Enzymological detection method

A detection method and enzymological technology, applied in the field of medical testing, can solve the problems of inability to apply clinical large-flow automatic analysis, high price, complicated operation, etc., and achieve high scientific value and economic value, simple operation, and sensitive detection effect

Inactive Publication Date: 2015-06-17
SHAOXING INST OF TECH COLLEGE OF ENG PEKING UNIV BIOENG CENT
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  • Claims
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Problems solved by technology

[0007] The purpose of the present invention is to overcome the defects of the existing detection method for asymmetric dimethylarginine, which is time-consuming, complicated to operate, requires special instruments, cannot be applied to the automatic analysis of clinical large flow rate, and is expensive, and provides a Enzymatic detection method, this method has the characteristics of sensitive, rapid, high accuracy, and low price, and can realize the detection of low-concentration asymmetric dimethylarginine and citrulline, etc. Symmetrical dimethylarginine makes clinical application possible and has good scientific and economic value

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Embodiment 1

[0056] 1. Extraction of Pseudomonas aeruginosa genome

[0057] Pseudomonas aeruginosa Pseudomonas aeruginosa (Schroeter) Purchased from the Microbial Strain Collection Center of Guangdong Institute of Microbiology (strain preservation number: 132405), Pseudomonas aeruginosa was inoculated and shaken overnight, and the genome was extracted with a bacterial genome extraction kit (commercially available, Tiangen Biochemical Technology Co., Ltd.). The integrity was detected by agarose gel electrophoresis, and the concentration and purity were detected by a multi-functional microplate reader (TECAN infinite M200). The result was suitable as a PCR template.

[0058] 2. PCR amplification of ornithine carbamoyltransferase (ArcB) and carbamoyl phosphate kinase (ArcC)

[0059] According to the sequences of ArcB (gene accession number NP_253859.1) and ArcC (gene accession number NP_253860.1) in GenBank, primers were designed, and the upstream primer of ArcB was 5'-CGC CATATG GCTTTCAA...

Embodiment 2

[0117] The operating steps of this embodiment refer to Example 1, which is different from Example 1 in that different detection systems are adopted for different target detection substances:

[0118] The concentration curve of arginine detected by chemiluminescence

[0119] 100 μL system contains (100mmol / L PBS pH 7.0, 1 μmol / L ADP, 40 μmol / L MgCl 2 , 100 μg / mL ArcB, 100 μg / mL ArcC, 100 μg / mL ADI (arginine deiminase), serially diluted arginine solution), after reacting at 37 ℃ for 20 min, add to the ATP detection kit 10 μL of the detection reagent was used to detect the relative luminescence intensity with a multifunctional microplate reader.

[0120] Chemiluminescent detection of the concentration of arginine in the analyte

[0121] 100 μL system contains (100mmol / L PBS pH 7.0, 1 μmol / L ADP, 40 μmol / L MgCl 2 , 100 μg / mL ArcB, 100 μg / mL ArcC, 100 μg / mL ADI, to be detected), after reacting at 37 ℃ for 20 min, add 10 μL of the detection reagent in the ATP detection kit, and d...

Embodiment 3

[0123] The operating steps of this embodiment refer to Example 1, which is different from Example 1 in that different detection systems are adopted for different target detection substances:

[0124] Concentration Curve of Chemiluminescent Detection of Ornithine Carbamoyltransferase

[0125] 100 μL system contains (100mmol / L PBS pH 7.0, 1 μmol / L ADP, 40 μmol / L MgCl 2 , serially diluted ornithine carbamoyltransferase, 100 μg / mL ArcC, 100 μmol / L L-citrulline), after reacting at 37 ℃ for 20 min, add 10 μL of the detection reagent in the ATP detection kit, and use the multifunctional enzyme The standard instrument detects the relative luminous intensity.

[0126] Chemiluminescent detection of the concentration of ornithine carbamoyltransferase in the test substance

[0127] 100 μL system contains (100mmol / L PBS pH 7.0, 1 μmol / L ADP, 40 μmol / L MgCl 2 , the substance to be detected, 100 μg / mL ArcC, 100 μmol / L L-citrulline), after reacting at 37 ℃ for 20 min, add 10 μL of the dete...

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Abstract

The present invention relates to the technical field of inspection and measurement. Disclosed is an enzymology detection method for detecting concentration of citrulline and asymmetric dimethylarginine (ADMA) in a reaction system by using an ATP detection method. The basic principle of the reaction is mainly as follows: ADMA is hydrolyzed by using dimethylarginine dimethylaminohydrolase to obtain citrulline; when ADP and Mg2+ exist, the citrulline is coupling-catalyzed by using ornithine carbamoyltransferase and carbamoyl phosphate kinase to finally obtain ornithine, carbon dioxide, ammonia gas, and ATP, wherein the released ATP can be detected by using a reagent. The present invention has the advantages of flexible and quick reaction, high accuracy, and low cost; ADMA or citrulline with the concentration of 0.1μmol / L at lowest can be detected, and the detection result approaches the concentration of ADMA in the normal serum and is far less than the concentration of citrulline in the normal serum, thereby providing a new applicable method for clinical ADMA detection.

Description

technical field [0001] The invention relates to the technical field of medical testing, in particular to an enzymatic detection method using ATP chemiluminescence method for detection. Background technique [0002] Asymmetric dimethylarginine (ADMA) is a methylated protein degradation product, it is a nitric oxide synthase inhibitor, it can competitively inhibit the activity of nitric oxide synthase, and reduce the production of nitric oxide , and can directly induce oxidative stress, as an endogenous relaxation factor, nitric oxide has the function of relaxing blood vessels, and plays an important role in diseases of the cardiovascular system. ADMA widely exists in human tissues, cells, plasma, and urine. It is mainly produced by the methylation of intracellular proteins under the action of arginine methyltransferase, and then hydrolyzed. 80% of ADMA in the human body ADMA is metabolized and inactivated by dimethylarginine dimethylamine hydrolase (DDAH), and only a small p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 胡广张明屠慧惠
Owner SHAOXING INST OF TECH COLLEGE OF ENG PEKING UNIV BIOENG CENT
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