Primer and probe for detecting mutant KRAS genes

A technology of probes and probe sequences, applied in the field of biotechnology and diagnostic research, can solve problems such as difficult detection of Kras gene mutations

Inactive Publication Date: 2015-04-08
HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of KRAS gene mainly includes ordinary sequencing method and PCR based on simple typing probe, wherein the sensitivity of ordinary sequencing metho

Method used

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  • Primer and probe for detecting mutant KRAS genes
  • Primer and probe for detecting mutant KRAS genes
  • Primer and probe for detecting mutant KRAS genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] PCR reactions were performed using the following items.

[0072] (1) The target to be tested (i.e. the DNA template in the PCR reaction)

[0073] Prepare eight groups of targets to be tested, which are 1% 12DKRAS mutant + 99% wild type, 1% 13DKRAS mutant + 99% wild type, 1% 12VKRAS mutant + 99% wild type, 1% 12CKRAS mutant + 99% Wild type, 1% 12SKRAS mutant + 99% wild type, 1% 12AKRAS mutant + 99% wild type, 1% 12RKRAS mutant + 99% wild type, 100% wild type and no template control.

[0074] (2) Primers

[0075] Select any forward primer, select F1 in this example,

[0076] Select any reverse primer, select R3 in this embodiment,

[0077] (3) Probe

[0078] Randomly select a group of probes. In this embodiment, group A probes are selected, in which the 5' end of the probe is labeled with the fluorescent substance Fam, and the 3' end of the probe is MGB NFQ.

[0079] Fam is carboxyfluorescein, 5-Fam and 6-Fam can be used, preferably 5-Fam.

[0080] MGB is the minor ...

Embodiment 2

[0091] A kit for detecting KRAS gene mutation

[0092] The kit in this embodiment includes: PCR reaction solution, PNA reagent, enzyme digestion solution, sequencing reaction solution, magnetic beads and product purification solution. Wherein the PCR reaction solution includes PCR buffer, dNTPs, PCR primers, probes and DNA polymerase.

[0093] Preferably, in the PCR primers in this embodiment, the forward primer is F2, the reverse primer is R1, and the probe is a group B probe, that is, the fluorophore at the 5' end of the probe is FAM, and the fluorophore at the 3' end is FAM. for BHQ1.

[0094] The PNA is PNA02.

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Abstract

The invention provides a primer and probe for detecting mutant KRAS genes and further provides a method and kit for detecting mutant KRAS genes by using the primer and the probe. By adopting the primer and probe provided by the invention, the amplification of wild KRAS genes can be inhibited, and the combination of the wild KRAS genes with the probe can be also inhibited, thus less than 1% of mutant KRAS genes can be detected.

Description

technical field [0001] The invention belongs to the field of biotechnology and diagnosis research, and relates to a primer and a probe for detecting KRAS mutation, and a method and a kit for detecting whether a sample contains KRAS mutation by using the primer and probe. Background technique [0002] There are three genes related to human tumors in the RAS gene family-H-RAS, KRAS and N-RAS, which are located on chromosome 11, 12 and 1, respectively. The RAS gene, which is a proto-oncogene, becomes an oncogene with oncogenic activity after being activated, and the RAS gene is activated by mutation. [0003] KRAS is one of the RAS genes that undergo mutations in a variety of cancers. The KRAS gene can be normal (called wild type) or abnormal (mutant). Under normal physiological conditions, when cells are activated by external stimuli and other signaling pathways, wild-type KRAS is temporarily activated after being phosphorylated by tyrosine kinases such as active growth fact...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 吕宁陈一友
Owner HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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