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Kit for detecting colorectal cancer and precancerous lesions and use method thereof

A technology for precancerous lesions and colorectal cancer, applied in the field of biotechnology and diagnostic research, can solve the problem of low screening specificity, low screening sensitivity and specificity of guaiac fecal occult blood test, and inability to detect intestinal distant End lesions and other problems, to achieve the effect of reducing the rate of missed diagnosis, high accuracy, and convenient sampling

Pending Publication Date: 2018-12-28
HANGZHOU HEYI GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although digital rectal examination is easy to operate, it can only detect lesions within 8 cm from the anus and cannot detect distal intestinal lesions; the screening specificity of guaiac fecal occult blood test is low, there are a large number of false positives, and it is susceptible to Food and drug interference of ferrous ions; CEA and CA19-9 are broad-spectrum tumor markers, and the screening sensitivity and specificity are not high

Method used

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  • Kit for detecting colorectal cancer and precancerous lesions and use method thereof
  • Kit for detecting colorectal cancer and precancerous lesions and use method thereof
  • Kit for detecting colorectal cancer and precancerous lesions and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Stool sample collection and DNA extraction

[0043] 1. Stool sample collection and processing:

[0044] Immediately after the person excretes feces, collect 5g of feces samples and put them into the collection tube, which contains 15ml of feces preservation solution. Shake the collection tube fully to fully mix the feces samples and the feces preservation solution;

[0045] Reagent name

15ml system / part

0.5M EDTA, pH8.0, sterile

3ml

5M NaCl solution, sterile

30ul

1M Tris buffer, pH7.6

7.5ml

purified water

4.47ml

[0046] 2. Extraction of stool sample DNA:

[0047] (1) cell pellet

[0048] Draw 1ml of stool sample liquid into a centrifuge tube, pipette and mix well, and then centrifuge at 10,000rpm for 3 minutes to precipitate intestinal exfoliated cells to the bottom of the tube, retain the precipitate, and remove the supernatant;

[0049] (2) Cell cleavage releases DNA

[0050] Add 800ul of feces ...

Embodiment 2

[0064] Example 2: KRAS mutation detection in stool sample DNA

[0065] 1. Reagent preparation

[0066] (1) Take out the internal reference gene (ACTB) reaction solution and KRAS reaction solution from the refrigerator, equilibrate to room temperature, fully dissolve, and centrifuge quickly for 10 seconds; take out the PCR reaction premix reagent (master mix) from the refrigerator at 4°C, and spin for 10 seconds . The PCR premix reagent is a commercial quantitative PCR reaction mixed reagent; the reaction solution consists of probes (synthesized by a professional probe synthesis company), primers (synthesized by a professional primer synthesis company), water, KRAS and internal reference gene (ACTB) each There is a reaction solution. The probe and primer sequences are from the literature "Optimized Multiplex Detection of 7KRAS Mutations by Taqman Allele-SpecificqPCR". The specific probe and primer sequences are as follows:

[0067] KRAS:

[0068]

[0069] Remarks: Primer...

Embodiment 3

[0083] Example 3: Methylation detection of human DNA in stool samples

[0084] 1. Sulphite conversion

[0085] (1) Add 1 ug of fecal sample DNA to be treated in a 2.0 ml centrifuge tube, that is, the DNA-containing solution obtained in Example 1, the recommended dosage of 10 ng / ul concentration is 100 ul;

[0086] (2) Add 300ul of sulfite conversion solution and 50ul of protection solution to the centrifuge tube in step (1), cover the centrifuge tube tightly, vortex to mix, and centrifuge briefly;

[0087] The sulfite conversion solution is an aqueous solution containing 50% (m / v) sodium bisulfite and 15% (m / v) sodium sulfite, and the pH value is 5.5;

[0088] The protective solution is a mixture of quinol dimethacrylate and diethylene glycol dimethyl ether prepared in a ratio of 200mg: 1.5ml;

[0089] (3) Seal the centrifuge tube in step (2) and place it in a constant temperature shaking incubator, and incubate at 95°C for 20 minutes;

[0090] (4) Immediately take out the ...

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Abstract

The invention relates to a kit for detecting colorectal cancer and precancerous lesions and a use method thereof. The invention provides the kit for detecting colorectal cancer and precancerous lesions, and the kit comprises a nucleic acid separation and purification reagent, a DNA sulfite conversion reagent, a KRAS gene mutation detection reagent, an SDC2 and SFRP2 gene methylation detection reagent and a fecal occult blood detection reagent, wherein the nucleic acid separation and purification reagent is used for separating and purifying human DNA in a fecal sample; and the DNA sulfite conversion reagent is used for converting the partial purified human DNA into sulfite for subsequent detection of the methylation level of SDC2 and SFRP2 genes. The sensitivity of the kit for detecting thecolorectal cancer can reach up to 90% or more and the specificity is about 85%, thereby realizing high-sensitivity and high-specificity detection of the colorectal cancer; and the kit provided by theinvention can detect most precancerous lesions of the colorectal cancer to realize ultra-early detection of the colorectal cancer, and can greatly reduce missed diagnosis and missed detection rates of clinical existing methods when being used for assisting clinical diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology and diagnosis research, more specifically, it relates to a kit for detecting colorectal cancer and precancerous lesions and its application method. Background technique [0002] The morbidity and mortality of colorectal cancer rank third and fourth among all cancers in the world, with approximately 1.36 million new cases of colorectal cancer and 694,000 deaths from colorectal cancer each year. In China, the morbidity and mortality of cancer are increasing, and since 2010, cancer has become the leading cause of death. It is estimated that in 2015, there were 376,300 new cases of colorectal cancer and 191,000 deaths in China, ranking fifth among all cancers. Colorectal cancer mainly occurs in people over the age of 50, and the older you are, the higher the risk of cancer. The development of most colorectal tumors goes through multiple stages, including the gradual accumulation of a series of histologi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886G01N33/72
CPCC12Q1/6886C12Q2600/154C12Q2600/156C12Q2600/166G01N33/721
Inventor 王军一肖雯刘杰王巍舒平尚慧捷
Owner HANGZHOU HEYI GENE TECH
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