Method for identifying colorectal cancer biomarker and detection kit thereof

A technology for biomarkers and colorectal cancer, applied in the field of medicine, can solve the problems of unsuitability for routine purposes, expensive instruments, large volume of serum samples, etc., and achieve the effects of simplified operation, reasonable price of instruments, and short analysis time.

Inactive Publication Date: 2019-09-20
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, when using enzymatic method, gas-liquid chromatography and high-resolution liquid chromatography, in order to avoid the influence of high-concentration glucose, it needs to be removed, resulting in cumbersome pretreatment process; gas-liquid chromatography-mass spectrometry instruments are expensive and not Suitable for routine purposes; large serum sample volume required

Method used

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  • Method for identifying colorectal cancer biomarker and detection kit thereof
  • Method for identifying colorectal cancer biomarker and detection kit thereof
  • Method for identifying colorectal cancer biomarker and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) Precisely weigh mannose (Man), glucosamine (GlcN), galactosamine (GalN), glucuronic acid (GlcUA), glucose (Glc), galactose (Gal), xylose (Xyl), rock Add appropriate amount of algalose (Fuc), add deionized water to prepare two mixed standard solutions containing the above monosaccharide 0.1mg / mL, and prepare immediately for use;

[0053] (2) Create an alkaline environment: Take 40 μL of the mixed monosaccharide standard in a 1.5mL EP tube, add 40 μL of 0.3mol / L sodium hydroxide, and vortex to mix;

[0054](3) PMP derivatization: Add 60 μL 0.5mol / L 1-phenyl-5-methylpyrazolone (PMP) to each sample, vortex and mix well, and react in an oven at 70°C for 1 h after centrifugation;

[0055] (4) Neutralization reaction by adding acid: Take out the samples in the oven, let them cool down, add 40 μL of 0.3mol / L hydrochloric acid to each sample, and vortex to mix;

[0056] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chlorof...

Embodiment 2

[0080] (1) Accurately weigh the appropriate amount of mannose (Man), rhamnose (Rha) and glucose (Glc), add deionized water to prepare 5 parts of the same mixed standard solution containing the above monosaccharide 0.1mg / mL, and use it immediately match;

[0081] (2) Create an alkaline environment: Take 40 μL of the mixed monosaccharide standard in a 1.5 mL EP tube, add 40 μL of 0.3 mol / L sodium hydroxide, and vortex to mix;

[0082] (3) PMP derivatization: Add 60 μL 0.5mol / LPMP to each sample, vortex and mix well, and react in an oven at 70°C for 1 hour after centrifugation;

[0083] (4) Neutralization reaction by adding acid: Take out the samples in the oven, let them cool down, add 40 μL of 0.3mol / L hydrochloric acid to each sample, and vortex to mix;

[0084] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0085] (6) Centrifuge the sample at 13000r / min for 10min, take 80μ...

Embodiment 3

[0106] (1) Accurately weigh the appropriate amount of mannose and glucose, add deionized water to prepare the above monosaccharides containing 0.5mg / mL, 0.25 mg / mL, 0.1mg / mL, 0.05mg / mL, 0.01mg / mL, 0.005mg / mL, 0.0025mg / mL, 0.001 mg / mL, 0.0005mg / mL mixed standard solution, ready-to-use;

[0107] (2) Create an alkaline environment: Take 40 μL of the mixed monosaccharide standard in a 1.5mL EP tube, add 40 μL of 0.3mol / L sodium hydroxide, and vortex to mix;

[0108] (3) PMP derivatization: Add 60 μL 0.5mol / L PMP to each sample, vortex and mix well, and react in an oven at 70°C for 1 h after centrifugation;

[0109] (4) Neutralization reaction by adding acid: Take out the samples in the oven, let them cool down, add 40 μL of 0.3mol / L hydrochloric acid to each sample, and vortex to mix;

[0110] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0111] (6) Centrifuge the sample at 1...

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Abstract

The invention provides a method for identifying a colorectal cancer biomarker and a detection kit thereof. The biomarker is free mannose and glucose obtained by high performance liquid chromatography of pre-column 1-phenyl-5-methylpyrazolone (PMP) derivation in a serum. A detection method of the invention is high performance liquid chromatography of pre-column PMP derivation. The technical scheme of the invention has advantages that pre-treatment is simple; analysis time is short; an apparatus price is reasonable and an apparatus accords with routine use; operation steps are simple and are easy to learn; accuracy of a detection result is high; normal people and a patient with a colorectal cancer can be distinguished only through blood sampling; and very little serum is required, a blood collection amount is less than 1mL and so on. An obtained result shows that the analysis method can rapidly quantify the free mannose and the glucose in the serum of the patient with the colorectal cancer. The method is very important to study a relationship between the free mannose and the glucose of the serum and the colorectal cancer, and search a novel colorectal cancer clinical detection marker.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for identifying a biomarker of colorectal cancer and a detection kit thereof. Background technique [0002] Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer death worldwide. It is estimated that there were approximately 1.4 million new cases and 700,000 deaths worldwide in 2012. The 5-year survival rate of patients in the early stage after surgery is more than 90%, but at present, most patients lack obvious clinical symptoms when they are found in the advanced stage, resulting in increased mortality. Currently, diagnostic methods widely used clinically include: colonoscopy, ultrasonography, computed tomography (CT), MRI, fecal occult blood test (FOBT), CEA, CA19-9 and other traditional serological biomarkers. Colonoscopy, the standard test for the diagnosis of colorectal cancer, is highly sensitive and spec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 张丽娟黄晓丹田字彬邢晓明王雪玲
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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