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Kit and method for detection of mutation of T790M site of EGFR genes

A technology of T790M and site mutation, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low free DNA, the precision and sensitivity of ordinary kits that cannot meet the detection requirements, and achieve high-sensitivity detection, Stable and reliable data results

Inactive Publication Date: 2020-05-29
宁波胤瑞生物医学仪器有限责任公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, due to the extremely low content of free DNA in blood, which is about 70-200 bp in length, and the content of mutation-related free DNA is even less, accounting for about 1% of the total free DNA content, the accuracy and sensitivity of ordinary kits cannot be used for digital PCR detection of blood. Requirements for detection of EGFR gene T790M site mutation

Method used

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  • Kit and method for detection of mutation of T790M site of EGFR genes
  • Kit and method for detection of mutation of T790M site of EGFR genes
  • Kit and method for detection of mutation of T790M site of EGFR genes

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 Design and Synthesis of Primers and Probes for Detecting Lung Cancer EGFR Gene T790M Site Mutation

[0042] According to the missense mutation of the 790th codon of exon 20 of EGFR (i.e. the T790M mutation), the primers and probes of the present invention based on digital PCR technology for detecting the T790M mutation of the EGFR gene in lung cancer were designed and synthesized. The specific sequences are as follows:

[0043] Primer sequence:

[0044] Upstream primer: 5'-GGCATCTGCCTCACCTCCA-3';

[0045] Downstream primer: 5'-GACATAGTCCAGGAGGCAGC-3';

[0046] Probe sequence:

[0047] Mutant fluorescent probe: 5'-GAGCTGCATGATGAG-3';

[0048] Wild-type fluorescent probe: 5'-GAGCTGCGTGATGAG-3';

[0049] Both primers and probes were synthesized by Shanghai Sangon Company and purified by HPLC grade. The 5' end of the mutant fluorescent probe is connected with a fluorescent reporter group FAM; the 3' end is connected with a fluorescent quencher group BHQ1; the...

Embodiment 2

[0052] Example 2 Method for Detecting T790M Site Mutation of Human Lung Cancer EGFR Gene

[0053] Using the kit in Example 1, prepare a PCR reaction system according to Table 1, wherein, PCR Mix was purchased from NEB, and added a final concentration of 0.1% Triton-X-100, 1U thermostable pyrophosphatase, and 5 μg / μL of BSA , according to ddH 2 O, the order of PCR mix, probe, primer, and template DNA, add the above samples into a 0.2mL PCR tube according to the addition amount of 20μL in the reaction system in Table 1, mix the mixed system for 15s with gentle vortex, and pass through the reaction system for a short time. Centrifuge to collect the solution at the bottom of the tube. Load the prepared reaction systems with different ratios onto the PCR chip to form a micro-reaction unit. Put the chip into a digital PCR instrument, perform PCR reaction according to the PCR reaction conditions in Table 2, and select FAM and CY3 as the channels for fluorescence detection.

[0054...

Embodiment 3

[0059] The detection of embodiment 3 standard substance

[0060] The preparation method of the mutation-containing standard product is as follows: the EGFR gene T790M site mutant genomic DNA and wild-type genomic DNA are synthesized respectively to construct plasmids, and then the T790M site mutant genomic DNA: wild-type genomic DNA ratio is 1:10, 1 :50, 1:100, 1:200, 1:500, 1:1000, 1:2000, 1:3000 were mixed, and then the resulting mixed plasmid was broken into 180bp fragmented DNA close to the ctDNA fragment; wild type The preparation method of the standard product is as follows: the EGFR gene T790M wild-type DNA plasmid is interrupted into a 180bp fragmented DNA close to the ctDNA fragment.

[0061] The 8 mutation standard products obtained by the above method were detected by the method in Example 2, and the experiment was repeated 3 times. The experimental results are as described in Table 3, and Table 3 is the standard substance (0.033%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2% a...

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Abstract

The invention provides a kit and method for detection of mutation of a T790M site of EGFR genes, and belongs to the field of nucleic acid detection. The kit includes an upstream primer and downstreamprimer which are used for detecting the T790M site and a mutant fluorescent probe, a wild-type fluorescent probe, a reaction premix, a positive quality control substance and a negative quality controlsubstance which are used for detecting the T790M site. According to the kit, the primers and probes with specific sequences are designed for the T790M site of the human EGFR genes, and the reaction system of the primers and probes are optimized; and through a digital PCR platform method, high-sensitivity detection of mutation of the T790M site of the EGFR genes is achieved, and the mutation abundance is obtained at the same time. The kit and method can be used for detecting nucleic acids in samples of multiple sources which include tumors, blood, saliva and serum, the application range of thekit and detection method is expanded, and medication guidance is provided for treatment of patients with lung-cancer T790M mutation.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and in particular relates to a kit and a method for detecting the T790M site mutation of the EGFR gene. Background technique [0002] Lung cancer is the malignant tumor with the highest morbidity and mortality rate in the world. Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancer patients. The most common cause of non-small cell lung cancer in Chinese population Disease mutations come from the EGFR gene (about 20% to 40%). Among the EGFR mutations, they mainly focus on the deletion of exon 19 (about 45%) and the L858R mutation of exon 21 (about 40%). Epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) T790M mutation is the main factor leading to acquired drug resistance during TKI (tyrosine kinase inhibitor) treatment. After 9-13 weeks of Gefitinib or Afatinib, this mutation will appear in about 60% of patients. Therefore, the detection of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6851C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 刘一博金鑫浩任鲁风张未来于军
Owner 宁波胤瑞生物医学仪器有限责任公司
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