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Primer, probe, kit and detection method for detecting ESR1 gene expression

A gene expression and kit technology, applied in the field of medical molecular biology, can solve problems such as wrong treatment plans, and achieve the effects of simple operation, small human error, and simple and objective interpretation

Inactive Publication Date: 2021-04-13
杭州联川基因诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, according to a joint study by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP), due to the limitations of immunohistochemical technology itself, one in five breast cancer patients may receive an error due to inaccurate IHC detection. treatment plan

Method used

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  • Primer, probe, kit and detection method for detecting ESR1 gene expression
  • Primer, probe, kit and detection method for detecting ESR1 gene expression
  • Primer, probe, kit and detection method for detecting ESR1 gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 A kind of detecting ESR1 gene expression primer, probe and RT-qPCR kit

[0059] Design specific primers according to the target gene sequence, and the designed primers and probes can be artificially synthesized according to existing methods. The specific primers are as follows:

[0060] ESR1-Fo: TCCACCTTCTAGAATGCCT (SEQ ID NO. 1)

[0061] ESR1-Re: CCAGTTGATCATGTGAACCAG (SEQ ID NO. 2)

[0062] ACTB-Fo: TTCTACAATGAGCTGCGTGTG (SEQ ID NO. 3)

[0063] ACTB-Re: GGTGTTGAAGGTCTCAAACATGA (SEQ ID NO. 4)

[0064] PGK1-Fo: TCCTTAGAGCCAGTTGCTGTAG (SEQ ID NO. 5)

[0065] PGK1-Re: GCACAGGCTTTCTCCACTT (SEQ ID NO. 6)

[0066] The probe sequences are as follows:

[0067] ESR1-Pr: CCACAAAGCCTGGCACCCCTCTTCG (SEQ ID NO. 7)

[0068] Wherein, the 5' end is labeled with FAM, and the 3' is labeled with BHQ1.

[0069] ACTB-Pr: CCGTGCTGCTGACCGAGGCC (SEQ ID NO. 8)

[0070] Wherein, the 5' end is labeled with VIC, and the 3' is labeled with BHQ2.

[0071] PGK1-Pr: TCTCTGCTGGG...

Embodiment 2

[0082] Example 2 ESR1 gene expression RT-qPCR detection method

[0083] The expression of ESR1 gene was detected by immunohistochemical kit and method and the above-mentioned RT-qPCR kit and RT-qPCR detection method respectively,

[0084] RT-qPCR detection includes the following steps:

[0085] (1) RNA extraction and sample RNA quantification of the tumor sample to be tested;

[0086] Tumor tissues were taken to extract RNA according to the instructions of commercial extraction kits. The extracted RNA was quantified, and the concentration was not lower than 10ng / μL, and the sample RNA was directly used as a PCR template.

[0087] (2) Preparation of reaction mixture

[0088] All components in the kit prepared in Example 1 were equilibrated to room temperature and melted, and the reaction mixture was calculated and prepared according to the number of samples to be tested. The 20 μL system preparation table is shown in Table 2.

[0089] Table 2 Reaction mixture preparation ta...

Embodiment 3

[0106] Example 3 Practical application of RT-qPCR detection kit and RT-qPCR detection method

[0107]The RT-qPCR detection kit of Example 1 and the RT-qPCR detection method of Example 2 were used to detect the expression of ESR1 gene in the tumor tissue of breast cancer patients, and the samples used were paraffin-embedded breast cancer tumor tissue samples.

[0108] The detection method of immunohistochemistry is as follows:

[0109] Commercialized anti-estrogen receptor antibody reagent—anti-estrogen receptor (SP1) rabbit monoclonal antibody reagent (immunohistochemical method) (National Food and Drug Administration (Jin) Zi 2013 No. 3404985, Roche), VENTANA The detection kit (ultraView DAB Detection kit, Roche) was used to detect the estrogen receptor (ER) antigen expressed in the paraffin-embedded tissue section samples of breast cancer tumors on the VENTANA BenchMark XT automatic section stainer, according to the operating instructions in the kit To detect:

[0110] (1)...

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Abstract

The invention discloses a primer pair combination for detecting ESR1 gene expression, and belongs to the technical field of medical molecular biology. The primer pair combination comprises a first primer pair for amplifying an ESR1 gene, and further comprises a second primer pair and a third primer pair which are used for amplifying reference genes ACTB and PGK1 respectively. The invention further discloses a probe combination which comprises a first probe for targeting an amplification product of the first primer pair, and further comprises a second probe for targeting an amplification product of the second primer pair and a third probe for targeting an amplification product of the third primer pair. The invention further discloses a kit prepared by utilizing the primer pair combination or the primer pair combination and the probe combination, and a method for detecting the ESR1 gene. When the ESR1 gene expression is detected by utilizing the primer pair combination, the sample type is not limited, the detection result is more accurate, the detection period is short, the human error is small, the repeatability is good, and the primer pair combination is more sensitive and reliable compared with IHC.

Description

technical field [0001] The invention belongs to the technical field of medical molecular biology, and in particular relates to a primer, a probe, a kit and a detection method for detecting ESR1 gene expression. Background technique [0002] The status of ERα, PR, HER-2 and Ki-67 is an important indicator for judging the molecular type of early invasive breast cancer (non-special type), which can be divided into Luminal type (Luminal A and Luminal B), HER -2 overexpression type or basal-like type (Basal-like). Existing studies have shown that compared with the primary tumor, the ERα, PR, and HER2 status of some advanced breast cancer metastases may change, resulting in changes in the treatment strategy for advanced breast cancer. Statistics show that the incidence rates of ER, PR, and HER2-positive primary lesions and negative metastatic lesions are 12%-24%, 22%-41%, and 3%-16%, respectively. The proportion of positive metastases was 5%-9%, 7%-19%, 1%-5%. Due to the hetero...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6886C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/16C12Q2600/166C12Q2537/143C12Q2531/113C12Q2563/107C12Q2545/101C12Q2545/113C12Q2521/107
Inventor 杨欢欢潘石玄伟李璐璐梁洪郎秋蕾盛苨晶
Owner 杭州联川基因诊断技术有限公司
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