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HER2 gene amplification detection probe, kit and method

A detection kit and gene amplification technology, applied in the field of molecular biology, can solve problems such as result judgment and interpretation variation, and achieve the effects of high accuracy, good specificity and convenient operation

Inactive Publication Date: 2015-04-29
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with FISH, IHC is cost-effective and simple, but it is easily affected by tissue processing methods and fixation time, and there are large variations in the judgment and interpretation of results, while FISH has a high degree of repeatability and reliability.

Method used

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  • HER2 gene amplification detection probe, kit and method
  • HER2 gene amplification detection probe, kit and method
  • HER2 gene amplification detection probe, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The detection probe and kit for detecting HER2 gene amplification described in the present embodiment are designed as follows:

[0031] 1. Design the amplification primers

[0032] Two sets of amplification primers were designed for the HER2 gene and the centromere region of human chromosome 17, the first set of amplification primers for HER2, and the second set of amplification primers for the centromere region of human chromosome 17, The corresponding amplification products respectively constitute the first group of probe libraries and the second group of probe libraries. The amplification products obtained by each set of amplification primers can be used as rearranged FISH probes, do not contain repetitive sequences, can avoid cross-reactions, and perform specific recognition and hybridization with the HER2 target detection fragment in the chromosome.

[0033] The following primers were artificially synthesized, and the amplification primers and their corresponding ...

Embodiment 2

[0088] Example 2 Detection of clinical samples using the HER2 gene amplification detection kit in Example 1:

[0089] Using the HER2 gene amplification detection kit of Example 1 (including twenty probes of SEQ NO.41-50 and SEQ NO.51-60) to carry out on 20 paraffin-embedded tissues provided by the hospital, the steps are as follows. 1. Sample pretreatment process:

[0090] Preparations: Turn on the slicer (45°C-60°C); turn on the water bath (37±1°C); preheat the pepsin buffer in a 37±1°C water bath, use pepsin and preheated pepsin Prepare a working solution of pepsin with a concentration of 1.0 mg / mL in the buffer solution, and place the working solution in a water bath at 37±1°C to preheat (preheat for at least 1 hour).

[0091] Section dewaxing: Submerge the section in xylene at room temperature, dewax for 10 min, replace with fresh xylene and repeat dewaxing twice (a total of 3 dewaxing). The xylene-treated slices were submerged in absolute ethanol for 5 min, and repeated...

Embodiment 3

[0116] The influence of the selection of embodiment 3 primer pair quantity on sample detection result

[0117] A probe library was constructed for the HER2 gene and the centromere region of human chromosome 17, and different numbers of primer pairs were selected for amplification of the HER2 gene (the first group) and the centromere region of the human chromosome 17 (the second group). 4 groups of experiments, in order to study whether the detection effect of the corresponding probe library composed of different numbers of amplification products (i.e., probes) is consistent, wherein the experimental group 1 is to select 1 pair of amplification primers; the experimental group 2 is to select 3 pairs of amplification primers. pair of amplification primers; in experiment 3, 5 pairs of amplification primers were selected; in experiment 4, 10 pairs of amplification primers were selected. The specific test arrangements are shown in Table 10. The synthesis of probes, the construction ...

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Abstract

The invention discloses an HER2 gene amplification detection probe, a kit and a method. The detection probe comprises a first group of probes for labeling fluorescent dye and targeted to an HER2 gene, and a second group of probes targeted to the centromere region of human chromosome 7, wherein the first group of the probes are selected from at least one of probes as shown in SEQ ID NO.41-SEQ ID NO.50; the second group of the probes are selected from at least one of probes as shown in SEQ ID NO.51-SEQ ID NO.60; and the fluorescent dyes labeled by the two groups of the probes are different in color. A FISH probe in the HER2 gene amplification detection kit provided by the invention is free from a repeated sequence, and capable of avoiding a cross reaction and undergoes hybridization recognition with an HER2 target detection fragment in the chromosome; and the probe has the advantages of high accuracy, good specificity and low false positive rate.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a HER2 gene amplification detection probe, a kit and a method. Background technique [0002] The human HER-2 / neu gene (Her-2 gene) gene is located on chromosome 17q21, and its encoded product is a 185kD transmembrane protamine p185, which consists of 1255 amino acids, and the 720-987 position belongs to the tyrosine kinase region. Its structure Schematic such as figure 1 shown. Her-2 / neu protein is a transmembrane protein with tyrosine protein kinase activity and is a member of the EGFR family. Her-2 / neu protein is composed of three parts: the extracellular ligand-binding region, the single-chain transmembrane region and the intracellular protein tyrosine kinase region. Body. It is mainly through other members of the family including EGFR (HER1 / erbBI), HER3 / erbB3. HER4 / erbB4 forms heterodimers to bind to their respective ligands. Her-2 / ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/112C12Q2600/158
Inventor 胡文晖吴诗扬廖传荣黄萌
Owner SUREXAM BIO TECH
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