Method and reagent kit for detecting human EGFR gene T790M and L858R mutations

A technology of T790M and L858R, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve problems affecting hydrogen bond formation, EGFR-TKI cannot bind to tyrosine kinase, etc.

Pending Publication Date: 2019-06-07
DALIAN GENTALKER BIO-TECH CO LTD
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Problems solved by technology

Once threonine is replaced by methionine, a larger amino acid side chain is introduced at this site to form a steric hindrance. The formation of this steric hindrance a

Method used

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  • Method and reagent kit for detecting human EGFR gene T790M and L858R mutations
  • Method and reagent kit for detecting human EGFR gene T790M and L858R mutations
  • Method and reagent kit for detecting human EGFR gene T790M and L858R mutations

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1 Design of primers and probe combinations for the double digital PCR detection system of human EGFR gene.

[0060] According to the mutation sites T790M and L858R of the human EGFR gene and adjacent sequences, the following primer and probe combinations (SEQ ID NO.1-8) for the double digital PCR detection system of the human EGFR gene were designed:

[0061] T790M upstream primer: 5'-GCCTGCTGGGCATCTG-3' (SEQ ID NO.1)

[0062] T790M downstream primer: 5'-TTTGTGTTCCCGGACATAGTC-3' (SEQ ID NO.2)

[0063] T790M mutant probe: 5'-FAM-TGAGCTGCATGATGA-MGB-NFQ-3' (SEQ ID NO.3)

[0064] T790M wild-type probe: 5'-VIC-TGAGCTGCGTGATGA-MGB-NFQ-3' (SEQ ID NO.4)

[0065] L858R upstream primer: 5'-CGCAGCATGTCAAGATCACAGAT-3' (SEQ ID NO.5)

[0066] L858R downstream primer: 5'-CTCCTTCTGCATGGTATTTCTTTCTC-3' (SEQ ID NO.6)

[0067] L858R mutant probe: 5'-FAM-AGTTTGGCCCGCCCAA-MGB-NFQ-3' (SEQ ID NO.7)

[0068] L858R wild-type probe: 5'-VIC-AGTTTGGCCAGCCCAA-MGB-NFQ-3' (SEQ ID NO.8) ...

Embodiment 2

[0069] Example 2 Establishment of Human EGFR Gene Double Digital PCR Detection System and Method

[0070] 1.1 Experimental materials

[0071] The wild-type gene DNA standard product used in this example was prepared by mixing equal volumes of leukocyte genomic DNA from 100 normal people. The T790M and L858R mutant plasmids were purchased from Origene, USA, and their article numbers were RC400289 and RC400290, respectively. The wild-type genome, the T790M mutant plasmid and the L858R mutant plasmid were mixed according to a certain ratio to form T790M / L858R double mutant standard products with double mutation rates of 25% and 10%, respectively. The specific preparation method of the T790M / L858R double mutant standard with a mutation rate of 25% for both T790M and L858R is as follows: first, the L858R plasmid stock solution obtained by fluorescent PCR was diluted to 10 -5 The gradient is about 10 times that of the 30ng / μL wild-type genome, and the T790M plasmid stock solution ...

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Abstract

The invention provides a primer and probe combination for a digital PCR detection system for double mutations of human EGFR gene T790M and L858R, the digital PCR detection system for the double mutations of the human EGFR gene T790M and L858R, a digital PCR detection reagent kit for the double mutations of the human EGFR gene T790M and L858R, and a detection method of the double mutations of the human EGFR gene T790M and L858R. High sensitivity detection of the T790M and L858R is realized by the method of the digital PCR platform in the dual system, and so the cost is reduced and the consumption of samples is reduced, especially for ctDNA with scarce samples. Moreover, the detection of other sample types such as tumor tissues is facilitated, and certain medication guidance is provided forpatients with non-small cell lung cancer.

Description

technical field [0001] The invention belongs to the field of biomedical technology, in particular to a combination of primers and probes for a digital PCR detection system for human EGFR gene T790M and L858R double mutations, a digital PCR for human EGFR gene T790M and L858R double mutations The detection system includes a digital PCR detection kit for human EGFR gene T790M and L858R double mutations, and a digital PCR detection method for human EGFR gene T790M and L858R double mutations. Background technique [0002] For a long time, lung cancer has been one of the malignant tumors with the highest morbidity and mortality worldwide. According to the cancer report released by the International Agency for Research on Cancer of the World Health Organization, the number of new cases of lung cancer worldwide has exceeded 1.6 million, and the death rate can reach 1.38 million, accounting for 13% and 18% of the incidence and mortality of malignant tumors. Ranked first among malig...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
Inventor 赵金银刘琦赵月杨兰邢晓星
Owner DALIAN GENTALKER BIO-TECH CO LTD
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