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Reagent system and kit for detection of colorectal cancer spindle assembly checkpoint and application

A kit and spindle technology, applied in the field of colorectal cancer spindle assembly checkpoint target gene detection, can solve the problems of low accuracy, limited application, time-consuming and laborious, and achieve low misdiagnosis rate, high sensitivity and specificity. strong effect

Active Publication Date: 2020-01-24
刘鹏飞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The technical problem solved by the present invention is: the existing colorectal cancer gene detection is only limited to DNA detection of mutated susceptibility genes and intestinal flora. application is limited

Method used

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  • Reagent system and kit for detection of colorectal cancer spindle assembly checkpoint and application
  • Reagent system and kit for detection of colorectal cancer spindle assembly checkpoint and application
  • Reagent system and kit for detection of colorectal cancer spindle assembly checkpoint and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The steps to obtain a DNA preservation solution for a stool sample include:

[0061] 1. Collect fecal samples from subjects exposed for less than 24 hours, and pick 170-180 mg of samples on ice with a sterile razor blade.

[0062] 2. Add 3.0ml of feces lysate to the sample, centrifuge after fully lysing, take the supernatant and add 250mg of bovine serum albumin, centrifuge to obtain the supernatant, add 30μl of proteinase K to the supernatant and place it at 60°C The digestion reaction was carried out for 2 hours to obtain the digestive juice.

[0063] The composition of the stool lysate is: 30mmol / L Tris-Cl, 3mmol / L EDTA, 0.6mmol / L NaCl and 0.1% (W / V) sodium lauryl sulfate.

[0064] 3. Cool the digested solution obtained above to room temperature, add 300 μl protein precipitation solution to precipitate protein, mix well, remove protein impurities by centrifugation, and obtain supernatant, which is DNA extract.

[0065] The protein precipitation solution is prepared...

Embodiment 2

[0069] The steps to obtain a DNA preservation solution for a stool sample include:

[0070] 1. Collect fecal samples exposed for less than 24 hours, and pick 200-220 mg of samples on ice with a sterile razor blade.

[0071] 2. Add 0.5ml of feces lysate to the sample, centrifuge after fully lysing, take the supernatant and add 250mg of bovine serum albumin, centrifuge to obtain the supernatant, add 30μl of proteinase K to the supernatant and place it at 60°C The digestion reaction was carried out for 2 hours to obtain the digestive juice.

[0072] The composition of the stool lysate is: 100mmol / L Tris-Cl, 10mmol / L EDTA, 5mmol / L NaCl and 1.0% (W / V) sodium lauryl sulfate.

[0073] 3. Cool the digested solution obtained above to room temperature, add 300 μl protein precipitation solution to precipitate protein, mix well, remove protein impurities by centrifugation, and obtain supernatant, which is DNA extract.

[0074] The protein precipitation solution is prepared from 2mol / L p...

Embodiment 3

[0078] Prepared kit as attached Figure 1-3 As shown, except for the packing box, it mainly includes two parts, the upper layer and the lower layer. The upper part is used to obtain the DNA preservation solution of the stool sample, wherein, 1 is equipped with a stool lysate, 2 is equipped with a silica gel membrane binding solution, 3 is equipped with a silica gel membrane washing solution, and 4 is equipped with a protein precipitation solution.

[0079] The lower part contains reagents for each gene detection, among which, 5 is a reagent for BubR1 detection, 6 is a reagent for Mad1 detection, 7 is a reagent for Mad2 detection, 8 is a reagent for PlK1 detection, 9 is a reagent for AURKB detection, 10 is a reagent for Cdc20 detection Use reagents. Each group of 5 PE tubes contains DNA amplification primers corresponding to six genes, PCR reaction components, TaqDNA polymerase, PCR product purification components and DNA probe sequencing reaction components.

[0080] After u...

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Abstract

The invention relates to a reagent system and a reagent kit for detecting target genes of colorectal cancer spindle apparatus assembly checking point and application thereof. The reagent system comprises a reagent used for detecting BubR1 gene, a reagent used for detecting Mad 1 gene, a reagent for detecting Mad 2 gene, a reagent for detecting AURKB gene, a reagent for detecting PIK 1 gene and a reagent for detecting Cdc20 gene. According to the reagent system and the reagent kit for detecting target genes of colorectal cancer spindle apparatus assembly checking point and the application thereof, specially designed probe and PCR amplification technology are adopted, and through DNA specific binding reaction, the probe and the PCR amplification technology conduct one-time detection on expression state of six genes related to spindle apparatus assembly checking point during a last phase of a mitosis process, thus filling the blank of colorectal cancer detection in genes related to the spindle apparatus assembly checking point. The reagent system and the reagent kit are comprehensive and reliable in detection result, quick and efficient in effect, simple to operate, high in sensitivity, strong in specificity, low in misdiagnosis rate, and is capable of conducting timely precise diagnosis on generation and development of the colorectal cancer in earlier days.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a reagent system and a kit for detection of a target gene of a spindle assembly checkpoint of colorectal cancer and an application thereof. Background technique [0002] Colorectal cancer is a malignant tumor of the digestive tract that occurs at the junction of the rectum and the sigmoid colon. It is one of the most common malignant tumors in the world, and most of them can only be diagnosed in the late stage. Colorectal cancer has always been a huge public health problem, with more than 1.3 million newly diagnosed cases worldwide every year, with the highest incidence in the 40-50 age group. At the same time, colorectal cancer is the second most common cancer in women and the third high-risk cancer in men after breast cancer. About 700,000 people die of colorectal cancer every year. The main early manifestations of colorectal cancer are indigestion, abdominal distension, abd...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/158
Inventor 刘鹏飞王菁蕊韩雪
Owner 刘鹏飞
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