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Pregnancy vitamin B12 deficiency risk assessment detection kit and an application method

A technology for detecting kits and vitamins, applied in the field of molecular biology, can solve the problems of vitamin B12 deficiency risk during pregnancy, the high price of gene polymorphism sequencing, etc., and achieve the effects 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 B12 deficiency risk prediction products during pregnancy, and provide a method based on MALDI-TOF System (Time-of-Flight Mass Spectrometry Biochip System) Detecting Vitamin B12 During Pregnancy Using the Detection Kit to Screen the Risk of Vitamin B12 Deficiency During Pregnancy

Method used

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  • Pregnancy vitamin B12 deficiency risk assessment detection kit and an application method
  • Pregnancy vitamin B12 deficiency risk assessment detection kit and an application method
  • Pregnancy vitamin B12 deficiency risk assessment detection kit and an application method

Examples

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Comparison scheme
Effect test

Embodiment 1

[0044] Primer composition design

[0045] The present invention is based on the FUT2 gene, the ACTL9 gene, the MUT gene, the MS4A3 gene, the FUT6 gene, the Clybl gene and the TCN1 gene are selected, and the characterization of the SNP site system is selected to design the PCR primer combination. For amplification of DNA fragments containing 9 SNP sites on different genes, the primer length range is 20 ~ 35 bp, and the PCR amplification product is 100 to 200 bp, and the GC content is preferably 40-60% as much as possible. Avoiding the primer itself produces a continuous arrangement of a hairpin structure and more than 5 or more purine or pyrimidine nucleotides. In addition, since mass spectrometry detection is a multi-PCR method, the number of primers in the same reaction system is much more, and therefore, it is necessary to pay more particularly, there is a complement of the primer between the primers, and it is necessary to absolutely avoid the complement of the SNP extension pr...

Embodiment 2

[0049] Next, the kit is taken from the kit of the present invention will be described in connection with Example 1, and Embodiment 2 is carried out in terms of the present invention, and the detailed embodiment and the specific operation process are given.

[0050] S1: DNA extraction:

[0051] 1. Sample treatment: 2x volumes of Buffer TBP is added to 200 ul pregnant women, mix well, 1 min at room temperature until the red blood cells are completely cleaved. 8,000 rpm, centrifugation 1min, discard it. The precipitate was resuspended with 500 μl of TE buffer, centrifuged for 1min, discarded, washed once to the precipitate with TE buffer, and then added 200 μL PBSSolution.

[0052] 2, add 20 μL Proteinase K, mix. The addition of 200 μl of Buffer DL, mixed with oscillating, 56 ° C water bath for 10 min. The mixed liquid is clear and transparent is complete.

[0053] 3. Add 200 μL of anhydrous ethanol to the above centrifugal tube, fully reversed.

[0054] 4. Put the suction column int...

Embodiment 3

[0106] In order to verify that this kit can accurately detect the 9 SNP bit polymorphisms of 7 genes, three samples to be tested simultaneously to be tested simultaneously by means of the sample of the kit according to the present invention.

[0107] A generation sequencing result consistent with the detection result of the kit according to the present invention (see Table 4), indicating that the patent of the present invention can accurately detect the polymorphism of the corresponding SNP site.

[0108] Table 4: Comparison of two detection methods:

[0109]

[0110]

[0111] * Patent: Use the kit detection result involved in the present invention;

[0112] # One generation: generation sequencing results.

[0113] Of the present invention Maldi-Tof System (Time Flight Mass Spectrometry Biococcal) is a genotyping system, which is exclusated and produced by SEQUENM, INC., Is also the only way to use flight time mass spectrometry directly to SNP typing. Detected equipment. Its ...

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Abstract

The invention discloses a pregnancy vitamin B12 deficiency risk assessment detection kit and an application method, and relates to molecular biology. A primer group for screening and predicting the deficiency risk of the vitamin B12 during pregnancy is used for amplifying single nucleotide polymorphism (SNP) sites of metabolic genes of the vitamin B12; 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 Flight Mass Spectrometry Biococcal Biococcal) Detects primers, kits and methods of gene site polymorphisms of pregnancy vitamin B12. Background technique [0002] Vitamin B12 (Vitb12) is a water-soluble vitamin of metal cobalt, and its physiological functions mainly include promoting erythrocytes, improves folic acid utilization, maintaining gene stability, and maintains normal operation of nervous systems. Vitamin B12 in the human body mainly comes from animal foods such as meat, eggs, fish, and milk, animals and plants cannot be synthesized. In the blood circulation system, vitamin B12 is mainly in the form of plasma binding protein, and free vitamin B12 cannot be absorbed by cells. The vitamin B12 is mainly used as a coenzide in the human body. When the vitamin B12 is lacking, the vitamin B12 is lacking, the folic acid content of human red blood cells is low, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6872C12N15/11
CPCC12Q1/6883C12Q1/6872C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2521/525C12Q2533/101
Inventor 叶辉铭苏志英李娟吴琦嫦曹佳莉邹悦
Owner 厦门市妇幼保健院
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