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

Features

  • 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

Products

PyroMark Q24 MDx经SFDA注册,可用于体外诊断。

Product Details

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。

Performance

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。

Principle

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

Procedure

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。

Applications

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。

Software

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。

Supporting data and figures

Specifications

FeaturesSpecifications
ConnectionsOne USB port (2.0)
Chemical resistancepH 4 to pH 9, common detergents, 0.5 M sodium hydroxide, ethanol
ApplicationsMethylation analysis, allele quantification, genotyping, sequence analysis
HumidityRelative humidity of 20–90% (noncondensing)
CE/FDA/IVD compatibleIn Europe
Instrument dimensions420 x 390 x 525 mm (16.5 x 15.4 x 20.7 in.)
Kits designed for this instrumentIVD-labeled therascreen Kits
Operating temperature15–32°C (59–90°F)
Overvoltage categoryII
Place of operationFor indoor use only
Pollution level2
Power100–240 V AC, 47–63 Hz, 1.1–0.45 A (grounded). From external power supply to instrument: 12 VDC and 24 VDC nominal
Process temperature28°C (82.4°F) ± 1°C
Process timeDepends on the number of nucleotide dispensations (20 dispensations take 24 minutes)
Samples per run (throughput)1–24
SoftwarePyroMark Q24 MDx Software 2.0
TechnologyPyrosequencing
Weight27.5 kg (60.6 lb)
AltitudeUp to 2000 m (6500 ft)

Resources

下载文件 (15)
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0002
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0014
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0003
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0006
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0004
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0005
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0012
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0009
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0008
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0001
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0007
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0011
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0013
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0010
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0015

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

Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE healthcare with the catalog no 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the catalog no 974203.

FAQ ID -2850
Can I order the nucleotides from PyroMark Gold Reagents separately?
The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.
FAQ ID -2827
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
In the range of 3-5 bases can be resolved depending on the sequence context and base. If it is possible sequencing of a homopolymer of more than 3-5 nucleotides should be avoided by resetting the sequencing primer.
FAQ ID -2871
What is a PyroMark instrument method or instrument code?

An instrument method or instrument code encodes the individual pulse time settings of specific cartridge lot batch. These pulse time settings change when e.g. a new batch of capillaries is used with slight variations in the needle diameter. For larger diameters, the pulse settings are lowered to dispense the correct volume of liquid. In addition, the viscosity of enzyme and substrate mixes can change which influences dispensing volumes.

The individual instrument method/code number is printed on the cartridge label. The corresponding methods/code settings can be downloaded as a file from the respective instrument webpage and opened in the PyroMark application software.

FAQ ID -2941
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.
FAQ ID -2879
When do I have to change the pulse settings/methods in a pyrosequencing run setup?
Always check for the actual method/code number printed on the cartridge label. Make sure that you choose this method/code number when setting up the pyrorun in the application software. If this method cannot be selected automatically in the application software, you can download the method/code file from the instrument webpage.
FAQ ID -2942
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures which are extended or the primers itself form dimmers which serve as template. Perform accurate sequencing controls (e.g. PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.
FAQ ID -2877
How many times can vacuum troughs be re-used with the PyroMark Vacuum Preparation Stations?
There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
Can I reinstall the PyroMark Q24 software on my new computer or following an operating system upgrade, or do I need to purchase a new license?

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

What concentration should be used for the sequencing primer in pyrosequencing?

Usually the sequencing primer is used at 0.3µM in annealing buffer but some assays might require additional optimization of the sequencing primer concentration.

 

For PyroMark Q24 and PyroMark Q96 MD the final concentration of the sequencing primer is 0.3µM and for PyroMark Q96 ID 0.4µM.


The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8µM, but may be adapted to optimize assays.

 

FAQ ID -2826
Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified whereas the other primers require standard desalting only.  Pyrosequencing primers can be ordered here.
FAQ ID -2832
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and fall down at any time. or exchanged.
FAQ ID -2881
What is the reason for signals ceasing in the middle of a pyrosequencing run?
The cartridge needle can be blocked or damaged causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case template conditions should be optimized.
FAQ ID -2875
What kind of reading length can I expect when using Pyrosequencing technology for sequence analysis?

Typical reading length using Pyrosequencing technology is 40-60 bases. However, as with any sequencing technology, the maximum read length will depend on template secondary structure, base content, quality of PCR-product, and other parameters.

Depending on the sequence to be analyzed, highly accurate read lengths of 140 or more bases can be obtained in just a single reaction with the Q48 PyroMark Autoprep.

 

 

FAQ ID -2216
Can unused wells in a pyrosequencing plate be used in the next run?
In principle it’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878