Primer composition, kit and method for detecting EGFR gene mutations

A primer composition and composition technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as high cost and cumbersome operation, and achieve the effect of improving convenience and flexibility

Inactive Publication Date: 2017-12-19
LIAONING KEJUN BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, for EGFR gene detection, there are many methods for library construction and detection through high-throughput sequencing platforms, but they all have disadvantages such as high cost and cumbersome operation. Therefore, existing detection methods still need to be improved

Method used

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  • Primer composition, kit and method for detecting EGFR gene mutations
  • Primer composition, kit and method for detecting EGFR gene mutations
  • Primer composition, kit and method for detecting EGFR gene mutations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The specific library construction method of the application is as follows:

[0060] (1) The first round of PCR

[0061](1) The first round of multiplex PCR amplification: A single portion needs to be amplified simultaneously according to the PCR amplification reaction systems of library A and library B (as shown in Table 4 and Table 5 below, respectively), (25 μl system):

[0062] Table 4:

[0063] A library component (1╳)

Volume (μl)

PCR Enzyme Mix

16.5

Primer Mix A

2

template DNA

X

Nuclease-free ultrapure water

6.5-X

[0064] Attachment: Primer mixture A is a mixture of SEQ ID NO:1-10.

[0065] table 5:

[0066] B library components (1╳)

Volume (μl)

PCR Enzyme Mix

14.5

Primer Mix B

2

template DNA

X

Nuclease-free ultrapure water

8.5-X

[0067] Attachment: Primer mixture B is a mixture of SEQ ID NO: 11-18.

[0068] (2) PCR amplification, put each reac...

Embodiment 2

[0090] Purchase traceable positive cell lines or FFPE samples of 6 EGFR gene mutation sites and EGFR gene mutation-negative cell lines from the market. Or FFPE DNA and EGFR gene mutation-negative cell line DNA are mixed in a certain proportion to make positive reference substances with different mutation frequencies. The specific information of the positive reference substances used in the following examples of this application is shown in Table 8:

[0091] Table 8:

[0092]

[0093] The method of Example 1 was used to detect the reference products of different positive mutation types, and the detection was repeated 6 times (L1-L6). The test results are shown in Table 9 below. The results showed that only the corresponding mutation types were positive, and the coincidence rate was 100%.

[0094] Table 9:

[0095]

[0096]

Embodiment 3

[0098] The method of Example 1 was used to detect 10 reference samples (N1-N10) of negative mutation types, and the test results are shown in Table 10 below. The results showed that they were all negative (neg), and the coincidence rate was 100%.

[0099] Table 10:

[0100]

[0101]

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PUM

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Abstract

The invention discloses a primer composition, a kit and a method for detecting EGFR gene mutations, which aim to improve the convenience and the flexibility of EGFR gene mutation detection. The primer composition comprises a first composition A, wherein the first composition A comprises a first primer pair for amplifying a No.18 exon and a second primer pair for amplifying a No.19 exon; the first primer pair comprises A_E18-CS1-TS_1F as shown in SEQ ID NO:1 and A_E18-CS2-TS_1R as shown in SEQ ID NO:2. The primer composition simultaneously comprises the primer compositions for amplifying the No.18 exon and the No.19 exon, and the length of each amplicon obtained through amplification is smaller than 150bp, so that mutational hotspot areas in the two exons can be detected conveniently at the same time, an appropriate sample source is conveniently selected according to a sample state, and the convenience and the flexibility of EGFR gene mutation detection are improved.

Description

technical field [0001] The invention relates to the field of gene mutation detection, in particular to a primer composition, kit and method for detecting EGFR gene mutation. Background technique [0002] The detection of gene mutations (including point mutations, insertions, deletions, fusions, etc.) has guiding significance for targeted drugs. The second-generation high-throughput sequencing technology developed in recent years provides strong technical support for the treatment of targeted drugs. High-throughput sequencing can simultaneously perform high-sensitivity quantitative detection of multiple samples, multiple genes, and multiple loci, which is a good supplement to traditional detection methods. At present, there are mainly the following methods for detecting these mutations, and their respective characteristics, advantages and disadvantages are shown in Table 1 below. [0003] Table 1: [0004] [0005] Studies have shown that the expression of EGFR is up-re...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2535/122C12Q2563/159
Inventor 焦慧
Owner LIAONING KEJUN BIOLOGICAL
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