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
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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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