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Method and kit for detecting gene point mutation based on digital PCR platform

A point mutation and digital technology, applied in the field of biology, can solve the problems of inability to meet clinical detection requirements, poor sensitivity, and inability to directly obtain digital objective results, so as to reduce the possibility of false negative and false positive interpretation and speed up Effect

Inactive Publication Date: 2014-07-09
上海涌泰生物医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The objective results of digitalization cannot be obtained directly
[0010] 3. The sensitivity is relatively poor. The sensitivity of the current method is preferably 1%, which cannot meet some specific clinical detection needs.

Method used

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  • Method and kit for detecting gene point mutation based on digital PCR platform
  • Method and kit for detecting gene point mutation based on digital PCR platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 L858R point mutation on exon 21 of EGFR (human epidermal growth factor receptor) gene

[0046] (1) Prepare samples: DNA containing the wild-type EGFR gene, a sample mixed with mutation-positive DNA of the L858R point mutation on EGFR exon 21 and wild-type DNA (the content ratio of the mutant to the wild type is 1 / 1, 1 / 100, 1 / 1000, 1 / 2500). The source of DNA can be serum, plasma, peripheral blood, oral mucosa, pleural effusion, body fluid or tissue, etc. Among them, the mutant DNA template is derived from a cell line carrying the L858R point substitution mutation on EGFR exon 21 (identified by PCR sequencing), and the mutation site is at position c.2573 on EGFR exon 21.

[0047] (2) Melt PCR primers and corresponding TaqMan probes at room temperature.

[0048] The sequences of the forward and reverse PCR primers are:

[0049] F1:5'-AAAACACCGCAGCATGTCAA-3'

[0050] R1: 5'-CTTCTGCATGGTATTCTTTCTCTTC-3'

[0051] The sequences of the TaqMan probes are:

[0052...

Embodiment 2

[0086] Example 2 T790M point mutation on exon 20 of EGFR gene

[0087] Adopt the same condition and operation method as embodiment 1, difference is to use following PCR primer and TaqMan probe:

[0088] The sequences of the forward and reverse PCR primers are:

[0089]Forward F1: 5'-TCTGCCTCACCCTCCACCG-3'

[0090] Reverse R1: 5'-AGGCAGCCGAAGGGCA-3'

[0091] The fluorophore and quenching group connected to the TaqMan probe combined with wild-type DNA are VIC and MGB, and its partial nucleotide sequence is: PW3:5'-TGAGCTGCGTGAT-3'.

[0092] The fluorophore and quenching group connected to the TaqMan probe combined with the mutant DNA are FAM and MGB, and its partial nucleotide sequence is: PM4:5'-GCTGCATGATGAG-3'.

[0093] The result is as figure 2 , with the above method, the T790M point mutation located on exon 20 of EGFR at c.2573 can be detected, and the detection limit reaches 1 / 2500. Quantitative analysis error is generally 0.01% to 1%. In the test results of samples...

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Abstract

The invention relates to the field of molecular biology and especially relates to a method and a kit for detecting gene point mutation based on a digital PCR platform. Digital PCR is used as a platform, PCR primers and TaqMan probes are added into a reaction system, the TaqMan probes labeled with different types of fluorescent light groups are used for detecting wild and mutational DNA templates, and according to fluorescent light types, types and an amount ratio of the DNA templates in a sample are determined.

Description

technical field [0001] The invention relates to the fields of biology and nucleic acid detection, in particular to a method for detecting gene point mutations and a kit thereof. Background technique [0002] Point mutation is a common type of gene variation, which will bring many changes to the biological traits of microorganisms, plants and animals, and human physiological traits; including partial or complete inactivation of protein functions, formation of inactive fragments, etc., and may also change biological Gene regulation and other functions of the body. The study of gene mutation is of great significance to the inheritance, evolution and transformation of organisms. [0003] At present, there are mainly direct sequencing method (also known as Sanger sequencing method) and ARMS method for detection of gene variation. They are briefly introduced as follows: [0004] The Sanger sequencing method, that is, the Sanger (Sanger) dideoxy chain termination method was inve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/101C12Q2563/159
Inventor 王贻锘
Owner 上海涌泰生物医药科技有限公司
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