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Quantitative detection kit and quantitative detection method for phenylalanine

A technology for quantitative detection of phenylalanine, applied in the field of disease diagnosis, can solve the problems of narrow detection linear range and low sensitivity, and achieve the effects of short detection time, high detection sensitivity and simple operation

Inactive Publication Date: 2013-07-10
BEIJING YUANDE BIO MEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Clinically, the diagnosis of phenylketonuria and the screening of neonatal phenylketonuria are mainly carried out by detecting the content of blood phenylalanine. Amino acid dehydrogenase quantitative method, chemical fluorescence method, these methods have the shortcomings of narrow detection linear range and low sensitivity

Method used

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  • Quantitative detection kit and quantitative detection method for phenylalanine
  • Quantitative detection kit and quantitative detection method for phenylalanine
  • Quantitative detection kit and quantitative detection method for phenylalanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The detection linear range determination of embodiment 1 kit of the present invention

[0035] Detection steps:

[0036] (1) Preparation of enzyme reaction solution:

[0037] Prepare phenylalanine dehydrogenase solution and coenzyme β-NAD solution with 0.05mol / L Gly-NaOH buffer solution at pH 10.0, so that the final concentrations are 0.1U / mL and 5mmol / L respectively;

[0038] With 0.1mol / L K of pH7.0 2 HPO 4 -KH 2 PO 4 Prepare coenzyme FMN solution and bacterial luciferase (product of Sigma company, its product number is L8507) solution containing NAD(P)H:FMN-oxidoreductase respectively in the buffer, so that the final concentrations are 0.2mmol / L and 5mg / L respectively. mL;

[0039] (2) Elution:

[0040] Use a special puncher to punch a series of calibrator filter paper dry blood slices with a diameter of 3 mm and a concentration of phenylalanine in the range of 0.16 to 50.55 mg / dL, put them into the microwells of the corresponding transparent microporous plate...

Embodiment 2

[0046] The detection sensitivity measurement of embodiment 2 kit of the present invention

[0047] Detection steps:

[0048] (1) Preparation of enzyme reaction solution: same as Example 1;

[0049] (2) Elution:

[0050] Use a special puncher to punch calibrators with a diameter of 3 mm of phenylalanine concentrations of 0.65, 1.98, 4.22, 5.50, 7.13, and 19.48 mg / dL filter paper dry blood slices, and 8 phenylalanine concentrations of 0 mg / dL. Put dL sample blood slices into the microwells of the corresponding transparent microwell plate, add 100 μL of 3% trichloroacetic acid to each well, shake and elute at room temperature at 300~500rpm for 45min;

[0051] (3) Dehydrogenation reaction of phenylalanine

[0052] After adding 0.5mol / L NaOH 25μL to each well of a new white microwell plate, add 75μL eluent, shake and mix well, then add 50μL of temporarily prepared 0.1U / mL phenylalanine dehydrogenase, 5mmol / L Coenzyme β-NAD50μL, shaking reaction at room temperature for 60min;

...

Embodiment 3

[0063] The intra-assay and inter-assay precision measurement of embodiment 3 kit of the present invention

[0064] (1) Preparation of enzyme reaction solution: same as Example 1;

[0065] (2) Elution:

[0066] Use a special puncher to punch 3 mm diameter quality control filter paper dry blood slices with phenylalanine concentrations of 2.3, 4.5, 11.5, and 16.7 mg / dL respectively, and put them into the corresponding microwells of the transparent microporous plate. Add 100 μL of 3% trichloroacetic acid to the solution, shake and elute at 300~500 rpm for 45 minutes at room temperature, repeat 8 times in the plate, and make 8 plates in total;

[0067] (3) Dehydrogenation reaction of phenylalanine

[0068] After adding 0.5mol / L NaOH 25μL to each well of a new white microwell plate, add 75μL eluent, shake and mix well, then add 50μL of temporarily prepared 0.1U / mL phenylalanine dehydrogenase, 5mmol / L Coenzyme β-NAD50μL, shaking reaction at room temperature for 60min;

[0069] (4...

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Abstract

The invention discloses a quantitative detection kit for of phenylalanine. The kit comprises phenylalanine dehydrogenase, coenzyme Beta-NAD, coenzyme FMN, long-chain aliphatic aldehyde, FMN-NADH oxidoreductase, bacteriofluorescein, a Gly-NaOH buffer with a concentration of 0 .05 mol / L and a pH value of 10.0 and a K2HPO4-KH2PO4 buffer with a concentration of 0.1 mol / L and a pH value of 7.0. The invention also discloses a quantitative detection method for phenylalanine. The method comprises the following steps: subjecting phenylalanine and Beta-NAD to a dehydrogenation reaction under the action of phenylalanine dehydrogenase so as to produce Beta-NADH; allowing Beta-NADH and FMN to undergo electron transfer under the action of bacteriofluorescein and FMN-NADH oxidoreductase so as to generate blue-green light; and calculating the content of phenylalanine in a sample according to luminous intensity. The detection method has the advantages of high sensitivity, a wide linear range, high precision and accuracy, short detection time, capacity of high-throughput detection, etc.

Description

technical field [0001] The invention relates to the technical field of disease diagnosis, in particular to a kit for quantitative detection of phenylalanine and a method for quantitative detection of phenylalanine using the kit. Background technique [0002] Phenylketonuria is mainly caused by the lack or decrease in activity of phenylalanine hydroxylase or its cofactor tetrahydrobiopterin in the liver, which causes phenylalanine to not be metabolized into tyrosine normally, resulting in a large amount of amphetamine and its bypass Metabolites accumulate in the body, causing irreversible damage to the patient's nervous system, causing mental retardation, abnormal skin color, microcephaly, language barriers, epilepsy and other symptoms of the most common congenital abnormal amino acid metabolism diseases. After a normal person takes in protein normally, the level of phenylalanine in the blood is generally 0.6~2mg / dL (36~120μmol / L). Phenylketonuria can be divided into classic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
Inventor 刘宾杨晓林孙旭东孙中锋李鹏聪张鑫李瑞娟
Owner BEIJING YUANDE BIO MEDICAL ENG
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