Primer probe for detecting breast cancer recurrence and metastasis gene HER2 amplification and application thereof

A technology of recurrence and metastasis, primer probes, applied in the determination/examination of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of cumbersome material collection, inability to perform dynamic detection, etc., to avoid PCR false negatives Effect

Pending Publication Date: 2021-08-24
SHENGZHEN CHINA GENE TECH COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods rely on tumor tissue, cumbersome materials, and cannot achieve dynamic detection

Method used

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  • Primer probe for detecting breast cancer recurrence and metastasis gene HER2 amplification and application thereof
  • Primer probe for detecting breast cancer recurrence and metastasis gene HER2 amplification and application thereof
  • Primer probe for detecting breast cancer recurrence and metastasis gene HER2 amplification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Design of Specific Primers and Probes for Detecting Breast Cancer Recurrence and Metastasis Gene HER2 Amplification

[0034] With human HER2 as the target gene and human EFTUD2 gene as the internal reference gene, specific primers and probes suitable for ddPCR were designed. The sequences of the primers and probes are as follows:

[0035]Target gene upstream primer sequence HER2-F: 5'-GTGTGGCAGTTCAGCCCTAT-3'SEQ ID NO: 1;

[0036] Target gene downstream primer sequence HER2-R: 5'-AATGTGTGCCACGAAACTGC-3'SEQ ID NO: 2;

[0037] Target gene probe sequence HER2-P: 5'-FAM-GGCTGATGGCTCGCGCTGGGCA-BHQ1-3'SEQ ID NO: 3;

[0038] Internal reference gene upstream primer sequence EFTUD2-F: 5'-CTCAAAGTGCGGGGACTGAT-3'SEQ ID NO: 4;

[0039] Internal reference gene downstream primer sequence EFTUD2-R: 5'-GGCATCAGGGTGACTCCAAA-3'SEQ ID NO: 5;

[0040] Internal reference gene probe sequence EFTUD2-P: 5'-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ1-3'SEQ ID NO: 6;

Embodiment 2

[0041] Example 2 Detection of breast cancer recurrence and metastasis gene HER2 amplification using digital PCR detection primers and probes

[0042] 1. DNA extraction from plasma

[0043] The NucleoSpin Plasma XS kit (product number: 740901.50) produced by MNG Company was used to extract nucleic acid from plasma samples. Take 240 μl of plasma, perform nucleic acid extraction according to the instructions of the extraction kit, and finally resuspend with 20 μl of eluent.

[0044] 2. Digital PCR amplification reaction

[0045] (1) Prepare digital PCR reaction solution; 20 μl of digital PCR reaction solution is formulated as follows: 4 μl of one-step reaction buffer, 2 μl of enzyme mixture, 1.2 μl each of 10 μM HER2-F, HER2-R, EFTUD2-F and EFTUD2-R primers, 0.6 μl each of 10 μM HER2-P and EFTUD2-P, 8 μl of the DNA template of the sample to be tested;

[0046] (2) On the generation chip of droplet PCR, add 20 μ L of droplet PCR reaction system in step 1), then add 40 μ L of dr...

Embodiment 3

[0052] Embodiment 3 breast cancer sample digital PCR primer probe sensitivity experiment

[0053] In this experiment, breast cancer positive samples were used for serial dilution. Detect by the detection method given in Example 2. The results show that this system can detect the copy number ( Figure 3-Figure 4 ).

[0054] Table 2. HER2 gene and EFTUD2 gene detection results of positive samples at the critical concentration

[0055]

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Abstract

The invention provides a primer probe for detecting breast cancer recurrence and metastasis gene HER2 amplification and application of the primer probe. Primer probe sequences are respectively a target gene upstream primer sequence FZD2-F: SEQ ID No: 1 5'-GTGTGGCAGTTCAGCCCTAT-3', a target gene downstream primer sequence FZD2-R: SEQ ID No: 2 5'-AATGTGTGCCACGAAACTGC-3', a target gene probe sequence FZD2-P: SEQ ID No: 3 5'-FAM-GGCTGATGGCTCGCGCTGGGCA-BHQ1-3', an internal reference gene upstream primer sequence EFTUD2-F: SEQ ID No: 4 5'-CTCAAAGTGCGGGGACTGAT-3', an internal reference gene downstream primer sequence EFTUD2-R: SEQ ID No: 5 5'-GGCATCAGGGTGACTCCAAA-3', and an internal reference gene probe sequence EFTUD2-P: SEQ ID No: 6 5'-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ1-3'. The primer probe is used for a digital PCR kit, and the kit can rapidly, specifically and sensitively detect HER2 gene amplification. The kit is simple in one-step amplification method and low in cost, is very suitable for gene copy number variation conditions in various samples such as tumor tissues, circulating tumor cells and circulating tumor DNA, and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention belongs to the technical field of tumor molecular detection, and in particular relates to a primer probe for detecting breast cancer recurrence and metastasis gene HER2 amplification and application thereof. Background technique [0002] Breast cancer is the most common malignant tumor in women, ranking first in the incidence of female tumors and the number one cause of death among female residents, causing huge mental and economic burdens to families and society. There are great differences in the therapeutic effects of targeted drugs and the resulting prognosis in different tumor patients. Therefore, the dynamic detection of relevant gene mutations in tumor patients can accurately guide the clinical selection of appropriate targeted drugs. [0003] HER2 (ERBB2), a member of the human epidermal growth factor receptor (EGFR) family, is a proto-oncogene. HER-2 gene amplification / overexpression is widespread in more than 30% of human tumo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2600/166C12Q2600/118
Inventor 盛司潼张晓英黄思强朱涛
Owner SHENGZHEN CHINA GENE TECH COMPANY
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