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Probe combination for detecting circulating tumor DNA of human thyroid cancer and capturing and sequencing system thereof

A technology for thyroid cancer and human detection, which is applied in the field of disease diagnosis and treatment, can solve the problems of inability to detect mutation types and low detection rate of detection methods, and achieve the effects of reducing medical costs, convenient collection, and high sensitivity

Inactive Publication Date: 2019-02-15
浙江天远生物科技有限公司
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Problems solved by technology

However, most of the current research is limited to a few sites of a few tumor-related genes, and most of them use first-generation sequencing or PCR amplification. In particular, there are only a few reports on ctDNA studies of thyroid tumors, which involve a single site of individual genes , such as BRAFV600E, for situations with highly heterogeneous characteristics like tumor tissues, the existing detection methods have problems such as low detection rate and inability to discover new mutation types

Method used

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  • Probe combination for detecting circulating tumor DNA of human thyroid cancer and capturing and sequencing system thereof
  • Probe combination for detecting circulating tumor DNA of human thyroid cancer and capturing and sequencing system thereof
  • Probe combination for detecting circulating tumor DNA of human thyroid cancer and capturing and sequencing system thereof

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preparation example Construction

[0038] 2. Preparation of probes: Combine the sequences in the above table 1 to obtain a probe combination composed of different thyroid cancer driver gene exon sequences and hot spot mutation region sequences, and hand it over to IDT (Integrated Device Technology, Inc. .) Complete the preparation of the probe.

[0039] A capture sequencing system for diagnosing human thyroid cancer, comprising the probe combination, hybridization system, sequencing device (Roche Nimblegen) and circulating free DNA extraction kit (QI Aamp Circμlating Nucleic Acid Kit) as claimed in claim 1;

[0040] The capture sequencing platform used in the present invention is Roche Nimblegen; the circulating cell-free DNA extraction kit is QI Aamp Circμlating Nucleic Acid Kit; the sequencing analysis is completed by Beijing Zhiyin Oriental Translational Medicine Center.

[0041] The biological materials used in the present invention are all from the Oncology Department of the First Affiliated Hospital of We...

Embodiment 1

[0043] 1. Extract plasma free circulating tumor DNA (using QI Aamp Circμlating Nucleic Acid Kit):

[0044] 1. Draw 10ml of blood from the patient into an EDTA anticoagulant blood collection tube containing DNA protection agent, centrifuge at 3000rpm for 10 minutes, transfer the supernatant to a new sterile centrifuge tube, centrifuge at 20000×g for 10 minutes, and separate the supernatant for later use.

[0045] 2. Add 100μl Proteinase K into a 50ml centrifuge tube.

[0046] 3. Take 1ml of separated plasma and add it to the centrifuge tube.

[0047] 4. Take 0.8ml Buffer ACL (containing 1.0ug carrier RNA) to the centrifuge tube, cover the lid, shake and mix for 30 seconds.

[0048] Incubate at 5.60°C for 30 minutes.

[0049] 6. Open the cap of the centrifuge tube, add 1.8m1Buffer ACB, then close the cap, shake and mix for 15-30 seconds.

[0050] 7. Place the centrifuge tube on ice for 5 minutes.

[0051] 8. Install the QI Aamp Mini column on the Vacconnector of the Ql Avac ...

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Abstract

The invention provides a probe combination for detecting circulating tumor DNA of human thyroid cancer and a capturing and sequencing system thereof. The probe combination comprises a thyroid cancer driving gene exon sequence and a hot spot mutation region sequence. The capturing and sequencing system comprises the probe combination, a hybridizing system, a sequencing device and a circulating freeDNA extracting kit, and performs detection according to the following steps: S1, extracting circulating free DNA from plasma; S2, repairing the tail end of the DNA fragment and adding a connector tothe tail end so as to construct a sequencing library; S3, performing PCR amplification and purification on the library before capturing; S4, hybridizing the amplified sample library with a probe; S5,removing an unbound sequence and eluting a captured sequence; S6, performing PCR amplification and purification again; and S7, performing high-throughput sequencing analysis. According to the capturing and sequencing system, the thyroid cancer can be diagnosed in an early state objectively, sensitively, specifically and stably, and a tumor load is monitored and evaluated so as to achieve timely and pertinent treatment.

Description

technical field [0001] The invention relates to the technical field of disease diagnosis and treatment, in particular to a probe combination for detecting human thyroid cancer circulating tumor DNA and a capture sequencing system thereof. Background technique [0002] The thyroid is a very important gland in the human body. The physiological functions of the thyroid hormone secreted by it are mainly: 1. Promote metabolism, increase the oxygen consumption of most tissues, and increase heat production; 2. Promote growth and development, which is crucial to the development and growth of long bones, brain and reproductive organs Important, especially in infancy, at this time the lack of thyroid hormones will cause cretinism; 3. Improve the excitability of the central nervous system. In addition, it also has the functions of strengthening and regulating other hormones, increasing heart rate, strengthening cardiac contractility, and increasing cardiac output; the thyroid gland al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 胡咏武王彤文黄必胜吴生成
Owner 浙江天远生物科技有限公司
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