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Method for detecting EGFR L858R locus in lung cancer by ddPCR and application thereof

A technology of L858R and detection method, which is applied in ddPCR detection and application field of EGFR L858R locus in lung cancer, which can solve the problems of complex procedures, complex positive interpretation methods, low sensitivity, etc., and achieve the effect of high detection sensitivity

Inactive Publication Date: 2019-05-17
铭时医疗科技(宁波)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The low sensitivity makes the above method only suitable for the detection of advanced tumor patients
[0011] 2) poor specificity
The results of the blood test do not match well with the test results of the tumor tissue of the corresponding patient. The blood test is required before the tissue test, and the procedure is complicated.
[0012] 3) The positive interpretation method is complex
[0019] In the prior art, in the detection of the EGFR gene L858R mutation, there are problems such as pathological tissue as the starting material, low accuracy, and low sensitivity

Method used

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  • Method for detecting EGFR L858R locus in lung cancer by ddPCR and application thereof
  • Method for detecting EGFR L858R locus in lung cancer by ddPCR and application thereof
  • Method for detecting EGFR L858R locus in lung cancer by ddPCR and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1: The method for detecting EGFR L858R mutation by ddPCR method;

[0087] 1) Screening of primer pairs;

[0088] According to the sequence of exon 21 of EGFR, design 5 pairs of primers (primers were synthesized from Shanghai Sangon Biotechnology Co., Ltd.), the specific sequences are as follows:

[0089]

[0090] Screening PCR system: 0.2ul of Taq HS polymerase, 2ul of 10×HS PCR buffer, 1.6ul of dNTP mixture, 1ul of primer 1, 1ul of primer 2, 5ul of tgDNA (DNA template), and make up the system to 20ul with water. Screening PCR program: 95°C for 10 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 20s). After PCR screening, primer pair 1 (1-858-F / 1-858-R) had the highest amplification efficiency for the target fragment and did not produce primer dimers. The primer pair is used as a detection primer pair for the L858R mutation of the EGFR gene.

[0091] 2) Optimization of the ddPCR reaction program;

[0092] According to the EGFR gene L858R mutation s...

Embodiment 2

[0101] Embodiment 2; ddPCR method to detect the method verification of EGFR L858R mutation;

[0102] According to the result of ddPCR using 1975 as a template in Example 1, the copy number of the FAM channel and the total copy number of 1975 were calculated, the two were divided and multiplied by 100%, and the mutation rate of 1975 or the ratio of mutant template was 66%.

[0103] The genome copy numbers of 1975 and tgDNA were calculated according to Example 1, and both were diluted to 4000 copies / ul with TE. 1975 was incorporated into tgDNA so that the ratios of the L858R mutation templates were 0.0625%, 0.125%, 0.25%, 0.5%, and 1%, respectively, and the gradient dilution templates were made.

[0104] Validate the L858R mutation detection method as follows. Verify the ddPCR system: 2×ddPCR master mix (without dUTP) 11ul, primer 1 (1-858-F) 1.1ul, primer 2 (1-858-R) 1.1ul, probe 1 (WTp-L858R) 0.55ul , probe 2 (MTp-L858R) 0.55ul, template 5.5ul, add water to make up to 22ul. ...

Embodiment 3

[0107] Example 3: The L858R mutation detected by the ddPCR method guides the administration of tumor-targeted drugs;

[0108] Cases TP009 and TP034 were stage IV patients with lung adenocarcinoma. By collecting venous blood, centrifuge twice to separate plasma (the first low-speed centrifugation, 4°C, 1900g, 10min, the second high-speed centrifugation, 4°C, 16000g, 10min), and carefully pipette 1ml of the supernatant into a 50ml centrifuge tube. Plasma free nucleic acid was extracted by QIAGEN free nucleic acid extraction kit. The extracted nucleic acid was quantified by Qubit. After quantification, it was stored in a -20°C refrigerator in the sample preparation room.

[0109]The ddPCR method of the present invention detects the EGFR L858R mutation. Detection PCR system: 2×ddPCR master mix (without dUTP) 11ul, primer 1 (1-858-F) 1.1ul, primer 2 (1-858-R) 1.1ul, probe 1 (WTp-L858R) 0.55ul , probe 2 (MTp-L858R) 0.55ul, template 5.5ul, add water to make up to 22ul.

[0110] ...

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Abstract

The invention discloses a method for detecting an EGFR L858R locus in lung cancer by ddPCR and an application thereof. According to the sequence of an EGFR gene, a pair of amplified primers includingan upstream primer and a downstream primer are designed; according to the mutation sequence of L858R (2573T>G, COSM6224), a probe for detecting a wild type sequence and a probe for detecting a mutation sequence are designed. The primers and the probes are used for ddPCR and used for detecting and analyzing important loci related to targeted medication for lung cancer in ctDNA. Firstly, DNA is extracted from peripheral plasma of lung cancer patients, and the mutation condition of the mutation locus L858R of EGFR is detected by the ddPCR technology, so as to guide the patients to use drugs. Themethod can be used for diagnosis of early-stage lung cancer such as stage I, II and III A of tumor, can detect 0.0625% EGFR gene L858R mutation, and has the sensitivity reaching 10 times or more thatof conventional methods.

Description

technical field [0001] The invention discloses a ddPCR detection method and application technology of EGFR L858R locus in lung cancer, which belongs to the field of medicine and biotechnology, and relates to the improvement technology of the measurement or detection method of C12Q containing enzyme or microorganism in IPC classification, which can be used to guide the gene detection of tumor medication , especially highly sensitive detection of EGFR gene L858R site mutation in peripheral plasma / serum, and further guide the epidermal growth factor receptor-tyrosine inhibitor (EGFR -TKIs) targeted drugs. Background technique [0002] Due to the difficulty in sampling tumor tissue, blood detection of EGFR gene mutations will become an important supplement or alternative for screening patients who are eligible for epidermal growth factor receptor-tyrosine inhibitors (EGFR-TKIs). However, there are huge challenges in the application of blood testing. The reason is that the conte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6886
Inventor 陈苗苗黄昕江萤童慧娟
Owner 铭时医疗科技(宁波)有限公司
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