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Primer set for detecting mutation of ESR1 gene, reagent, kit and method thereof

A primer set and kit technology, applied in the field of biomedicine, can solve problems such as difficulty, inability to meet the needs of blood sample detection, complex operation, etc., and achieve the effects of enhancing overall performance, accurately detecting low-copy mutations, and improving sensitivity

Pending Publication Date: 2019-11-15
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its detection sensitivity is low, and only more than 10-15% of mutations can be detected in tissue samples, and it cannot be detected in samples with lower abundance mutations, and it cannot meet the needs of blood sample detection.
The next-generation sequencing method has high detection sensitivity and can achieve 1% or even 0.1% mutation detection. However, the instruments used are expensive, the operation is complicated, the detection cycle is long, and there are great difficulties in clinical promotion.
[0012] At present, there is no better and more convenient method in clinical practice that can achieve very good results in the detection of ESR1 gene mutations in plasma samples, and there is no better method that is compatible with the detection of two types of samples, tissue and plasma, to meet clinical needs pain points and difficulties

Method used

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  • Primer set for detecting mutation of ESR1 gene, reagent, kit and method thereof
  • Primer set for detecting mutation of ESR1 gene, reagent, kit and method thereof
  • Primer set for detecting mutation of ESR1 gene, reagent, kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] (1) This embodiment provides a primer set for detecting ESR1 gene mutation, which includes: primer set 1, primer set 2 and primer set 3.

[0118] Primer combination 1 includes: the downstream primer shown in SEQ ID NO.12 (named ESR1-R1), the specific probe shown in SEQ ID NO.15 (named ESR1-P1) and SEQ ID NO.1-9 The upstream primers shown in SEQ ID NO.1-9 are named in sequence: D538G-F2, Y537N-F2, Y537S-F2, Y537C-F2, L536R-F2, L536H-F2, L536P-F2 , L536Q-F2, V534E-F2.

[0119] Primer combination 2 includes: the downstream primer shown in SEQ ID NO.13 (named ESR1-R2), the specific probe shown in SEQ ID NO.16 (named ESR1-P2) and the primer shown in SEQ ID NO.10 The upstream primer (named E380Q-F2);

[0120] Primer combination 3 includes: the downstream primer shown in SEQ ID NO.14 (named ESR1-R3), the specific probe shown in SEQ ID NO.17 (named ESR1-P3) and the primer shown in SEQ ID NO.11 The upstream primer (named S463P-F2).

[0121] Wherein, each of the above-mention...

Embodiment 2

[0188] Kit configuration and assembly

[0189] Prepare a positive control solution and a blank control solution. The positive control solution contains 11 kinds of plasmid DNAs, and these 11 kinds of plasmid DNAs contain 11 kinds of DNA fragments of the ESR1 gene respectively. The selection and design of the plasmids are well known to those skilled in the art. Both are 2000copies / μl; the blank control solution is Tris-HCl (10mM) buffer solution.

[0190] PCR system master mix preparation:

[0191] Prepare the quality control PCR system master mix according to the following table 3:

[0192] Table 3 Quality Control PCR System Master Mix Components

[0193]

[0194] According to the following table 4, the preparation of 11 mutation detection PCR system premixes was carried out:

[0195] Table 4 Components of Mutation Detection PCR System Master Mix

[0196]

[0197] Carry out the preparation of enzyme mixture according to following table 5:

[0198] Table 5 Enzyme Mixtu...

Embodiment 3

[0204] (1) Preparation of evaluation reference products

[0205] In order to more accurately evaluate the performance of the primer set in Example 1, including indicators such as specificity, accuracy, detection limit, and repeatability, use plasmids, cell line genomic DNA, tissue sample DNA, and plasma sample ctDNA to prepare relevant reference products. For the performance evaluation of the primer set, the specific implementation is as follows:

[0206] According to the 11 mutant sequences of the ESR1 gene in the GENE bank database, 11 DNA fragments were synthesized, and the 11 mutant DNA fragments were respectively cloned and connected to the pEASY-T1 (Transgen) plasmid vector (for the structure, see figure 2 ). The plasmid was transformed into the DH5α strain, and the plasmid was extracted after the strain was cultured. The A260 / A280 value was between 1.6 and 2.0, and the sequence was confirmed to be correct by sequencing.

[0207] The 11 mutant DNA sequences are shown ...

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Abstract

The invention discloses a primer set for detecting mutation of an ESR1 gene, a reagent, a kit and a method thereof, which relate to the field of biological medical science. The primer set for detecting the mutation of the ESR1 gene disclosed in the present invention includes any one or more of the following primer combinations: a primer combination 1, a primer combination 2, and a primer combination 3. The primer set can accurately detect 11 mutations of the ESR1 gene, and has the advantages of high sensitivity and good specificity, and can accurately detect the mutation of 0.1% in a total of20 ng DNA samples. The primer set can be applied to the detection of tissue samples and plasma samples, and can flexibly adapt to the needs of clinical testing. The detection method is simple in operation and the detection results are clear and easy to analyze.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a primer set, reagent, kit and method for detecting ESR1 gene mutation. Background technique [0002] Breast cancer is one of the malignant tumors that seriously threaten the health of women in my country. On March 23, 2018, the National Cancer Center released the latest data on breast cancer in Chinese women, estimating the incidence and death of breast cancer in Chinese women in 2014. The results show that , breast cancer ranks first in the incidence of female malignant tumors. According to the data released by the National Cancer Center, in 2014, there were about 278,900 new cases of female breast cancer nationwide, accounting for 16.51% of female malignant tumors, ranking first in the incidence of female malignant tumors. [0003] Globally, China accounts for 12.2% of newly diagnosed breast cancer cases and 9.6% of breast cancer deaths. According to the global cancer report releas...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 张喆程德勇吴亚蔡从利
Owner WUHAN YZY MEDICAL SCI & TECH
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