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Method for identifying biomarkers of pneumonia and detection kit thereof

A technology of biomarkers and kits, applied in the field of medicine, to achieve the effects of accurate detection, simplified operation, and simple pretreatment

Inactive Publication Date: 2019-01-15
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the PMP derivatization method of monosaccharides is widely used in various fields, there is no report on the application of this method to the detection of free mannose and glucose in the serum of patients with pneumonia.

Method used

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  • Method for identifying biomarkers of pneumonia and detection kit thereof
  • Method for identifying biomarkers of pneumonia and detection kit thereof
  • Method for identifying biomarkers of pneumonia and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Technical scheme of the present invention comprises the following steps:

[0054] (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;

[0055] (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;

[0056] (3) PMP derivatization: Add 60 μL of PMP (1-phenyl-5-methylpyrazolone) to each sample, vortex and mix well, and react in an oven at 70°C for 1 h after centrifugation;

[0057] (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;

[0058] (5) Extraction: add 500 μL ...

Embodiment 2

[0083] (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;

[0084] (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;

[0085] (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;

[0086] (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;

[0087] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0088] (6) Centrifuge the sample at 13000r / min for 10min, take 80μL su...

Embodiment 3

[0110] (1) Accurately weigh the appropriate amount of mannose and glucose, add deionized water to prepare the above monosaccharides containing 0.5mg / mL, 0.25mg / mL, 0.1mg / mL, 0.05mg / mL, 0.01mg / mL, 0.005mg / mL, 0.0025mg / mL, 0.001mg / mL, 0.0005 mg / mL mixed standard solution, ready-to-use;

[0111] (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;

[0112] (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;

[0113] (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;

[0114] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0115] (6) Centrifuge the sample at 13...

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Abstract

The invention provides a method for identifying biomarkers of pneumonia and applications thereof. The biomarker is a free glucose and a mannose obtained by high performance liquid chromatography (HPLC) derived from 1-phenyl-5-methylpyrazolone (PMP) from the serum of the patient. The method for identifying biomarkers of pneumonia has the advantages of being simple in pretreatment, short in analysistime, reasonable price of the instrument, in line with normal use, easy to learn, high in detection result accuracy, only blood sampling for HPLC analysis can distinguish between normal people and patients, and small in required serum amount, less than 1 mL in blood collection amount and the like. The results show that the analysis method can analyze the free mannose and the glucose in the serumquickly, and can be calculated to obtain the mannose, the glucose sugar concentrationm and the peak area, and has a very important significance in researching the relationship between the serum free mannose and the glucose and the diseases, and finding the biomarker for clinical detection of novel pneumonia diseases.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a biomarker for identifying pneumonia and its detection application. Background technique [0002] Pneumonia is a respiratory infection affecting the lungs, which refers to inflammation of the terminal airways, alveoli, and interstitium of the lungs, which can be caused by disease microorganisms, physical and chemical factors, immune damage, allergies, and drugs. Bacterial pneumonia is the most common pneumonia and one of the most common infectious diseases. The pathogens that cause pneumonia are complex, including bacteria, viruses, mycoplasma, etc., as well as physical and chemical factors such as radiation and inhaled foreign bodies. Clinical manifestations mainly include fever, cough, sputum production, dyspnea, lung X-ray shows inflammatory infiltrate shadow, may be accompanied by chest pain or dyspnea, etc. In childhood pneumonia, the symptoms are often not o...

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 张丽娟郝翠矫文捷
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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