Pregnancy vitamin A deficiency risk assessment detection kit and application method

A technology for detecting kits and vitamins, which is applied in the field of molecular biology, can solve the problem of expensive vitamin A gene polymorphism sequencing during pregnancy, and achieve the effect of low cost of consumables and low sample consumption

Active Publication Date: 2021-09-03
厦门市妇幼保健院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The third purpose of the present invention is to solve the problem of high price of gene polymorphism sequencing in the existing vitamin A deficiency risk prediction products during pregnancy,

Method used

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  • Pregnancy vitamin A deficiency risk assessment detection kit and application method
  • Pregnancy vitamin A deficiency risk assessment detection kit and application method
  • Pregnancy vitamin A deficiency risk assessment detection kit and application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Primer Composition Design

[0053]The present invention is based on the optimization of 17 SNP sites screened out from ALDH1A2 gene, CRABP2 gene, CYP26B1 gene, BCO1 gene, RBP4 gene, ABCA1 gene, ELOVL2 gene, CXCL8 gene, ISX gene, RPE65 gene, PKD1L2 gene and SOD2 gene Selection, combined with the characteristics of its SNP site system, design PCR primer composition to amplify DNA fragments containing 17 SNP sites on different genes, the length of primers is mostly 20-35bp, and the length of PCR amplification products is 100~ Between 200 bp, the GC content should be 40% to 60%, and try to avoid the hairpin structure of the primer itself and the continuous arrangement of more than 5 purine or pyrimidine nucleotides. In addition, since mass spectrometry detection uses multiple PCR methods, there are many primers in the same reaction system, so special attention should be paid to the complementarity between primers, and it is necessary to absolutely avoid complementarity betw...

Embodiment 2

[0057] The usage of the kit of the present invention will be described in detail below in conjunction with Example 1, and Example 2 is implemented under the technical premise of the present invention, providing detailed implementation methods and specific operating procedures.

[0058] S1: DNA extraction:

[0059] 1. Sample processing: Add 2 times the volume of Buffer TBP to 200ul pregnant women's whole blood, mix well, and place at room temperature for 1min until the red blood cells are completely lysed. Centrifuge at 8,000rpm for 1min and discard the supernatant. Resuspend the pellet with 500 μl TE Buffer, centrifuge at 8,000 rpm for 1 min, discard the supernatant, wash once with TE Buffer until the precipitate is white, and then add 200 μl PBSsolution.

[0060] 2. Add 20 μl Proteinase K and mix well. Then add 200 μl Buffer DL, vortex and mix well, and bathe in water at 56°C for 10 minutes. The mixed solution becomes clear and transparent, indicating complete lysis.

[0...

Embodiment 3

[0114] In order to verify that the kit can accurately detect polymorphisms of 17 SNP sites in 12 genes, three samples to be tested that have been tested using the kit involved in the patent of the present invention were simultaneously verified by first-generation sequencing.

[0115] The first-generation sequencing results are consistent with the detection results using the kit involved in the patent of the present invention (see Table 3), indicating that the patent of the present invention can accurately detect the polymorphism of the corresponding SNP site.

[0116] Table 4 Comparison of the results of two detection methods

[0117]

[0118]

[0119] * Patent: use the kit test results involved in the present invention;

[0120] # Generation: Generation sequencing results.

[0121] The present invention MALDI-TOF System (Time-of-Flight Mass Spectrometry Biochip System) is a genotyping detection system exclusively developed and produced by Sequenom, Inc. of the Unit...

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Abstract

The invention discloses a pregnancy vitamin A deficiency risk assessment detection kit and an application method, and relates to molecular biology. A primer group for screening and predicting the risk of vitamin A deficiency during pregnancy is used for amplifying single nucleotide polymorphic sites of vitamin A metabolism genes; and the length of the primers is 18-30 bp. The application method comprises the following steps of adding an SNP sequence specific extension primer into a product subjected to multiple PCR amplification, extending one basic group on an SNP site, extending different basic groups on different genotypes, exciting by using instantaneous nanosecond (10-9s) intense laser, separating in a non-electric field drift region according to the mass-to-charge ratio, wherein different genotypes are distinguished according to different time of flight to a detector in a vacuum tubule due to different masses of extension bases.

Description

technical field [0001] The present invention relates to molecular biology, in particular to MALDI-TOF System (time-of-flight mass spectrometry biochip system) detection kit and application method for vitamin A deficiency risk assessment during pregnancy. Background technique [0002] Vitamin A (vitamin A, VA) is a group of fat-soluble vitamins with all-trans retinol biological activity, mainly including retinol, retinoic acid, retinal and esters. The main form of transport; retinoic acid and retinal are the active forms of vitamin A to exert biological effects in the body; retinyl esters are the main storage form of vitamin A in the body. The physiological functions of vitamin A mainly include maintaining the function of skin epithelial cells, maintaining vision and reproductive function, maintaining lung function, and it is also closely related to the development and function of the central nervous system. [0003] According to statistics in 2011, nearly 9.8 million preg...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2600/16C12Q2600/172C12Q2531/113C12Q2537/143C12Q2521/525C12Q2533/101C12Q2565/627
Inventor 叶辉铭李娟苏志英吴琦嫦曹佳莉邹悦
Owner 厦门市妇幼保健院
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