Tandem mass spectrometry kit and detection method for simultaneous detection of pku, cah and g-6pd deficiency

A technology of G-6PD and tandem mass spectrometry, which is applied in the field of tandem mass spectrometry kits for simultaneous detection of PKU, CAH and G-6PD deficiency, which can solve cross-reaction, low sensitivity and specificity, blood sample volume and high detection cost , Increased false positive rate of test results and other issues, achieving good stability at room temperature, improving screening efficiency and throughput, and reducing the risk of pathogen infection

Active Publication Date: 2021-08-17
易达精准(杭州)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the relatively low level of steroid hormones in the blood, the requirements for detection methods are high. Although the above methods can achieve a certain accuracy, there are still cross-reactions between different hormones, and the sensitivity and specificity are relatively low. The false positive rate increases, and different kits must be used for the determination of each hormone, and the required blood sample volume and detection cost are high
Moreover, the current commercially available kits can only detect a single indicator in sequential experiments, and cannot simultaneously detect three markers of phenylalanine, 17-α-hydroxyprogesterone, and glucose-6 phosphate dehydrogenase in one experiment

Method used

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  • Tandem mass spectrometry kit and detection method for simultaneous detection of pku, cah and g-6pd deficiency
  • Tandem mass spectrometry kit and detection method for simultaneous detection of pku, cah and g-6pd deficiency
  • Tandem mass spectrometry kit and detection method for simultaneous detection of pku, cah and g-6pd deficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. The kit includes:

[0059] Table 1 Kit composition and main components

[0060]

[0061]

[0062] 2. The method for simultaneously detecting PKU, CAH and G-6PD deficiency using a kit, comprising the following steps:

[0063] (1) Incubation: The puncher rolls off a blood piece with a diameter of about 6 mm on the dry blood piece of the filter paper of the sample to be tested, and puts every two blood pieces into the wells of the 96-well plate reaction plate together, and the calibrator wells, quality control wells and Add 160uL of enzyme diluent and 200uL of incubation solution to the sample wells to be tested, cover and seal the slide, shake for 30 seconds, place in a constant temperature incubation shaker, and incubate at 37°C for 30 minutes with vibration at 100rpm.

[0064] 2. Pipetting and nitrogen blowing: Take out the incubated solution and quickly add 400uL of enzyme quencher to quench the reaction. After sealing, centrifuge at 10000r / min for 10min in a...

Embodiment 2、3

[0099] Compared with Example 1, the enzyme quencher was replaced by methanol, acetonitrile:methanol=1:1 (V / V).

[0100] Take the same quality control sample, adopt different enzyme quenchers respectively, detect the content of glucose-6-phosphate dehydrogenase according to the method of Example 1, the enzyme quenchers of Examples 2 and 3 can obtain ideal detection results, and It does not affect the detection results of phenylalanine and 17-α-hydroxyprogesterone.

Embodiment 4～10

[0102] Compared with Example 1, the reconstitution agent in the kit was replaced with the formula shown in Table 6.

[0103] Take the same quality control sample, use the reconstitution solvent of the formula shown in Table 6 respectively, and detect the content of phenylalanine, 17-α-hydroxyprogesterone and 6-phosphate glucose dehydrogenase according to the method of Example 1, and all can obtain ideal The detection effect is good, and the repeatability of the experiment is good.

[0104] The composition formula of table 6 different reconstitution solvents

[0105] Example Reconstitution agent components and their proportions Example 1 Acetonitrile: water = 1:1 Example 4 Acetonitrile Example 5 Methanol Example 6 Methanol: water = 1:1 Example 7 Ethanol: water = 1:1 Example 8 Acetonitrile: water = 4:1 Example 9 Methanol: water = 4:1 Example 10 Ethanol: water = 4: 1

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Abstract

The invention discloses a tandem mass spectrometry kit for simultaneously detecting PKU, CAH and G-6PD deficiency, comprising: a calibrator, an isotope internal standard, an incubation solution, a diluent, an enzyme quencher, a reconstitution solvent and a mobile phase; Calibrator contains phenylalanine, 17‑α‑hydroxyprogesterone, and glucose‑6‑phosphate dehydrogenase; isotopic internal standard contains phenylalanine‑d8 and 17‑α‑hydroxyprogesterone‑d8; Consists of water, buffer salts, glucose‑6‑phosphate, nicotinamide adenine dinucleotide phosphate, and surfactant; diluent consists of water, buffer salts, and surfactant; mobile phase consists of mobile phase A and mobile phase B ; Mobile phase A is an aqueous solution of ammonium formate and / or ammonium acetate; Mobile phase B is methanol and / or acetonitrile. The invention also discloses a method for simultaneously detecting PKU, CAH and G-6PD deficiency using the tandem mass spectrometry kit. The kit of the invention can detect multiple substances at the same time, and has the characteristics of high sensitivity and strong specificity.

Description

technical field [0001] The invention relates to the field of detection of substances related to genetic metabolic diseases, in particular to a method for simultaneously detecting PKU (phenylketonuria), CAH (congenital adrenal hyperplasia) and G-6PD deficiency (glucose-6-phosphate dehydrogenase deficiency) Syndrome) tandem mass spectrometry kit and detection method. Background technique [0002] Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the deficiency or insufficient activity of phenylalanine hydroxylase (PAH) in the liver or its coenzyme tetrahydrobiopterin (tetrahydrobiopterin, BH4) Lack of phenylalanine (phenylalanine, Phe) can not be converted into tyrosine according to the normal metabolic pathway. Due to the blocked formation of tyrosine, phenylalanine in blood cannot be hydrolyzed, so the concentration of phenylalanine in blood, cerebrospinal fluid, various tissues and urine is extremely high, and a large amount of phenylpyruvate and ph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/88
CPCG01N30/06G01N30/88
Inventor 李士敏袁京群吴筱丹葛志伟朱亚尔李家睿
Owner 易达精准(杭州)科技有限公司
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