PyroMark Q24 MDx

使用焦磷酸测序技术进行IVD验证的突变和甲基化分析

Products

PyroMark Q24 MDx经SFDA注册，可用于体外诊断。

特点

经SFDA注册，符合中国医疗器械相关法规
序列信息确保发现稀有突变
一次运行可完成多个分析
最快15分钟内分析1–24个样本

产品详情

PyroMark Q24 MDx实时定量焦磷酸序列分析仪应用经验证的焦磷酸测序技术，进行基于实时序列的定量检测分析，在欧洲和中国用于体外诊断。这个创新的平台非常适合用于突变的基因分型、评估疾病相关的DNA甲基化模式、验证生物标记物以及其他诊断相关的分析。

绩效

焦磷酸测序技术可对遗传学和表观遗传学的DNA变化进行准确、灵敏的定量分析，提供高度可靠的序列数据。可鉴定新的突变，并检测低水平的DNA甲基化模式异常。

原理

焦磷酸测序技术基于合成测序原理，可在数分钟内提供序列的定量数据。PyroMark Q24 MDx实时定量焦磷酸序列分析仪是提供 real-time测序信息的完整体系，高度适用于遗传学和表观遗传学分析。该体系包括PyroMark Q24 MDx实时定量焦磷酸序列分析仪、 PyroMark Q24 MDx Vacuum Workstation、PyroMark Q24 MDx Software 2.0、PyroMark Gold Q24 Reagents、PyroMark Control Oligo和PyroMark Q24 Validation Oligo。同时提供样本制备方案，可使用PyroMark Q24 MDx Vacuum Workstation制备单链DNA。

焦磷酸测序步骤

步骤1：扩增DNA片段，作为焦磷酸测序模板的单链是生物素标记的。变性后，分离生物素标记的单链PCR扩增子，与测序引物杂交。杂交的引物和单链模板同各种酶，包括DNA聚合酶、ATP硫酸化酶、荧光素酶、三磷酸腺苷双磷酸酶以及底物、腺苷酰硫酸（APS）和荧光素一起孵育（参见"Principle of Pyrosequencing — step 1"）。

步骤2：第一个脱氧核苷三磷酸（dNTP）加入反应中，在DNA聚合酶的作用下，dNTP结合到DNA链上与其互补的碱基位。每次结合都会释放与核苷酸相等摩尔量的焦磷酸脂（PPi）（参见"Principle of Pyrosequencing — step 2"）。

步骤3：在存在APS的条件下，ATP硫酸化酶将PPi转化为ATP。在这个ATP的驱动下，荧光素酶将荧光素转化为氧化荧光素，并产生可见光，可见光的量与ATP的量成正比。通过电荷耦合（CCD）相机即可检测到荧光素酶催化反应产生的可见光，并在原始数据输出（Pyrogram）中显示为一个峰，每个峰的高度（光信号）与结合的核苷酸数量成正比（参见"Principle of Pyrosequencing — step 3"）。

步骤4：三磷酸腺苷双磷酸酶，是一种核苷酸降解酶，会降解未结合的核苷酸与ATP。完全降解后，加入另一个核苷酸（参见"Principle of Pyrosequencing — step 4"）。

步骤5：持续添加dNTP。α-硫代脱氧腺苷三磷酸（dATPαS）在反应中用于替代天然脱氧腺苷三磷酸（dATP），这是因为DNA聚合酶可高效的应用前者，而不被荧光素酶识别。当该过程连续进行时，就会形成互补的DNA链，可根据Pyrogram追踪的信号峰确定核苷酸的序列（参见"Principle of Pyrosequencing — step 5"）。

专用的IVD验证分析

QIAGEN提供与PyroMark Q24 MDx实时定量焦磷酸序列分析仪配套的IVD验证分析。目前，这些试剂盒包括一些重要的癌症相关突变。


产品	用于定量检测的突变
therascreen KRAS Pyro Kit	人KRAS基因密码子12、13和61
therascreen BRAF Pyro Kit	人BRAF基因密码子600和464-469
therascreen EGFR Pyro Kit	人EGFR基因密码子719、768、790、858和861以及外显子19
therascreen NRAS Pyro Kit	人NRAS基因密码子12、13和61

程序

从PCR产物到直接用于测序的单链模板，应用PyroMark Q24 MDx Vacuum Workstation可在15分钟内同时制备24个样本。该工作站操作简单，且手动时间少于5分钟。

在焦磷酸测序之前，形成了一个生物素标记的PCR产物，该产物结合到覆盖链霉亲和素的琼脂糖凝胶珠上。这些胶珠会被真空仪器捕捉并彻底清洗，随后变性，产生适合于焦磷酸测序的单链DNA。该模板DNA放入含测序引物的焦磷酸测序反应板中，随后引物退火，将该板放入PyroMark仪器中。PyroMark Gold试剂包含用于焦磷酸测序反应的酶、核苷酸和底物，将这些试剂移入配药管或筒（取决于仪器），按照软件中提供的量放入仪器中，用于焦磷酸测序。

应用

PyroMark Q24 MDx实时定量焦磷酸序列分析仪适合体外诊断应用，用于检测与临床相关的特定可变DNA位点的变化。

软件

PyroMark Q24 MDx Software装载在PC机上，能够全面的分析您的结果。该软件包含2个分析模块：CpG和AQ（等位定量）。2个模块可分析在同一孔板中的样品，不同类型的样品可同时分析。AQ模块用于分析单个和di-、tri-和tetra-多个等位突变。CpG模块用于分析多个连续的CpG位点并提供经亚硫酸氢盐转化的内参。

辅助数据和图表

GGT to GAT mutation in base 2 of codon 12.

Nucleotide 35 is indicated with an arrow.

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".

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)

资源

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