Detection kit for autosomal dominant inheritance polycystic kidney disease

A detection kit and autosome technology, which is applied in the field of medical molecular biology detection, can solve the problems of complex structure, lower detection specificity, lack of uniformity and repeatability, etc., and achieve the effect of increasing the number of primers and preventing errors

Inactive Publication Date: 2003-08-06
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex structure of the pathogenic gene of autosomal dominant polycystic kidney disease, the type I pathogenic gene has multiple homologous sequences, which directly reduces the specificity of detection
Affected by it, most current studies on pathogenic gene mutations are limited to some regions of type I pathogenic genes, even though individual foreign research groups have initially carried out their full-gene mutation detection ( Rossetti S., Strmecki L., Gamble V., et al. Mutation analysis of the entire PKD1 gene: genetic and iagnostic implications. Am J Hum Genet, 2001, 68: 46-63), but there are many kinds of molecular biology techniques used, lack of uniformity and reproducibility

Method used

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  • Detection kit for autosomal dominant inheritance polycystic kidney disease
  • Detection kit for autosomal dominant inheritance polycystic kidney disease
  • Detection kit for autosomal dominant inheritance polycystic kidney disease

Examples

Experimental program
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Effect test

preparation example Construction

[0054] 1. Preparation of each solution of the primer mix

[0055] (1) 0.8mol / L Tris solution

[0056] Add 96.9 g of tris to 800 ml of deionized ultrapure water, adjust the pH to 8.9 with concentrated hydrochloric acid, and dilute to 1 L with deionized ultrapure water.

[0057] (2) 1.7mol / L potassium acetate solution

[0058] Add 166.8 g of potassium acetate to 800 ml of deionized ultrapure water, and after fully dissolving, dilute to 1 L with deionized ultrapure water.

[0059] (3) Dimethyl sulfoxide solution

[0060] The dimethyl sulfoxide solution with a concentration greater than 99% is dispensed in 10ml / bottle for use.

[0061] (4) 20% Octylphenoxypolyethoxyethanol

[0062] Take 1.88 ml of commercially available octylphenoxypolyethoxyethanol (Shanghai Huamei Biotechnology Co., Ltd.) with a concentration of 106.5% (w / v), and dilute to 10 ml with deionized ultrapure water.

[0063] (5) Deoxynucleoside triphosphate solution

[0064] A commercial deoxynucleoside triphosp...

Embodiment 1

[0074] Example 1: Preparation of Primer Mix I-S-1

[0075] Take 20 μl of 0.8 mol / L tris hydroxymethyl aminomethane solution, 20 μl of 1.7 mol / L potassium acetate solution, 20 μl of dimethyl sulfoxide solution, 2 μl of 20% octylphenoxy polyethoxyethanol, 10 mmol / L deoxygenated nucleus Glycoside triphosphate solution 24μl, 250mmol / L magnesium acetate solution 2μl, enzyme activity 4U / μl thermostable deoxyribonucleotide polymerase 20μl, 50μmol / LI-S-1 forward and reverse primers 8μl each, mix well , dilute to 200 μl with sterilized deionized ultrapure water, put in a 0.5ml thin-walled tube and store at -20°C for later use. At this time, the final concentrations are: Tris 80mmol / L, potassium acetate 170mmol / L, dimethyl sulfoxide 10%, octylphenoxypolyethoxyethanol 0.2%, deoxynucleoside triphosphate 1200μmol / L , magnesium acetate 2.5mmol / L, thermostable deoxyribonucleotide polymerase 80U, forward and reverse primers 2μmol / L each.

[0076] The preparation method of primer mixture I-H...

Embodiment 2

[0077] Example 2: Preparation of Primer Mix I-S-2

[0078] Take 10 μl of 10 mmol / L deoxynucleoside triphosphate solution, 4 μl of thermostable deoxynucleotide polymerase with an enzyme activity of 4 U / μl, and the amounts of other components are the same as in Example 1. After thorough mixing, dilute to 200 μl with sterilized deionized ultrapure water, put in a 0.5ml thin-walled tube and store at -20°C for later use.

[0079] The preparation method of primer mixtures I-S-3 to I-S-8 and I-H-2 and I-H-8 is the same as that in Example 2.

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Abstract

The present invention relates to the field of detection technology in medical molecular physiology, it is a kit for detecting autosomal dominant inheritance polycystic kidney disease. According to the structure property of pathogenic gene type A and type B of patient said invention can synthesize correspondent primer, adopt molecular physiological techonlogy and combine it with single-strand conformation polymorphism analysis method or high-pressure liquid-phase chromatography analysis method to make detection, and can directly detect out the mutant site and mutation type of pathogenic gene of the autosomal recessive inheritance polycystic kidney disease for preventing and curing said disease.

Description

technical field [0001] The invention relates to the technical field of medical molecular biology detection, and is a kit for detecting autosomal dominant polycystic kidney disease. Background technique [0002] Autosomal dominant polycystic kidney disease is a very common genetic-related kidney disease. The organ function is abnormal, and most patients eventually die of renal failure. The clinical diagnosis of the disease is mainly made by using imaging technology and family genetic medical history. Although this diagnostic method is simple and direct, it can only be diagnosed when multiple renal cysts have appeared in the body, and it is difficult to diagnose early. Treat early. The molecular diagnosis of the disease mainly uses microsatellite linkage analysis technology to make an indirect diagnosis by detecting the changes of several genetic markers closely linked to the pathogenic gene of autosomal dominant polycystic kidney disease in the family. This detection techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N27/447G01N33/48
Inventor 梅长林张树忠张殿勇
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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