A tandem mass spectrometry kit for the detection of pku and cah
A technology of kits and quality control products, which is applied in the field of detection of phenylalanine and 17-hydroxyprogesterone, can solve the problems of low sensitivity, low work efficiency, and easy contamination, so as to reduce the demand for blood collection, reduce management costs, Remove the effect of low sensitivity
Active Publication Date: 2019-05-14
GUANGZHOU DARUI BIOTECH
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Problems solved by technology
[0004] Both phenylalanine and 17-hydroxyprogesterone belong to small molecular metabolites. At present, the commercially available kits for detecting phenylalanine on newborn blood collection filter paper mainly use fluorescence analysis method, which uses phenylalanine and indene The reaction of triketone and dipeptide to generate fluorescent substances for quantitative detection mainly has problems such as low sensitivity, poor specificity, short half-life of fluorescent substances, easy contamination, and high false positives; commercially available reagents for detecting 17-hydroxyprogesterone on neonatal blood collection filter paper The kit is mainly based on the time-resolved immunofluorescence method. This method uses the antigen-antibody "competition method" to detect the content of 17-hydroxyprogesterone. There are inevitable problems in the immune "competition method": low specificity, different antigens or antibodies. The test results have a great influence, the test values of reagents from different manufacturers vary greatly, and the accuracy is low.
Moreover, at present, all commercially available kits can only detect a single indicator in one experiment, and cannot simultaneously detect two markers, phenylalanine and 17-hydroxyprogesterone, in one experiment. Not only the work efficiency is low, but also the requirements for blood collection volume are increased.
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Embodiment 1
[0034] Embodiment 1: kit composition and its components
[0035]
[0036]
Embodiment 2
[0037] Embodiment 2: the operation method of kit (schematic diagram as figure 1 )
[0038] 1. Preparation of internal standard working solution: Dilute internal standard 100 times according to internal standard: extraction solution = 1:99, and prepare internal standard working solution (1.0mL internal standard + 99mL extract);
[0039]2. Incubation: Use a puncher to roll off small blood spots (about 3 mm in diameter each) on filter paper dry blood spots (calibrators, quality controls, and samples to be tested), and move the pierced blood spots into 96 wells in sequence Add 100 μL of internal standard working solution to each well of a microwell plate (U-shaped bottom, no coating), cover with a seal, and incubate at 37°C for 45 minutes with shaking.
[0040] 3. Pipetting: Transfer 75 μL of the extracted solution from each plate well to the wells of a 96-well microplate (V-bottom, no coating).
[0041] 4. On-machine detection: Select 20 μL of the extracted liquid to enter the ...
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The invention discloses a kit capable of detecting phenylalanine and 17alpha- hydroxyprogesterone in neonatal dried blood spots by using a high-throughput liquid chromatography-tandem mass spectrometry at the same time, and capable of detecting two neonatal hereditary metabolic diseases of phenylketonuria and congenital adrenal hyperplasia at the same time. The invention provides a relatively simple experimental operation method, comprising four steps: preparation of an internal standard working liquid, sample incubation, liquid relief and computer detection. The experiment operation process is simplified and the detection efficiency is improved. The kit is high in throughput, accuracy and repeatability and simple in operation; the work efficiency can be greatly improved; and the requirements on the blood sampling quantity is reduced.
Description
technical field [0001] The invention relates to the technical field of detection of phenylalanine and 17-hydroxyprogesterone, in particular to a high-throughput liquid chromatography tandem mass spectrometry method for simultaneous detection of phenylalanine and 17-hydroxyprogesterone in dried blood slices of newborn filter paper finished kits. Background technique [0002] Phenylketonuria (PKU) is a disorder of phenylalanine metabolism in newborns caused by normal chromosome defects in the human body. Its pathogenesis is the lack of phenylalanine hydroxylase (phenylalaninehydroxylase, PAH) in newborns. This enzyme can hydrolyze phenylalanine in protein into tyrosine, which is convenient for human body to absorb and utilize. Because the formation pathway of tyrosine is blocked, phenylalanine in blood cannot be hydrolyzed, so the concentration of phenylalanine in blood, cerebrospinal fluid, various tissues and urine is extremely increased, and a large amount of phenylalanine...
Claims
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IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 吴英松周健伟董志宁龙允麟
Owner GUANGZHOU DARUI BIOTECH