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Kit and method for detecting human-derived KRAS gene mutation in excrement

A detection reagent and human-derived technology, applied in the medical field, can solve problems such as low detection or detection sensitivity, interference with KRAS gene mutation detection, etc., to achieve the effect of improving detection sensitivity and reducing interference

Inactive Publication Date: 2018-07-06
WUHAN AIMISEN LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the presence of a large number of microbial genomes in stool samples, it interferes with the detection of human KRAS gene mutations, resulting in inability to detect or low detection sensitivity

Method used

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  • Kit and method for detecting human-derived KRAS gene mutation in excrement
  • Kit and method for detecting human-derived KRAS gene mutation in excrement
  • Kit and method for detecting human-derived KRAS gene mutation in excrement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 KRAS gene capture reagent preparation

[0048] For the KRAS gene sequence, specific oligonucleotide probes are designed and synthesized by professional companies. The specific steps of each magnetic bead coating are as follows:

[0049] 1. Invert the magnetic beads up and down or vortex to mix the magnetic beads thoroughly, pipette 20 μl of magnetic beads into a 1.5mL sterilized centrifuge tube;

[0050] 2. Add 1mL magnetic bead rinsing solution, mix by inversion, wash magnetic beads, magnetic field adsorption for 2min, discard supernatant; repeat washing three times;

[0051] 3. Add biotin-labeled specific nucleic acid probes and an equal volume of coupling solution, resuspend the magnetic beads, shake gently, and incubate at room temperature for 10-15 minutes;

[0052] 4. Magnetic field adsorption for 2-3 minutes, discard the supernatant;

[0053] 5. Add 500 μL of magnetic bead washing solution to wash the magnetic beads for 2-3 times, adsorb to the magn...

Embodiment 2

[0055] Example 2 Stool sample human KRAS gene capture

[0056] 1. Add 1-10ml of fecal cell lysate to 1-5g of fresh feces sample, mix well and lyse, transfer the supernatant to a new clean centrifuge tube by centrifugation at 12000rpm.

[0057] 2. Add a piece of PVPP adsorbent to the supernatant, and vortex to mix the sample thoroughly.

[0058] 3. Centrifuge at 12000rpm for 2-5min, and transfer the supernatant to a new centrifuge tube.

[0059] 4. Add proteinase K and RNase to the nucleic acid sample obtained after preliminary treatment to completely remove protein and RNA.

[0060] 5. Add the supernatant obtained in the previous step to the binding solution CB, and mix well by inverting up and down.

[0061] 6. Add the prepared gene capture reagent to the above solution, mix by inversion, and incubate at room temperature for 1-2 hours.

[0062] 7. After incubation, transfer the centrifuge tube to the magnetic stand to absorb the magnetic beads, and discard the supernatant....

Embodiment 3

[0067] Embodiment 3, the experiment that detects KRAS gene mutant type in wild-type plasmid

[0068] KRAS gene mutation solutions with different concentration gradients were prepared, and 10 5 Copies / μL of the template contained 100% mutant gene, 10% mutant gene, 1% mutant gene, 0.1% mutant gene and 0% mutant gene in KRAS solution. Taking 34G>A as an example, the preparation method of each concentration is listed as follows:

[0069] (1) KRAS 34G>A 100% mutation solution preparation: 10 5 Copies / μL of 34G>A mutant plasmid

[0070] (2) Preparation of KRAS 34G>A 10% mutation solution: equal volume 2×10 4 Copies / μL KRAS 34G>A gene mutant plasmid with 1.8×10 5 Copies / μL of the wild-type plasmid was mixed evenly, shaken to mix, and centrifuged briefly;

[0071] (3) Preparation of KRAS 34G>A 1% mutation solution: equal volume 2×10 3 Copies / μL KRAS 34G>A gene mutant plasmid with 1.98×10 5 Copies / μL of the wild-type plasmid was mixed evenly, shaken to mix, and centrifuged brief...

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Abstract

The invention relates to the field of biotechnology and medical science, in particular to a kit and a method for detecting human-derived KRAS gene mutation in excrement. The invention specifically relates to a detecting method for detecting mutation of 12nd and 13rd codons of the KRAS gene by taking an excrement sample as a specific detecting sample type. The kit relates to an excrement human-derived genome separating and purifying reagent and a KRAS gene mutation detecting reagent. The excrement human-derived genome separating and purifying reagent is used for obtaining human-derived genomesthrough separation and purification; the KRAS gene mutation detecting reagent is subsequently adopted for detecting the gene mutation. The excrement human-derived genome separating and purifying reagent is used for removing microbial genomes in the excrement and obtaining the human-derived genomes through purification and enrichment, and interference of non-human-derived genomes to detection is reduced; a primer and a probe are capable of inhibiting amplification of wild type KRAS and combination with the probe, increasing detection sensitivity of seven kinds of gene mutation types and detecting a sample containing 0.1 percent of KRAS gene mutation DNA (Deoxyribonucleic Acid).

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a kit and method for detecting human-derived KRAS gene mutation in feces. Background technique [0002] Colorectal cancer is the third most common malignancy in the world. In recent years, the incidence of colorectal cancer in China has been increasing year by year. It is estimated that there are about 400,000 new cases each year, ranking second among malignant tumors of the digestive system in my country. The 5-year survival rate of patients with early colorectal cancer can be as high as 90%, while the 5-year survival rate of patients with middle and advanced stages is only about 10%. However, about 80% of clinically diagnosed colorectal cancers are middle and late stages, which leads to the highest mortality rate. one of the key factors. The incidence rate of patients with early colorectal cancer after effective treatment is reduced by 60%, and the mortality rate is reduced ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 张良禄董兰兰姚希辉魏照云
Owner WUHAN AIMISEN LIFE TECH CO LTD
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