copy of the experiment.

Start the run (QuantStudio™ 5 Real Time PCR Instrument)

Start the run in the AccuSEQ™ Software v3.1 or later.

1

Monitor the run

During the instrument run, you can monitor the run from the following places:

•On the instrument touchscreen.

•In the Monitor the Run pane of the AccuSEQ™ Software (Home) screen.

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4

5

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6

1Instrument name

2Instrument status (Ready, Running, Offline)

3Calibration status

–Calibrated—All required calibrations are "Current". Required calibrations include: Background, ROI/Uniformity, and Dyes.

–Not calibrated—None of the required calibrations are complete.

–Requires calibration—One or more dyes are not calibrated.

4Time lapsed (if a run is in progress)

5Total run time

6Experiment name

•In the Open Existing Experiments pane of the AccuSEQ™ Software (Home) screen. The experiment being run is the first experiment listed. Status is Run.

Note: You cannot start another run while the instrument status is Running.

When the run is complete, the status changes to Analysis and the status bar displays as complete.

1

2

1Open Existing Experiments pane

2Experiment status

•In the Run tab of the AccuSEQ™ Real Time PCR Software. You can perform the following actions.

–Select wells in the plate layout to highlight respective curves in the plot.

–Hover over curves in the plot for well information.

–(Optional) Change what is displayed in the table wells, by selecting Sample Name, Sample Color, or Target in the View dropdown list.

On run completion, the Post Run Summary displays the run length, user and instrument information, and a list of any errors that occurred.

When the run is complete, unload the plate from the instrument.

Analyze the results

IMPORTANT! The acceptance criteria that are provided in this section are based on our current knowledge of assay performance in detection of Mycoplasma recovered from a wide variety of unknown sample matrices. We recommend that you qualify and validate the assay internally using samples that are specific to your process and manufacturing environment (raw materials, bioreactor, or cell line samples) to ensure that these criteria are appropriate.

For specific sample types, it may be necessary to make slight changes to the acceptance criteria based on specific results. We can provide you with one-on-one support during this process.

Workflow: Review MycoSEQ™ experiments

“View the Result Summary” on page 19

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“Evaluate the overall shape of the Amplification Plot curves” on page 20

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“View and evaluate the Melt Curve Plot” on page 22

See the AccuSEQ™ Real Time PCR Software v3.0 User Guide (Pub. No. 100084348) for more information on analyzing results.

View the Result Summary

The Result tab is populated when the run is complete.

Calls are made based on the customizable Rule Settings within the Analysis Settings for MycoSEQ™ experiments. See the AccuSEQ™ Real Time PCR Software v3.1 User Guide (Pub. No. 100094287) for more information.

1.View the Plate Call.

a.View the calls for the positive controls (POS).

b.View the calls for the no template controls (NTC).

The number inside the circle indicates the number of samples that passed or failed. The overall Plate Status (VALID or INVALID) is determined by the POS and NTC calls.

Note: One passing POS and NTC call are required for a Plate Status to be VALID. This requirement is not editable.

1Plate Call

2Positive controls (POS)

3Plate Status

4No Template Controls (NTC)

2.View the Well Calls (Unknown).

a.View the total number of wells for each call—Present, Absent, Review, or Fail.

b. Use the Legends to view the calls in the plate

Figure 3 Example MycoSEQ™ Results (Grid View)

1Results pane

2Well call

3Flags—the number within the triangle indicates the number of QC flag calls in the well; review in QC Summary.

4Grid View

Evaluate the overall shape of the Amplification Plot curves

You can evaluate the overall shape of the Amplification curves in the Result tab. Evaluating the amplification curve in wells with a Review call can help determine if the sample should be rerun, or whether to edit the call to Present or Absent.

Ensure that the experiment is open in the AccuSEQ™ Software.

1.In the Results Analysis pane of the Result tab, click Amplification in the horizontal scroll bar.

Note: If no data are displayed in the Result Analysis pane, then click Analyze.

1 Amplification Plot

The Amplification Plot is displayed for the selected wells in the (Grid View) .

2.Ensure that the Target selected is Mycoplasma.

3.(Optional) Click (Settings) , then make the following selections:

•Plot Type: ΔRn, Rn, or CT

•Graph Type: Log or Linear

•Show: Legend, Cq Mark (the cycle at which the curvature of the amplification curve is maximal), or Unselected

•Plot Color: Target, Sample, Well, or Flag_Status

•Threshold: Select Auto or specify a threshold, then decide whether to Show Threshold.

•Baseline: Decide whether to Show Baseline.

4.(Optional) Adjust the (Plot Properties).

a.(Optional) In the General tab, add a Plot Title, adjust the Font and Color, then click Apply.

b.In the X Axis or Y Axis tabs, you can:

•Add a Label

•Select whether you want Tick Marks

•Select Auto-adjust range or enter minimum and maximum values Note: The minimum value must be greater than 0.

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4

Figure 4 Typical Amplification Plot (4 phases)

View and evaluate the Melt Curve Plot

You can view and evaluate the Melt Curve Plot in the Result Analysis window in the Result tab.

1Melt Curve plot

2Melt Curve Stage dropdown

1.In the Result Analysis window, select Melt Curve Plot from the horizontal scroll bar.

2.Keep the default Melt Curve Stage.

Custom experiments with multiple data collections in the Melt Curve Stage, can select the stage that they want displayed.

1Stage 3 data collection point

3.Click to configure the plot, then make the following selections:

•Targets: Select Mycoplasma.

• Plot Settings: Select the Plot Type and Plot Color.

• Plot Properties: Edit Plot Title, change fonts, colors, and labels.

• Save Image: Save the image (PNG or SVG).

•Use the select, pan, and zoom options to interact with the plot.

The Melt Curve Plot is displayed for data points that are selected in the plot settings. The data points for selected wells in the Grid View or Table View are highlighted in the plot.

4.(Optional) View the default Melt Curve Stage.

5.Confirm that amplification in the no template control and IC control wells is as expected. Use one of the following options:

•Select control wells in the Grid View or Table View, then confirm the location of the data points in the Melt Curve Plot.

•View the amplification plots for the no template controls.

6.In the Melt Curve Plot, view the signal intensity and calls for the unknown samples.

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2

Figure 5 Example Melt Curve Plot

1Melting temperature (Mycoplasma)

2Melting temperature (positive control)

Guidance for unknown samples

The table shows criteria for positive and negative calls. A positive call indicates that at least one genome copy of Mycoplasma DNA was present in the unknown reaction and the sample is positive for the presence of Mycoplasma.

Note: Tm and DV assay acceptance criteria are only relevant if Ct value for present acceptance criteria are met. The AccuSEQ™ Software v3.1 flags these as "Review".

Table 2 Example acceptance criteria for unknown samples: AccuSEQ™ Software v3.1 or later

Figure 6 Decision tree for unknown sample calls (with or without an inhibition control [IC])