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Primer, probe and kit for HRAS G13R mutation detection

A mutation detection and kit technology, which is applied in DNA/RNA fragmentation, recombinant DNA technology, and the determination/inspection of microorganisms, etc., can solve the problems of sensitivity and specificity that cannot meet the detection requirements of trace samples and low cfDNA content.

Pending Publication Date: 2021-04-30
山东康华生物医疗科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the content of cfDNA in blood is low. Current detection technologies mainly include next-generation sequencing (NGS), digital PCR, ARMS-PCR and Sanger sequencing to detect HRAS point mutations, but their sensitivity and specificity cannot meet the needs of trace samples. Testing needs

Method used

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  • Primer, probe and kit for HRAS G13R mutation detection
  • Primer, probe and kit for HRAS G13R mutation detection
  • Primer, probe and kit for HRAS G13R mutation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The sample to be tested is: thyroid cancer C643 cell line;

[0036] The detection method is as follows:

[0037] Step 1: Use a commercial cell / tissue DNA extraction kit to extract the genomic DNA of the cell line according to its instructions. In this example, Biotech Cell / Tissue DNA Extraction Kit (spin column type) was used to extract genomic DNA from thyroid cancer C643 cell line. The specific steps are as follows:

[0038] 1) a. Collect about 10 5 -10 6 Suspend the cells into a 1.5 ml centrifuge tube. For adherent cells, they should be digested with trypsin and collected by pipetting.

[0039] b. Centrifuge at 13,000 rpm for 10 seconds to pellet the cells. Discard the supernatant, leaving the cell pellet and about 10-20 μl residual liquid.

[0040] c. Add 200 μl buffer TB to resuspend and wash the cells, repeat the above step 1b, discard the supernatant and resuspend the cells in 200 μl buffer TB.

[0041] d. Add 200 μl of binding solution CB, immediately i...

Embodiment 2

[0067] The sample to be tested is the colorectal cancer SW480 cell line;

[0068] The detection method is as follows:

[0069] Step 1: Use a commercial cell / tissue DNA extraction kit to extract the genomic DNA of the cell line according to its instructions. In this example, Biotech Cell / Tissue DNA Extraction Kit (spin column type) was used to extract the genomic DNA of the colorectal cancer SW480 cell line. The specific steps are as follows:

[0070] 1) a. Collect about 10 5 -10 6 Suspend the cells into a 1.5 ml centrifuge tube. For adherent cells, they should be digested with trypsin and collected by pipetting.

[0071] b. Centrifuge at 13,000 rpm for 10 seconds to pellet the cells. Discard the supernatant, leaving the cell pellet and about 10-20 μl residual liquid.

[0072] c. Add 200 μl buffer TB to resuspend and wash the cells, repeat the above step b, discard the supernatant and resuspend the cells in 200 μl buffer TB.

[0073] d. Add 200 μl of binding solution C...

Embodiment 3

[0099] Samples to be tested: blood samples from 2 patients with thyroid cancer (i.e. blood free DNA---cfDNA)

[0100] Step 1: Use EDTA K2 anticoagulant tubes to collect blood, centrifuge at 2000-4000 rpm for 15 minutes at 4°C, and absorb the upper layer of plasma for enrichment of cfDNA. Use commercial kits or existing methods to enrich free DNA in plasma.

[0101] Step 2: Carry out the first round of PCR reaction, and use the cfDNA product obtained in Step 1 as a template to perform the following PCR reaction. The reaction system is as follows:

[0102]

[0103] Upstream primer: 5'-TCCTTGGCAGGTGGGGCA-3' (corresponding to the 7th sequence in the sequence list)

[0104] Downstream primer: 5'-CGCACTCTTGCCCACAC-3' (corresponding to the 8th sequence in the sequence list)

[0105]Probe: FAM-5'-TGAGGAGCGATGACGGAAT-3'-MGB. (corresponding to the 9th sequence in the sequence list)

[0106] The reaction conditions are as follows:

[0107] 94°C for 3 minutes; 94°C for 30 seconds,...

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Abstract

The invention relates to the technical field of biology, particularly to a primer, a probe and a kit for HRAS G13R mutation detection. The kit comprises allele specific primers, probes, allele non-specific primers, allele non-specific probes and PCR technology common reagents, wherein the PCR common reagents, the primers and the probes form a PCR reaction solution. The kit can effectively and accurately detect the mutation of HRAS G13R in a sample, and has high specificity and sensitivity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer, a probe and a kit for HRAS G13R mutation detection. Background technique [0002] Mutations in the RAS gene were first reported in cancer more than 30 years ago, and numerous studies since then have demonstrated that mutated RAS is a driver of tumor initiation and maintenance (Cox and Der, 2010). Three human RAS genes, the Kirsten murine sarcoma viral oncogene homolog (KRAS), the neuroblastoma RAS viral oncogene homolog (NRAS), and the Harvey rat sarcoma viral oncogene homolog (HRAS), encode four RAS protein. RAS proteins (KRAS4A, KRAS4B, NRAS, and HRAS) act as GDP-GTP-regulated binary switches that regulate cytoplasmic signaling networks that control various normal cellular processes. The HRAS gene is responsible for encoding a protein called H-ras, which is part of the RAS / MAPK pathway and is responsible for controlling transcriptional activity and cell cycle, and is as...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858
Inventor 王振新杨致亭马立娜魏佳杨锋斌田永帅
Owner 山东康华生物医疗科技股份有限公司
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