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Primer pair and probe combined product, composition, and kit used for detecting KRAS mutation, and applications thereof

A combination product and primer pair technology, applied in the field of molecular biology, can solve the problems of LNA-MB detection method without KRAS gene mutation, and achieve the effect of high accuracy, high sensitivity and good reliability

Inactive Publication Date: 2018-08-07
求臻医学科技(北京)有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no LNA-MB detection method for KRAS gene mutation in the prior art

Method used

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  • Primer pair and probe combined product, composition, and kit used for detecting KRAS mutation, and applications thereof
  • Primer pair and probe combined product, composition, and kit used for detecting KRAS mutation, and applications thereof
  • Primer pair and probe combined product, composition, and kit used for detecting KRAS mutation, and applications thereof

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Experimental program
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Effect test

Embodiment

[0048] 1. Design of primers and probes

[0049] The wild-type and mutant gene sequences of the human KRAS gene were obtained according to Genbank query, and allele-specific amplification primers and hydrolysis probes were designed with the software PrimerPremier5. The 3' end of the allele-specific upstream primer is a mutant base and uses a locked nucleic acid. The hydrolysis probe is labeled with FAM at the 5' end and BHQ3 at the 3' end.

[0050] (1) KRAS sequence

[0051] The second exon and the surrounding sequence of the normal human KRAS gene are shown in SEQ ID NO:14.

[0052](2) Primers and probes

[0053] The common gene mutations of human KRAS are G12A, G12C, G12D, G12R, G12S, G12V and G13D. The corresponding forward and reverse primers are shown in the table below.

[0054]

[0055] Wherein, the first nucleotide at the 3' end of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 is a locked nucleic acid;

[0056] The 9th, 11th, 18th and 23rd nucleotides at the 5' end of SEQ ID N...

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Abstract

The invention belongs to the field of molecular biology, and more specifically relates to a primer pair and probe combined product, a composition, and a kit used for detecting KRAS mutation, and applications thereof. The combined product comprises a LNA fluorescence probe and primers used for detecting different KRAS gene 2th exon mutation types; wherein the forward primer sequence of the primer pair comprises at least one locked nucleic acid, and / or, the LNA fluorescence probe sequence comprises at least one locked nucleic acid. The primer pair and the probe composition is rapid and convenient in detection, is high in specificity, sensitivity, reliability, detection efficiency, and accuracy, low in detection cost, short in detection time, and low in false positive rate. The kit can be used for detecting KRAS mutation, is capable of providing guidance for clinical diagnosis and treatment of KRAS mutation cancer, and is promising in application prospect.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a combined product, composition, kit and application of a primer pair and a probe for detecting KRAS mutations. Background technique [0002] Molecular targeted therapy is one of the hot spots in the field of cancer treatment research, and epidermal growth factor receptor inhibitors are a class of targeted therapy drugs. Studies have shown that there is a clear correlation between the mutation status of the human KRAS gene and the efficacy of epidermal growth factor receptor inhibitors. [0003] Circulating tumor DNA (ctDNA) is a new type of tumor molecular marker, which has the characteristics of high detection rate, specificity, and high consistency with tissue detection results. ctDNA can be used to detect tumors in a non-invasive manner, which is helpful for clinical judgment of KRAS gene mutation status in tumor tissues. However, due to the low content of circulating free ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6858
CPCC12Q1/6858C12Q2525/117C12Q2563/185C12Q2563/107
Inventor 承康平段小红丁蕊杨春燕王东亮周启明
Owner 求臻医学科技(北京)有限公司
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