PyroMark Q24 MDx Accessories and Reagents

For use with the PyroMark Q24 MDx

Products

PyroMark Q24 MDx Accessories are intended for in vitro diagnostic use.

PyroMark Annealing Buffer (250 ml)

Cat. No. / ID: 979309

For annealing sequencing primer to single-stranded PCR product and for Pyrosequencing reaction

€59.90

Quantity

PyroMark Denaturation Solution (500 ml)

Cat. No. / ID: 979307

For denaturation of double-stranded PCR product into single-stranded template DNA

€59.90

Quantity

PyroMark Q24 Gold Reagents (5 x 24)

Cat. No. / ID: 971802

For 5 x 24 samples for use on the PyroMark Q24 MDx: Enzyme Mixture, Substrate Mixture, and nucleotides

€652.00

Quantity

PyroMark Q24 Cartridge (3)

Cat. No. / ID: 979302

Cartridge for dispensing nucleotides and reagents

€417.00

Quantity

PyroMark Q24 Control Oligo

Cat. No. / ID: 979303

For installation check of PyroMark Q24 MDx system

PyroMark Binding Buffer (200 ml)

Cat. No. / ID: 979306

For binding of biotinylated PCR product to Sepharose beads

€59.90

Quantity

PyroMark Q24 MDx Vacuum Workstation

Cat. No. / ID: 9001517

Vacuum Workstation (220 V) for preparing 24 samples in parallel, from PCR product to single-stranded template

Inquire

PyroMark Q24 Plate (100)

Cat. No. / ID: 979301

24-well sequencing reaction plate for use with PyroMark Q24 MDx; 100 plates in each package

€200.00

Quantity

PyroMark Q24 Validation Oligo

Cat. No. / ID: 979304

For performance check of PyroMark Q24 MDx system

PyroMark Wash Buffer concentrate (200 ml)

Cat. No. / ID: 979308

For washing of single-stranded DNA

€59.90

Quantity

PyroMark Q24 Vacuum Prep Troughs (12)

Cat. No. / ID: 979206

Reusable plastic troughs for use with all PyroMark Q24 Vacuum Workstations

€32.60

Quantity

PyroMark Vacuum Prep Filter Probe (100)

Cat. No. / ID: 979010

Reusable filter probes for PyroMark Vacuum Workstation Q96 and Q24

€293.00

Quantity

Features

CE-marked as compliant with EU IVD Directive 98/79/EC
Highly accurate results for in vitro diagnostics
Reliable quantification of allele representation
Sequence information enables discovery of rare mutations
1–24 samples can be analyzed in as little as 15 minutes

Product Details

The PyroMark Q24 MDx uses proven Pyrosequencing technology for real-time, sequence-based detection and quantification for in vitro diagnostic use in Europe. PyroMark Q24 MDx Accessories are used in combination with the PyroMark Q24 MDx instrument.

Supporting data and figures

CE-IVD marked.

Resources

Download Safety Data Sheets for QIAGEN product components.

For performing Pyrosequencing reactions on the PyroMark Q24 MDx

Publications

Genetic diagnostics of functional variants of the human dopamine D2 receptor gene.

Doehring A; Kirchhof A; Lötsch J;

Psychiatr Genet; 2009; 19 (5):259-68 2009 Oct PMID:19512960

[Quantitative pyrosequencing of heterozygous single nucleotide polymorphisms for rapid diagnosis of Down's syndrome].

Liu XQ; Wu HP; Bu Y; Zou BJ; Chen ZY; Zhou GH;

Zhonghua Yi Xue Yi Chuan Xue Za Zhi; 2009; 26 (3):331-5 2009 Jun PMID:19504451

Association between single nucleotide polymorphisms of surfactant protein D and allergic rhinitis in Chinese patients.

Deng YQ; Tao ZZ; Kong YG; Xiao BK; Chen SM; Xu Y; Wang Y; He Q;

Tissue Antigens; 2009; 73 (6):546-52 2009 Jun PMID:19493231

Global and gene-specific promoter methylation changes are related to anti-B[a]PDE-DNA adduct levels and influence micronuclei levels in polycyclic aromatic hydrocarbon-exposed individuals.

Pavanello S; Bollati V; Pesatori AC; Kapka L; Bolognesi C; Bertazzi PA; Baccarelli A;

Int J Cancer; 2009; 125 (7):1692-7 2009 Oct 1 PMID:19521983

BCL2-938C>A and CALCA-1786T>C polymorphisms in aseptic loosened total hip arthroplasty.

Wedemeyer C; Kauther MD; Hanenkamp S; Nückel H; Bau M; Siffert W; Bachmann HS;

Eur J Med Res; 2009; 14 (6):250-5 2009 Jun 18 PMID:19541585

FAQ

The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.

FAQ ID -2827

PyroMark Gold Q24 reagent and the PyroQ24 Advanced reagent can only be used on the intended instrument model. The chemistry in the PyroMark Q24 Advanced reagent has been improved. Using PyroMark Q24 reagent on a PyroMark Q24 Advanced instrument can lead to much lower peaks; while using PyroMark Q24 Advanced reagent on a PyroMark Q24 instrument can result in increased background and running out of enzyme and substrate prematurely.

FAQ ID -9070

All new assays have to be validated by the user. Interaction between primers or loops formed on single-strangled DNA can serve as priming sites for base incorporation by DNA polymerase. The following controls should be included when an assay is analyzed for the first time:

1. PCR without template DNA —shows if the primers interact to give a background signal in Pyrosequencing reactions

2. PCR with template DNA but with no sequencing primer — shows if the template can loop back on itself and give a background signal in Pyrosequencing reactions

3. Sequencing primer without any PCR product — shows if the sequencing primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

4. Biotinylated primer without any PCR product — shows if the biotinylated primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

5. Sequencing primer and biotinylated primer together without PCR product —shows if the sequencing primer and the biotinylated primer can form duplexes and give background signal in Pyrosequencing reactions

Programs from these controls ought not to show any significant peaks after any nucleotide addition.

FAQ ID -9066

This instruction is to retrieve run file for PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments

The data collected by the PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, and PSQ MA, and PSQ HS/HSA software is stored in an Oracle database. The original data are important for trouble-shooting. Follow these steps to export data from the Oracle database.

1. Launch the Pyrosequencing Data Exchange Tool application. Click File and select Login.

2. Uncheck Login to import database. Use “user” without the quotation marks for both user name and password. Click OK.

Note: The user name and password are case-sensitive.

3. On the next window, ensure that Environment variables and Analysis results options are checked. Click Export.

4. Select the run(s) that you want to export from the list on the left-side panel. You can export up to four runs in a single file. Ignore the field “Enter xsl file” file. Click the 3dot button next to the “Enter result file” field.

5. Select the location for the exported results file, and name the exported result file. Click Select and this window will close.

6. Click Export and wait for the message “Export done”. This may take a few minutes depending on the complexity of the assay and volume of data in the run(s). Click OK.

7. Repeat steps 3 through 6 if you want to export more runs. Otherwise, click File and select Exit to close the Pyrosequencing Data Exchange Tool application.

The exported result files typically are large. Please compress the files before sending them to QIAGEN Technical Service for further assistance.

FAQ ID -9048

When using the PyroMark Q24 v2.0, PyroMark Q24 Advanced v3.0, and PyroMark ID 2.5 software, the Sequence to Analyze can be modified if an unexpected mutation is detected. Enter the Sequence to Analyze that fits the detected mutation and apply the change to re-analyze the data.

When using the PyroMark ID 1.0 and PyroMark MD 1.0 software, the Sequence to Analyze cannot be modified. If an unexpected mutation is detected, you will need to re-run the assay with the new Sequence to Analyze.

FAQ ID -9045

The PyroMark Control Oligo is a self-priming oligo which can be used directly in the pyrosequencing reaction without an extra sequencing primer. It was designed for installation of the different PyroMark systems and can be used for general troubleshooting.

It can be used directly for sequencing without previous PCR setup and sample preparation by the vacuum prep tool. By this test, the performance of the instrument can be checked.

In addition, it can also be processed through the vacuum sample preparation to check the performance of this procedure. The control oligo run results in a defined pyrograms with certain peak heights for the individual instruments which are described in the handbook.

The PyroMark Control Oligo can be used on all PyroMark systems including the Q24, Q24 advanced, Q96 ID, and Q96 MD.

FAQ ID -9067

In the case of mistakes and instrument failure, the single stranded DNA can be re-used without re-run PCR. Perform the vacuum prep steps with the streptavidin beads and release them to a new Pyrosequencing plate for a fresh run.

FAQ ID -9063

Cracked waste bottle cap, dirty or wet inline filter between the waste bottle and the vacuum pump, and more than 100-time usage of the vacuum prep probes are the most common causes of slow suction.

Check the integrity of the waste bottle cap. Replace the dirty or wet inline filter. Each system comes with two inline filters. You can order extra inline filters from Millipore with catalog number SLFG05010. Replace the probes after 100 times of usage.

FAQ ID -9041

Mutation assays designed by QIAGEN detect the most common mutations. In addition, QIAGEN provides plug-ins to analyze more mutations and convenient reports for PyroMark KRAS, PyroMark EGFR, and PyroMark BRAF kits. The plug-ins can be obtained by emailing pyro.plugin@qiagen.com

If the mutation cannot be analyzed successfully, please send the original run files to QIAGEN Technical Service for further assistance.

FAQ ID -9065

This instruction is intended to retrieve log file for PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments

1. Power on both the operator’s computer and the PyroMark instrument. Wait until the Information LED on the instrument’s front panel is blinking.

2. On the operator’s computer, click the Start button and click Run…. A new window will open.

3. In the new window, type the string \\192.168.255.201\C$ and click OK. A login window will open.

4. Log in with user name Administrator and leave the password field blank. Some instruments may have the user name Pyroservice and password floang.

Note: User names and passwords are case sensitive.

5. In the new window, sequentially open the “Pyrosequencing”, the “Instrument”, and then the “Log” folders.

6. Copy the “PSQXXXLog.txt” file to the Operator’s computer and send it to QIAGEN Technical Service for further assistance.

FAQ ID -9047

Blocked reagent cartridge/tips, reagent issue, and incorrect pipetting are the common causes of the substrate peak being absent. Test cartridge/tips before adding reagent. Ensure reagent is pipetted to the correct positions and is collected at the bottom of the cartridge/tips.

If the substrate peak is present, loss of template during the vacuum prep steps, no or incorrect sequencing primer, and camera failure are the common causes. Ensure no residual vacuum pressure before releasing the streptavidin beads and that the correct sequencing primer is used. Perform camera function test, see FAQ ID 9046.

If the issue persists, please send the run files (see FAQ ID 9048) to QIAGEN Technical Service for further assistance.

FAQ ID -9042

The camera function test is intended for the PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments.

1. Setup a mock run without using PyroMark reagent and other consumables.

2. When nucleotide dispensation starts, open both instrument lids so that the 96-well block is exposed to ambient light.

3. Check the detected signal level on the Y-axis on the operator’s computer and report the value to QIAGEN Technical Service.

Note: The inner lid of the instrument moves around during nucleotide dispensation. The lid can still be lifted while it is moving.

For PyroMark Q24 and PyroMark 24 Advanced instruments, please contact QIAGEN Technical Service for the self-test software and instruction.

FAQ ID -9046

Pyrosequencing assay can detect unexpected/unknown mutations. Modify the Sequence to Analyze to analyze unexpected/unknown mutations. See How can I modify the Sequnce to Analyze post a run.

If the mutation cannot be analyzed successfully, please send the original run files to QIAGEN Technical Service for further assistance.

FAQ ID -9064

Insufficient amount of PCR template, loss of streptavidin bead during vacuum prep steps, incorrect instrument method, and incorrect reagent are the common causes of low peak signals.

Check the PCR product with QIAxcel instrument or agarose gel electrophoresis and determine the appropriate amount of PCR product to be used. Perform vacuum prep function test. Determine the vacuum prep station functionality using the PyroMark Control Oligo with and without the vacuum prep steps. Ensure the correct instrument method is used. Refer to Managing PyroMark Instrument Methods. Ensure the correct reagent is used for the specific instrument model.

FAQ ID -9069

Clogged cartridge/tips, and excessive PCR product are the most common causes. Test cartridge/tips before adding reagent. Avoid using filtered pipette tips because small particles from the filter can clog the cartridge/tips. Use appropriate PCR product for optimal single peak heights, also see FAQ ID 9058.

If the issue persists, please send the run files (see FAQ ID 9048) to QIAGEN Technical Service for further assistance.

FAQ ID -9043

Please see the Pyrosequencing Technology and Platform Overview.

FAQ ID -9068

Fill a PCR plate with 80– 100 µl of high-purity water. Apply vacuum pressure to the vacuum prep tool. Lower the vacuum prep tool to the PCR plate and wait for 10 seconds. If the wells are not empty in 10 seconds, the vacuum system needs to be checked. Check the integrity of the waste bottle cap. Replace the inline filter if dirty or wet. Replace the vacuum probes after they are used 100 times.

FAQ ID -9044

1. When the instrument is not processing, insert a USB stick into the USB port on the instrument.

2. Use the Up and Down buttons to select “Administration” in the menu and press OK.

3. Select “Copy Log Files” and press OK. When the instrument confirms that the log files have been saved to the USB stick, press Close and remove the USB stick.

For other PyroMark instruments, see FAQ ID -9047

FAQ ID -9049

The instruments can continue processing samples if there is no USB stick inserted in the USB port. The data are stored in an internal storage memory card. Follow these steps to retrieve data from the internal storage memory.

1. When the instrument is not processing, insert a USB stick into the USB port on the instrument.

2. Use the Up and Down buttons to select “Administration” in the menu and press OK.

3. Select “Copy Unsaved Runs” and press OK.

4. Use the Up and Down buttons to select the run file for retrieval and press Select.

5. When the instrument confirms that the run file has been saved to the USB stick, press Close and remove the USB stick.

FAQ ID -9050

Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.

FAQ ID -2844