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Method for detecting genotype of polymorphic sites of corneal dystrophy gene and its kit

A technology of gene polymorphism and polymorphism loci, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of judgment of unfavorable results and high background, achieve accurate and reliable judgment of results, reduce pollution, easy results

Active Publication Date: 2018-05-29
北京华大通瀛科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the concentration of human genomic DNA molecular template is generally high, the background of ARMS primers after selective amplification is still high, and for specific sequences, ARMS primers are not enough to form obvious amplification differences, which is not conducive to the judgment of results

Method used

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  • Method for detecting genotype of polymorphic sites of corneal dystrophy gene and its kit
  • Method for detecting genotype of polymorphic sites of corneal dystrophy gene and its kit
  • Method for detecting genotype of polymorphic sites of corneal dystrophy gene and its kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Detection of the TGFBI gene R555W point mutation genotype related to corneal dystrophy

[0060] The present invention designs internal reference β-actin primers to evaluate whether the amplification of extracted samples is normal in real-time fluorescent quantitative PCR, making the results easier to judge.

[0061] The present invention uses enhanced ARMS primers to reduce the amplification efficiency of wild-type enhanced ARMS primers to mutant samples and the amplification efficiency of mutant enhanced ARMS primers to wild-type samples in real-time fluorescent quantitative PCR, making the results easier to judge .

[0062] 1) β-actin primer design

[0063] The upstream and downstream primers of β-actin gene were designed in the experiment. The relevant parameters are: Tm value 55.0℃-60.0℃, GC value 40.0%-60.0%, primer size 20±3bp. The primer sequences are as follows:

[0064] Upstream primer: β-actin-F AGTGGCTTCCCCAGTGTGACATG (SEQ ID NO: 1)

[0065] Dow...

Embodiment 2

[0085] Example 2 Detection of the genotype of the TGFBI gene R124H point mutation associated with corneal dystrophy

[0086] The present invention designs internal reference β-actin primers to evaluate whether the amplification of extracted samples is normal in real-time fluorescent quantitative PCR, making the results easier to judge.

[0087] The present invention uses enhanced ARMS primers to reduce the amplification efficiency of wild-type enhanced ARMS primers to mutant samples and the amplification efficiency of mutant enhanced ARMS primers to wild-type samples in real-time fluorescent quantitative PCR, making the results easier to judge .

[0088] 1) β-actin primer design

[0089] The upper and lower primers of β-actin gene were designed in the experiment. The related parameters are: Tm value 55.0℃-60.0℃, GC value 40.0%-60.0%, primer size 20±3bp. The primer sequences are as follows:

[0090] Upstream primer: β-actin-F AGTGGCTTCCCCAGTGTGACATG (SEQ ID NO: 1)

[0091] ...

Embodiment 3

[0112] Example 3 Detection of TGFBI gene R124L point mutation genotype related to corneal dystrophy

[0113] The present invention designs internal reference β-actin primers to evaluate whether the amplification of extracted samples is normal in real-time fluorescent quantitative PCR, making the results easier to judge.

[0114] The present invention uses enhanced ARMS primers to reduce the amplification efficiency of wild-type enhanced ARMS primers to mutant samples and the amplification efficiency of mutant enhanced ARMS primers to wild-type samples in real-time fluorescent quantitative PCR, making the results easier to judge .

[0115] 1) β-actin primer design

[0116] The upper and lower primers of β-actin gene were designed in the experiment. The related parameters are: Tm value 55.0℃-60.0℃, GC value 40.0%-60.0%, primer size 20±3bp. The primer sequences are as follows:

[0117] Upstream primer: β-actin-F AGTGGCTTCCCCAGTGTGACATG (SEQ ID NO: 1)

[0118] Downstream prime...

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Abstract

The invention discloses a method for detecting a genotype of polymorphic sites of a corneal dystrophy gene and its kit. In a same reaction system, real-time fluorescence quantification PCR detection is respectively carried out on a same to-be-detected sample by an enhanced ARMS primer aiming at a gene wild type template for polymorphic sites of corneal dystrophy and an enhanced ARMS primer aimingat a mutant template; according to a Ct mutant type detected by the real-time fluorescence quantification detection by the enhanced ARMS primer aiming at the gene wild type template, a Ct wild type detected by the real-time fluorescence quantification detection by the enhanced ARMS primer aiming at the mutant template, and a difference delta-Ct value of the Ct mutant type and the Ct wild type, and the mutant genotype of the corneal dystrophy gene can be determined according to the types of the polymorphic site. The method has the advantages of simple operation and high accuracy.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method for detecting the genotype of corneal dystrophy gene mutation and a kit thereof. Background technique [0002] Corneal dystrophy is a group of primary, hereditary diseases with pathological histological changes in both eyes, and its common feature is the formation of deposits of various shapes in corneal tissue. At present, there are many genes related to corneal dystrophy reported, among which TGFBI gene is the first discovered and most common related gene, which is autosomal dominant inheritance. In the Chinese population, R555 and R124 are mutation hotspots on this gene, accounting for more than 80% of all mutations. [0003] Corneal dystrophies caused by TGFBI gene mutations can be divided into three categories according to clinical symptoms: 1) Lattice corneal dystrophy (LCD), including: LCDI, LCDIII / IIIA, LCDIV, and atypical LCD; 2) Granular corneal dystrophy Dystro...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6883
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2535/137C12Q2545/101C12Q2545/113C12Q2563/107
Inventor 郭李平赵相胜杨文辉陈路其他发明人请求不公开姓名
Owner 北京华大通瀛科技有限公司
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