SAA1 beta / beta homozygote genotype based detection method for prognosis and / or diagnosis of cirrhosis

A detection method and genotype technology, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve problems such as poor repeatability, difficulty in large-scale SNP genotype analysis, cumbersome steps, etc., to achieve accurate Effects of rapid detection, improved reliability and accuracy, and improved amplification efficiency

Active Publication Date: 2013-06-19
DIASYS DIAGNOSTIC SYST SHANGHAI
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  • Application Information

AI Technical Summary

Problems solved by technology

[0018] At present, the RFLP method is commonly used in the study of SAA1 gene polymorphism. This step is extremely cumbersome and has poor repeatability, and it is not easy to carry out SNP genotype analysis on a large scale.

Method used

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  • SAA1 beta / beta homozygote genotype based detection method for prognosis and / or diagnosis of cirrhosis
  • SAA1 beta / beta homozygote genotype based detection method for prognosis and / or diagnosis of cirrhosis
  • SAA1 beta / beta homozygote genotype based detection method for prognosis and / or diagnosis of cirrhosis

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Example 1 Establishment of real-time fluorescent quantitative allele-specific PCR and its clinical evaluation and application

[0063] 1. Establishment of real-time fluorescence quantitative allele-specific PCR method

[0064] Real-time fluorescence quantitative PCR technology has the characteristics of real-time monitoring, quantification and high throughput, and is easy to operate and high in sensitivity. Fluorescent quantitative PCR is divided into probe quantitative PCR and fluorescent dye quantitative PCR. SYBR Green I dye is added to the quantitative PCR of fluorescent dyes, which can specifically embed in double-stranded DNA molecules and emit fluorescence when combined with DNA molecules. When the DNA molecule is single-stranded, the dye is released and the fluorescence decreases. Therefore, in a reaction system, the intensity of fluorescence is positively correlated with the content of DNA double-strand molecules. Therefore, in each cycle, the fluorescence s...

Embodiment 2

[0109] Embodiment 2: detect SAA1 genotype with AS-PCR combined with RFLP method

[0110] At present, the RFLP method is generally used in the study of the polymorphism of the SAA1 gene. In this example, the detection method of the RFLP and AS-PCR is used to study the distribution of the SAA1 genotype in the healthy population. The reliability and accuracy of the method of the present invention are evaluated by comparing with the results obtained by real-time fluorescent quantitative allele-specific PCR.

[0111] RFLP refers to the difference in the length of restriction enzyme fragments between genotypes, which is caused by the insertion, deletion, rearrangement or point mutation of bases at the restriction enzyme site. RFLP can locate and separate a specific gene, and its polymorphism can be seen directly from the electrophoretic pattern after enzyme digestion. It is currently the most commonly used method for SNP research. This method can be used to determine the difference...

Embodiment 3

[0187] This embodiment provides a human serum amyloid A1 (Serum Amyloid A, SAA1) genome single nucleotide polymorphism SNP real-time fluorescent quantitative allele-specific PCR detection kit.

[0188] This kit can identify and detect the SAA1 allele α, β, and γ genotypes in the human genome, and is suitable for the typing of human SAA1 genotypes. The kit adopts the principle of SYBR Green I chimeric fluorescence method, including the most suitable SYBR Green I concentration for real-time fluorescence quantitative allele-specific PCR reaction, and uses pfu DNA polymerase and real-time fluorescence quantitative allele specificity The most matching buffer and primer combination for the PCR reaction can effectively inhibit non-specific PCR amplification and achieve the purpose of high-sensitivity, high-throughput real-time fluorescence quantitative allele-specific PCR amplification reaction. When conducting experiments, the preparation of PCR reaction solution is very convenient ...

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Abstract

The invention discloses an SAA1 beta / beta homozygote genotype based detection method for prognosis and / or diagnosis of cirrhosis. The detection method employs a nucleotide sequence based on three alleles, SAA1 alpha, beta and gamma, to synthesize a primer and conducts real-time fluorescence quantification allele specific PCR, so as to determine whether a target to be detected belongs to susceptible population of liver cirrhosis. The detection method provided by the invention has the advantages of simpleness, accuracy, rapidness and high throughput, and is suitable for research on SAA1 SNP and related diseases.

Description

technical field [0001] The present invention relates to a method for realizing the prognosis diagnosis and / or diagnosis of liver cirrhosis by discriminating the genotype of SAA1 (serum amyloid A1), and in particular relates to a method for liver cirrhosis based on the homozygous genotype of serum amyloid A1SAA1β / β. A detection method for cirrhosis prognosis diagnosis and / or liver cirrhosis diagnosis, including a kit suitable for clinical application. Background technique [0002] Human serum amyloid A1 (Serum Amyloid A1, SAA1) is an acute phase response protein consisting of 104 amino acids. Its molecular weight in natural state is about 12-14kDa, and its coding gene is located on human chromosome 11. Early studies believed that SAA1 is an acute inflammatory protein, because in acute inflammation, SAA1 can be synthesized by activated macrophages and fibroblasts in the liver, and the concentration is 100-1000 times the normal level (the normal value is generally 910±270 µg / ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 王荣芳吴峻张艳钱震斌
Owner DIASYS DIAGNOSTIC SYST SHANGHAI
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