Method for homogeneously detecting mutation index of exon of EGFR allele 19

An allele and exon technology, applied in the field of gene exon 19 mutation index detection, can solve the problems of complicated operation steps, high cost, easy pollution, etc., and achieve simple result analysis, low cost, and easy standardization. Effect

Inactive Publication Date: 2017-06-13
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

[0003] The object of the present invention is to provide a method for homogeneous detection of EGFR allele 19 exon mutation index by fluorescence polarization, which is used for EGFR allele 19 exon mutation index, that is, EGFR allele 19 exon wild type and The detection of the content ratio of the mutant type overcomes the problems of one-sidedness, high cost, cumbersome operation steps and easy pollution in the prior art

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Examples

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Embodiment

[0039] Detect the EGFR allele exon 19 mutation index and the sensitivity to EGFR targeting inhibitors of the following cell samples to be tested.

[0040] The reagents used in this example include: the amplification reaction is carried out in a 25uL system, 2.5μL of 10×PCR reaction buffer is added, and the concentration is 2.0mmol / L of dGTP, dCTP, dATP and dTTP, 1-2 units of DNA polymerase, 2.5mM MgCl 2 . 0.5uM EGFR19 exon forward and reverse primers, 0.07uM EGFR19 exon reverse primer, 0.5uM EGFR19 exon R110 labeled wild-type probe, EGFR19 exon FAM labeled mutant probe, template 5uL.

[0041] Add 5uL of negative control standard (pGEM-T-easy vector 10,000 copies / ml) to the negative control reaction, and 5uL of positive control standard (containing EGFR allele exon 19 wild gene product pGEM-T- wild-19 clone 1000 copies / ml and pGEM-T-delmutation-19 clone 1000 copies / ml containing EGFR allele exon 19 mutation gene product). 1 / 1 mixed cell sample to be tested: Human lung cance...

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Abstract

The invention relates to a method for homogeneously detecting a mutation index of an exon of an EGFR allele 19. The method comprises the following steps: amplifying a specific reagent; then performing FAM and R110 fluorescence polarization value detection on an amplified product; and judging the mutation index of the exon of the EGFR allele 19 of a to-be-detected specimen according to fluctuation and specific value of FAM and R110 fluorescence polarization values. The invention creatively provides the method for detecting the mutation index of the exon of EGFR allele 19 and the mutation index of the exon of the EGFR allele 19. The mutation index of the exon of EGFR allele 19 is a molecular identifier which is highly related to and more sensitive and specific to drug reactivity compared with absolute quantity and anomaly of the exon of EGFR allele 19. The method for detecting the mutation index of the exon of the EGFR allele 19 is more scientific and accurate compared with that of detecting anomaly and absolute quantity only, and the method performs detection of criterion and quantity and with a predictive value.

Description

technical field [0001] The invention relates to the technical field of detection of EGFR allele exon 19 mutation index, in particular to a method for homogeneously detecting the EGFR allele exon 19 mutation index. Background technique [0002] Allelic variation is the variation of a gene that controls a certain trait at the same position in the double-stranded DNA of a pair of homologous chromosomes in a cell. In the individual genome, allelic variation and wild type exist at the same time, mutually restricting the balance, affecting gene expression and function together, affecting biological responses, and determining phenotypes such as cell fate; allelic variation within individuals has heterogeneity and dosage Effect, different individuals have different relative quantitative levels of allelic variation and wild type at different stages, and the relative quantitative level of variation directly affects gene function and expression. EGFR allelic exon 19 variation and wild...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2537/165C12Q2545/113C12Q2547/101C12Q2561/119
Inventor 张菊张潍王晓栋王金燕姜英浩刘文超
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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