PyroMark Q24 MDx

For IVD-validated mutation and methylation analysis using Pyrosequencing technology

Products

The PyroMark Q24 MDx is intended for in vitro diagnostic use.

Features

Compliant with EU IVD Directive 98/79/EC
Reliable quantification of allele representation and methylation status
Sequence information enables discovery of rare mutations
More than one assay can be completed in a single run
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. This innovative platform is highly suited for genotyping mutations, evaluating disease-related DNA methylation patterns, validating biomarkers, and other diagnostic-related assays.

Performance

Pyrosequencing technology enables accurate and sensitive quantification of genetic and epigenetic DNA variations by providing highly reliable sequence data. It allows the identification of novel mutations, as well as detection of aberrant DNA methylation patterns present at low levels.

The therascreen KRAS Pyro Kit is a good example of the informative analysis provided by the therascreen Pyro Kits and the PyroMark Q24 MDx instrument. The KRAS gene is mutated in approximately 35% of metastatic colorectal cancer (CRC) patients. Studies have shown that KRAS mutation testing can better define which CRC patients will benefit from treatment with epidermal growth factor receptor (EGFR) inhibiting monoclonal antibodies, such as panitumumab and cetuximab.

The therascreen KRAS Pyro Kit consists of 2 assays: one for detecting mutations in codons 12 and 13 and the other for detecting mutations in codon 61. The two regions are amplified separately by PCR, using optimized PCR reagents and primers included in the kit, and then sequenced through the defined region (see figure " Normal genotype in codons 12 and 13"). Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis. Pyrosequencing technology on the PyroMark Q24 MDx enables identification of specific mutations (see figure " GGT to GAT mutation in base 2 of codon 12"), including less frequent mutations (see figures " GGT to AGT mutation in base 1 of codon 12" and " Reanalysis of the data in figure 'GGT to AGT mutation in base 1 of codon 12'"), as well as discovery of new mutations.

See figures

Normal genotype in codons 12 and 13.

GGT to GAT mutation in base 2 of codon 12.

GGT to AGT mutation in base 1 of codon 12.

Reanalysis of the data in figure "GGT to AGT mutation in base 1 of codon 12".

Principle

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 MDx is a fully integrated system that provides real-time sequence information and is highly suited for genetic and epigenetic analysis. The system includes the PyroMark Q24 MDx Instrument, PyroMark Q24 MDx Vacuum Workstation, PyroMark Q24 MDx Software 2.0, PyroMark Gold Q24 Reagents, PyroMark Control Oligo, and PyroMark Q24 Validation Oligo. Sample preparation solutions are also available to enable preparation of single-stranded DNA using the PyroMark Q24 MDx Vacuum Workstation.

Steps of the Pyrosequencing reaction:

Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure " Principle of Pyrosequencing — step 1").

Step 2: The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide (see figure " Principle of Pyrosequencing — step 2").

Step 3: ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated (see figure " Principle of Pyrosequencing — step 3").

Step 4: Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added (see figure " Principle of Pyrosequencing — step 4").

Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure " Principle of Pyrosequencing — step 5").

Dedicated IVD-validated assays

QIAGEN offers an expanding suite of IVD-validated assays to be used with the PyroMark Q24 MDx. Currently, these kits include important cancer-related mutations:

Product	For quantitative measurement of mutations in
therascreen KRAS Pyro Kit	codons 12, 13, and 61 of the human KRAS gene
therascreen BRAF Pyro Kit	codons 600 and 464-469 of the human BRAF gene
therascreen EGFR Pyro Kit	codons 719, 768, 790, 858, and 861 and exon 19 of the human EGFR gene
therascreen NRAS Pyro Kit	codons 12, 13, and 61 of the human NRAS gene

See figures

Principle of Pyrosequencing — step 1.

Principle of Pyrosequencing — step 2.

Principle of Pyrosequencing — step 3.

Principle of Pyrosequencing — step 4.

Principle of Pyrosequencing — step 5.

Procedure

From PCR product to single-stranded template ready for sequencing — up to 24 samples can be prepared in parallel using the PyroMark Q24 MDx Vacuum Workstation, in less than 15 minutes. The workstation ensures easy handling, and the actual hands-on time is less than 5 minutes.

Prior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Gold reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.

Applications

The PyroMark Q24 MDx is suitable for in vitro diagnostic applications in Europe.

Genetic analysis comprises multiple applications to analyze differences in genomic DNA, including mutation detection and SNP typing. PyroMark Q24 MDx facilitates accurate and highly sensitive mutational analysis of any gene of interest and enables quantification of allele representation in mixed cell populations. The system can be used for self-validated assays for oncology studies and for analysis of epigenetic markers in methylation studies, or it can be used with dedicated IVD-validated assays available from QIAGEN

Software

PyroMark Q24 MDx Software, installed on a PC, enables comprehensive analysis of your results. The software contains two analysis modes: CpG and AQ (allele quantification). Both modes can be used to analyze samples on the same plate, enabling different types of samples to be run at the same time. The AQ mode can be used for analyzing single and multivariable positions, as well as di-, tri- , and tetra- allelic mutations. The CpG mode enables analysis of multiple consecutive CpG sites and provides a built-in control for the bisulfite treatment.

Supporting data and figures

GGT to GAT mutation in base 2 of codon 12.

Nucleotide 35 is indicated with an arrow.

Specifications

Features	Specifications
Connections	One USB port (2.0)
Chemical resistance	pH 4 to pH 9, common detergents, 0.5 M sodium hydroxide, ethanol
Applications	Methylation analysis, allele quantification, genotyping, sequence analysis
Humidity	Relative humidity of 20–90% (noncondensing)
CE/FDA/IVD compatible	In Europe
Instrument dimensions	420 x 390 x 525 mm (16.5 x 15.4 x 20.7 in.)
Kits designed for this instrument	IVD-labeled therascreen Kits
Operating temperature	15–32°C (59–90°F)
Overvoltage category	II
Place of operation	For indoor use only
Pollution level	2
Power	100–240 V AC, 47–63 Hz, 1.1–0.45 A (grounded). From external power supply to instrument: 12 VDC and 24 VDC nominal
Process temperature	28°C (82.4°F) ± 1°C
Process time	Depends on the number of nucleotide dispensations (20 dispensations take 24 minutes)
Samples per run (throughput)	1–24
Software	PyroMark Q24 MDx Software 2.0
Technology	Pyrosequencing
Weight	27.5 kg (60.6 lb)
Altitude	Up to 2000 m (6500 ft)

Resources

Publications

Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues.

Dufort S; Richard MJ; de Fraipont F;

Anal Biochem; 2009; 391 (2):166-8 2009 May 21 PMID:19464247

Y chromosomal STR analysis using Pyrosequencing technology.

Edlund H; Allen M;

Forensic Sci Int Genet; 2009; 3 (2):119-24 2009 Jan 6 PMID:19215881

Amelogenin sex determination by pyrosequencing of short PCR products.

Tschentscher F; Frey UH; Bajanowski T;

Int J Legal Med; 2008; 122 (4):333-5 2008 Mar 20 PMID:18351373

Identification of mammal species using species-specific DNA pyrosequencing.

Karlsson AO; Holmlund G;

Forensic Sci Int; 2007; 173 (1):16-20 2007 Feb 28 PMID:17331687

More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA.

Malmström H; Svensson EM; Gilbert MT; Willerslev E; Götherström A; Holmlund G;

Mol Biol Evol; 2007; 24 (4):998-1004 2007 Jan 25 PMID:17255122

FAQ

Do not remove the USB stick while data are being copied. Corrupted data files are usually due to the interrupted copying process. Follow these steps to retrieve recently saved runs.

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 Recently Saved Runs” and press OK.

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

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

In addition, repeat the process with a different USB stick. If the issue persists, please contact QIAGEN Technical Service for further assistance.

FAQ ID -9053

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

FAQ ID -2827

Yes. The PyroMark Q24, PyroMark Q24 Advanced, and PyroMark Q96 v2.5 software provide multiple types of assay modes, which enable analyzing different types of samples at the same time.

FAQ ID -9057

The histogram is the theoretical peak pattern based on the Sequence to Analyze that you enter into the software, while the pyrogram is the experimental peak pattern that is detected by PyroMark instruments. The peak pattern in the pyrogram has to be in agreement with that in the histogram for a successful run. Overlaying the histogram on the pyrogram provides an invaluable tool for trouble-shooting Pyrosequencing runs. To do so, right click anywhere on the pyrogram and select Show Histogram on the pop-up menu.

FAQ ID -9056

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

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

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

Please see PyroMark software operating system compatibility and hardware requirements.

FAQ ID -9054

If you need to install the PyroMark Q24 software on a new computer replacing your old one, or after you reinstall or upgrade the operating system, you can reinstall the PyroMark Q24 software without purchasing a new license.

FAQ ID - 3473

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

PyroMark instruments dispense the correct volume of enzyme mix, substrate mix, and nucleotides using variable pulse times and dispensing pressures. The settings for pulse times and dispensing pressure are specific for each lot of PyroMark dispensing tips and cartridges and need to be updated in the PyroMark software being used to operate the instrument.

Dispensing pressures and pulse time settings must be checked and, if necessary, updated every time a cartridge from a new lot number is used. Check the details at Managing PyroMark Instrument Methods.

FAQ ID -9055

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

Sodium hydroxide carry-over, too much template, and incorrectly stored reagent are the most common causes of wide peaks. Ensure correct volume of denaturation buffer is used on the vacuum prep station. Use 5–20 µl of PCR product depending on instrument models. Store reagent as described in the handbook.

If the issue persists, please send the run files (see Instruction for run file export) to QIAGEN Technical Service for further assistance.

FAQ ID -9062

For PyroMark Q24, PyroMark Q24 Advanced, PyroMark Q96 ID v2.5, and PyroMark CpG software, data are file-based. The original run files can be attached to email and sent to QIAGEN Technical Service.

For PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software, data are stored in an Oracle database and need exporting (See Instruction for run file export). Exported data can be attached to email and sent to QIAGEN Technical Service.

The log files from the PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments provide valuable information. See Instruction for log file retrieval) for log file retrieval instruction

Please send the log file along with the original run file to QIAGEN Technical Service.

FAQ ID -9051

A single peak height is the signal produced from a non-variable single nucleotide. The single peak height is an important criterion for trouble-shooting.

The optimal single peak height is 30 RLU (minimum 20 RLU) (Relative Light Unit) for PyroMark Q24 and PyroMark Q24 Advanced, 20 RLU (minimum 10 RLU) for PyroMark Q96 ID, and 100 RLU (minimum 50 RLU) for PyroMark Q96 MD instrument, respectively.

FAQ ID -9058

The PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software requires a functional Oracle database, which cannot be installed on an office computer. You can install the PyroMark software on an office computer and access the Oracle database on the PyroMark operator’s computer via network. The operator’s computer needs to be powered on while you are accessing the Oracle database remotely. Your IT department is responsible for the network setup.

FAQ ID -9059

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