Typing kit, primer and typing method for vitamin K metabolism related genes

A typing method and kit technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the cost, time-consuming, personnel requirements, unsuitable gene SNP site detection, sensitivity Low, limited throughput and other issues, to achieve the effect of improving the efficiency of typing detection, high degree of automation, and reducing the amount of samples

Active Publication Date: 2022-05-06
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] PCR-direct sequencing method is recognized as the gold standard for genotyping detection, but it is not easy to popularize due to long detection cycle, low detection throughput, low sensitivity, high cost, special requirements for reagents and instruments
The detection sensitivity of PCR-restriction fragment length polymorphism analysis is also not high, the throughput is low, the operation steps are cumbersome, and it is only suitable for the typing of some SNP sites. The detection results still need to be verified again by the next generation sequencing method, especially when When the sample size is large, it is easy to cause cross-contamination of PCR products, and it is prone to insufficient or excessive digestion, resulting in false negative or false positive results
The PCR-gene chip method has high throughput, but the accuracy and repeatability of th...

Method used

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  • Typing kit, primer and typing method for vitamin K metabolism related genes
  • Typing kit, primer and typing method for vitamin K metabolism related genes
  • Typing kit, primer and typing method for vitamin K metabolism related genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] A test kit, its composition is as shown in table 10:

[0091] Table 10

[0092]

Embodiment 2

[0093] Embodiment 2 Typing method

[0094] 1. Genomic DNA extraction: For details, refer to the Standard Operating Procedure for Genomic DNA Extraction of Blood Cell Tissues (BJHH-SOP-PCR-QT-001).

[0095] 2. PCR amplification

[0096] 2.1 Primer Mix preparation, the specific amplification primer Mix configuration method is shown in Table 11, and the single base extension primer Mix configuration method is shown in Table 12:

[0097] Table 11

[0098]

[0099] Table 12

[0100]

[0101] 2.2 PCR amplification system

[0102] The details are shown in Table 13~Table 14:

[0103] Table 13: Multiplex PCR amplification system (Agena enzyme system)

[0104]

[0105] Note: Both the internal reference gene and A1 were amplified with this reaction system.

[0106] Table 14: Multiplex PCR amplification system (FastStart enzyme system)

[0107]

[0108] Note: B1 was amplified with this reaction system.

[0109] Due to the low efficiency of using the Agena enzyme system...

experiment example 1

[0153] This example is used to illustrate the accuracy of the above kit and detection method.

[0154] DNA samples from five normal people were used for detection (sample names were RJJ, LXJ, SLP, JYX and ZHF), homozygous mutation positive control (MUT), negative control (WT) heterozygous mutation positive control (Con ), using the above kit and detection method for detection.

[0155] 1. Use the above kits for liquid quality test results;

[0156] (1) The mass spectrometry results of tube A / tube C are shown in Table 21:

[0157] Table 21

[0158]

[0159]

[0160] " / " indicates that the corresponding MS molecular weight was not detected.

[0161] The sequencing results detected by multiplex PCR sequencing are shown in Table 22;

[0162] Table 22

[0163]

[0164] The results of tube B / tube D mass spectrometry are shown in Table 23:

[0165] Table 23

[0166]

[0167] " / " indicates that the corresponding MS molecular weight was not detected.

[0168] The se...

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Abstract

The invention relates to the technical field of gene detection, in particular to a typing kit, primers and a typing method for vitamin K metabolism related genes. The typing kit at least comprises a nucleic acid amplification reagent and a single-base extension reaction reagent, the nucleic acid amplification reagent contains PCR (Polymerase Chain Reaction) amplification primers as shown in SEQ ID NO: 1 to SEQ ID NO: 22; the single base extension reaction reagent contains single base extension primers as shown in SEQ ID NO: 23 to SEQ ID NO: 43. The typing method comprises the following steps: carrying out a PCR amplification reaction on the PCR amplification primer, carrying out alkaline phosphatase digestion, carrying out a single-base extension reaction on the single-base extension primer, carrying out desalination treatment, and carrying out high performance liquid chromatography-mass spectrometry detection. The kit and the detection method provided by the invention can improve the typing detection efficiency of the vitamin K metabolism related genes, and are simple, convenient, feasible, high in accuracy and high in automation degree.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a typing kit, primers and typing method for genes related to vitamin K metabolism. Background technique [0002] Vitamin K is an essential fat-soluble vitamin for blood clot formation. Naturally available in two natural forms: phylloquinone (VK1) synthesized by green plants and menaquinone (VK2) synthesized by bacteria. VK3 and VK4 are human-made synthetic products, which are water-soluble and have no activity themselves. They can be alkylated into MK4 (VK2) in the liver in vivo and have biological activity. Vitamin K is essential for the conversion of inactive precursors into functional clotting factors and is therefore essential for clot formation. Once vitamin K is utilized, it becomes inactive and must be reactivated in order to exert its biological effects. This vitamin K recycling process is known as the vitamin K cycle. Vitamin K is also important for bone healt...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 贾永娟周坤刘春冉倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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