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Specific primer, probe and kit for detecting EGFR gene L858R locus

A specific and probe technology, applied in the field of molecular detection, can solve the problem of not being suitable for the detection of free nucleic acid in blood circulation, and achieve the effect of high sensitivity and accurate detection

Inactive Publication Date: 2016-04-20
BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can detect samples containing 0.1-1% EGFR gene mutation DNA, but the mutation of free nucleic acid in blood circulation is sometimes lower than this frequency, so this technical solution is not suitable for the detection of free nucleic acid in blood circulation

Method used

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  • Specific primer, probe and kit for detecting EGFR gene L858R locus
  • Specific primer, probe and kit for detecting EGFR gene L858R locus
  • Specific primer, probe and kit for detecting EGFR gene L858R locus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 is used to illustrate the screening process of the specific primers of the present invention.

[0040] Primer design: Search the human EGFR (NM_005228.3) gene cds sequence from NCBI, use primer5 software to design upstream and downstream primers that can amplify and store the fragment containing the mutation site (c.2537), and select 6 pairs of primers The size of the amplified fragments ranged from 200bp to 50bp; the 6 pairs of primers were synthesized; different amplification lengths would affect the sensitivity of the experimental program, so the PCR technique was used to test the 6 pairs of primers.

[0041] QPCR experiments were performed on the obtained primers to verify the amplification efficiency of different primer sets. The QPCR results showed that under the same amplification system and amplification conditions, the CT value of QPCR appeared the earliest when the length of the primer set amplification product was 142bp, indicating that the amplificati...

Embodiment 2

[0046] Example 2 is used to illustrate the specific probes of the present invention.

[0047] For the specific primer screened in Example 1, the specific probe used in conjunction with it was designed by primerdesign, which can be used together when detecting the L858R mutation site of the EGFR gene.

[0048] Advantages of MGB probes: Since the MGB group has a high affinity for the DNA double-strand minor groove, in the PCR reaction, the Tm value of the MGB probe is much higher than that of the ordinary probe, usually 10-20 ℃. MGB probes have a high Tm value, which can well overcome the difficulty of probe design for templates with low GC content. The Tm values ​​of allelic mutation sequences are very different, which improves the specificity of detection. At the same time, the MGB probe includes a non-fluorescent quencher (NFQ), which can truly eliminate the background fluorescence generated by the traditional quencher, improve the signal-to-noise ratio, and thus improve the...

Embodiment 3

[0055] Example 3 is used to illustrate the application of the specific primers and probes of the present invention.

[0056] 1. Specificity and sensitivity experiments

[0057] The mutant plasmid (mutant) containing the EGFR gene L858R mutation site and the plasmid (WT) containing the wild type of the site were respectively constructed, and using these two plasmids as templates, the specificity of the probe was detected by the droplet digital PCR system. and sensitivity tests. The reaction system includes 2×mixbuffer10ul, template 4ul (WT0.01ng / 20ul; WT0.001ng / 20ul, WT0.0001ng / 20ul; mutant0.01ng / 20ul; mutant0.001ng / 20ul; mutant0.0001ng / 20ul; mutant0.00001ng / 20ul; 20ul), forward primer 1ul, reverse primer 1ul, WT probe 2ul, mutant probe 2ul, a total of 20ul reaction system. The reaction conditions are 95°C, 10 minutes; 94°C, 30 seconds, 60°C, 1 minute (40 cycles); 98°C, 10 minutes; 4 degrees.

[0058] Test results such as Figure 1 ~ Figure 3 shown. When the final concentr...

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Abstract

The invention relates to molecular detection, and particularly discloses a specific primer, probe and kit for detecting an EGFR gene L858R locus. A nucleotide sequence of the specific primer is shown as SEQ ID NO. 1 to 2, and a nucleotide sequence of the probe is shown as SEQ ID NO. 3 to 4. The specific primer and the MGB-Taqman probe provided by the invention adopt a probe method to detect the EGFR gene L858R, and are not only suitable for detecting nucleic acid from a tumor tissue and a paraffin-embedded tissue, but also suitable for detecting a mutant locus of the EGFR gene L858R in free circulating nucleic acid of blood; and moreover, by combining a detection system and a micro-drop digital PCR system, the mutation frequency of a nucleic acid sample can be accurately detected.

Description

technical field [0001] The invention relates to molecular detection, in particular to specific primers, probes and kits for detecting the L858R mutation site of the EGFR gene. Background technique [0002] Epidermal Growth Factor Receptor (EGFR) is a receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. EGFR is a member of the HER or ErbB receptor family, which includes EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). EGFR is also often referred to as HER1 or ErbB1. EGFR and its family members can regulate many physiological processes such as cell differentiation, apoptosis, cell movement, angiogenesis, invasion and metastasis, and are expressed in most human tumors. Therefore, EGFR has been proved to be effective in many tumors. It plays an important regulatory role in the process of occurrence, development and prognosis. [0003] EGFR is a typical receptor with tyrosine kinase (Tyrosine Kinase, TK) activity, and TK pl...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2563/107C12Q2563/159
Inventor 李俊博武会娟安云鹤田彦捷王金龙
Owner BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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