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Mass spectrum for detecting folic acidinheritancemetabolic capability and calcium absorptioninheritance

A technology for metabolic capacity and calcium absorption, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. It can solve the problems of not fully disclosing the detection technology, staying in tumor detection, complicated operation, etc., to achieve The effect of high degree of automation, low false positive and high detection sensitivity

Inactive Publication Date: 2017-06-13
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology still belongs to the traditional PCR amplification detection technology, and can only detect a single mutation site, and the operation is cumbersome
[0038] In summary, the current technical problems are: the lack of methods and products that can simultaneously detect multiple gene polymorphic sites related to folic acid and calcium absorption at one time, common detection technologies, such as sequencing, real-time fluorescent quantitative PCR, etc., all require Detect the sites one by one. When there are many sites, the operation is complicated and the cost is high
However, the existing mass spectrometry detection technology has not fully disclosed the detection technology for the above multiple sites at the same time, and these technologies still remain in the relevant aspects of tumor detection

Method used

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  • Mass spectrum for detecting folic acidinheritancemetabolic capability and calcium absorptioninheritance
  • Mass spectrum for detecting folic acidinheritancemetabolic capability and calcium absorptioninheritance
  • Mass spectrum for detecting folic acidinheritancemetabolic capability and calcium absorptioninheritance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1: Primer design and synthesis.

[0115] The SNPs related to folic acid are MTHFR gene rs1801131 (A1298C), MTHFR gene rs1801133 (C677T) and MTRR gene rs1801394 (A66G), and the SNPs related to calcium absorption are VDR gene rs1544410 (Bsm I ), VDR gene rs731236 site (Taq I), design corresponding specificity PCR primer core sequence (SEQ ID No: 1 to SEQ ID No: 10) and specific extension primer core sequence (SEQ ID No: 11 to SEQID No: 15).

[0116] Among them, in order to prevent the PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, a certain number of bases can be added to the core sequence (SEQ ID No: 1 to SEQ ID No: 10) at the 5' end of each PCR primer , common such as 10bp tag (ACGTTGGATG), to increase the molecular weight of the PCR primer, thereby exceeding the detection window of the mass spectrometer.

[0117] The relevant primers were synthesized in Shanghai Jierui Bioengineering Co., Ltd...

Embodiment 2

[0119] Embodiment 2: sample DNA extraction.

[0120] A total of 10 clinical patients were collected. Among them, sample collection, DNA extraction, etc. were collected in accordance with the requirements of the instructions, and human venous blood was collected with EDTA anticoagulant tubes. According to the instructions, the collected blood should not be stored at 2-8°C for more than one week, and at -20°C for no more than one month, and can be transported in a curling box with ice or a foam box with ice. It is recommended to use fresh blood as much as possible. Genomic DNA extraction. Since this kit does not provide human genomic DNA extraction reagents, a commercial nucleic acid extraction kit (such as: Tiangen Biochemical Technology (Beijing) Co., Ltd. Blood Genomic DNA Extraction Kit (spin column type)) was used to extract DNA from each patient. Human genomic DNA was extracted from 200 ul whole blood of the patient, quantified with NanoDrop 2000 (Thermo Company), and st...

Embodiment 3

[0121] Embodiment three: Biological experiment.

[0122] Using ABI 9700 PCR instrument, according to the instructions, 3 gene polymorphism sites related to folic acid genetic metabolism genes and 2 gene polymorphism sites related to calcium absorption were tested.

[0123] The components used in the kit for PCR, PCR product purification and single base extension are shown in Table 4:

[0124] Table 4. Kit composition

[0125] serial number component name main ingredient Packing Specifications 1 Reaction solution I dNTPs, Tris, MgCl 2

270μL / tube×1 tube 2 Enzyme I PCR enzyme, UNG enzyme 22μL / tube×1 tube 3 Amplification primer PCR primers 100μL / tube×1 tube 4 Reaction solution II Tris, MgCl 2

170μL / tube×1 tube 5 Enzyme II SAP enzyme 30μL / tube×1 tube 6 Reaction solution III ddNTPs, Tris, MgCl 2

100μL / tube×1 tube 7 Enzyme III elongase 4μL / tube×1 tube 8 extension primer extension...

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Abstract

The invention discloses a primer system for detecting folic acidinheritancemetabolic capability and calcium absorptioninheritance to detect related genepolymorphic site (SNP). A product prepared on the basis of the primer system is capable of detecting folic acidinheritancemetabolic capability and calcium absorption related genepolymorphic sites simultaneously. By using the product, folic acidinheritancemetabolic capability related genepolymorphic sites are detected, reference bases are provided to instruct women preparing for pregnancy and pregnantwomen to take folic acid, and meanwhile detection on calcium absorption related genepolymorphic sites can also instruct the pregnant women to scientifically and reasonably supplement calcium nutrients in the pregnancy period. By adopting the primer system, 5 genepolymorphic sites on different genes can be simultaneously detected within one reaction system, the cost is relatively low when being compared with that of techniques such as sequencing and real-time fluorescence quantification PCR (Polymerase Chain Reaction), the operation is relatively simple, and the accuracy and the sensitivity are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and product for determining polymorphic sites (SNPs) of genes related to genetic metabolism of folic acid and calcium absorption. Specifically, multiplex PCR technology, single base extension Technology and mass spectrometry, methods for detecting 3 genetic polymorphic sites of folic acid genetic metabolism and 2 genetic polymorphic sites related to calcium absorption, and corresponding kits. Background technique [0002] Folic acid is one of the B vitamins. It participates in the synthesis of DNA, RNA, protein and other important compounds. It plays an important role in the body's metabolic process. It is often used in nutritional supplements during pregnancy to prevent diseases such as neural tube defects. [0003] The top 5 birth defects in my country include: congenital heart disease, neural tube defects, cleft lip and palate, Down syndrome, and polydactyly (toes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/125C12Q2565/627
Inventor 张海燕杨宇凤马庆伟李清林燕
Owner BIOYONG TECH
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