Probe group and kit used to detect HER-2 gene proliferation level and application thereof

A HER-2 and gene amplification technology, which is applied in recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as time-consuming, difficult to evaluate false-negative results, and patients' missed opportunities for medication, etc., to achieve Accurate diagnosis and treatment, rapid hybridization effect

Pending Publication Date: 2019-08-27
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This judgment method is not easy to evaluate whether the false negative results caused by polysomy or amplification of the centromere region of the chromosome make the patient miss the best time for medication
In addition, the products currently on the market for detecting the expression level of HER-2 gene in patients take too long (about 6 hours), which makes patients wait longer for the diagnosis result

Method used

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  • Probe group and kit used to detect HER-2 gene proliferation level and application thereof
  • Probe group and kit used to detect HER-2 gene proliferation level and application thereof
  • Probe group and kit used to detect HER-2 gene proliferation level and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0023] In some embodiments, the present invention provides a method for preparing the above-mentioned probe, comprising: probe labeling (using phi29 DNA polymerase labeling )→Quantitative (#The sample volume and length of the probe will affect the hybridization effect)→Packing / stand-by.

[0024] In some embodiments, the present invention provides the optimization process of the probe hybridization system, which mainly involves the selection of hybridization promoters, and reduces the hybridization time to achieve rapid hybridization. The specific optimization process is as follows:

[0025] (1) Accelerator selection:

[0026] During the hybridization process, increasing the denaturant can affect the base stacking force and the formation of hydrogen bonds, increase the hydrophobicity of water and reduce the energy difference between double-stranded and single-stranded DNA, and reduce the Tm value. The current conventional hybridization buffer contains formamide, and the hy...

Embodiment 1

[0035] Embodiment 1 probe design and preparation

[0036] An embodiment of the probe set for detecting the amplification level of HER-2 gene of the present invention includes 3 probes, respectively targeting 3 sequence segments on the human genome, namely HER-2, RAI1 and 17 Centromere region of chromosome number.

[0037] 1) The probe design process includes:

[0038] BAC clone selection → purchase (commercialization) → labeling, testing → confirmation of the probe clone group (available for subsequent production).

[0039] For the method of BAC clone screening, please refer to p26-28 of Zhao Jia et al.

[0040] • BAC clone selection (see Table 2): Gene site probes are generally designed to detect probes that include the gene. Probe clone selection should pay attention to the specificity of clones, and select clones whose fluorescence intensity meets the requirements to prepare probes through verification. In order to ensure the brightness of the signal, the length of the ...

Embodiment 2

[0054] Embodiment 2 probe hybridization system

[0055] The labeled probes of Example 1 were prepared in the hybridization system as shown in Table 4:

[0056] Table 4 hybrid system

[0057] ingredients per test Green probe (50ng / μl) 0.3~0.6μl Red probe (50ng / μl) 0.3~0.5μl Cyan probe (50ng / μl) 0.2~0.4μl hybridization buffer 7μl COT Human DNA (1mg / ml)* 1μl h 2 o

[0058] *COT Human DNA is placental DNA, mainly 50 to 300bp in length, and rich in repetitive DNA sequences, such as Alu and Kpn family members, usually used to block non-specific hybridization and suppress repetitive DNA sequences.

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Abstract

The invention discloses a probe group used to detect HER-2 gene proliferation level. The probe group includes a probe targeted toward an HER-2 gene sequence and a reference probe. The reference probeincludes a probe targeted toward an RAI1 gene sequence and a probe targeted toward a CSP17 gene sequence, wherein the probe targeted toward an HER-2 gene sequence and the reference probe both adopt phi29DNA polymerase to label probes. The invention further closes a kit used to detect HER-2 gene proliferation level, including the probe group and hybridisation buffer, and the hybridisation buffer includes dimethyl sulfoxide acting as a promoter. The invention provides the kit used to accurately detect HER-2 gene proliferation level. Accurate interpretation of HER-2 proliferation and rapid hybridization within two hours of a tissue sample and a probe are realized; the adoption of the detection kit disclosed by the invention can help an existing method to quickly perform accurate layering, precise diagnosis and treatment on tumors.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a probe set, a kit and an application thereof for detecting the amplification level of HER-2 gene. Background technique [0002] Breast cancer is a major public health problem in current society. According to the 2009 breast cancer incidence data released by the National Cancer Center and the Ministry of Health's Bureau of Disease Control and Prevention in 2012, the incidence of breast cancer in the national tumor registration area ranks first among female malignant tumors, and the incidence (crude rate) of female breast cancer is the highest in the country. The total is 42.55 / 100,000, 51.91 / 100,000 in cities, and 23.12 / 100,000 in rural areas. And the incidence has a rising and younger trend. The treatment of breast cancer is a comprehensive treatment, including surgery, radiotherapy, chemotherapy, endocrine therapy, biological targeted therapy and other means. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/136
Inventor 陈绍宇何瑰
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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