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PKU (Phenylketnuria) gene detection kit

A gene detection and kit technology, applied in the field of kits for determining genotypes, can solve the problems of inability to determine mutation sites and mutation content, and achieve the effects of high accuracy, simplified operation process, and reduced operation steps.

Active Publication Date: 2014-12-24
GUANGDONG HUAMEI ZHONGYUAN BIOLOGICAL SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that it can only detect the existence of gene mutations, but cannot determine the mutation site and mutation content

Method used

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  • PKU (Phenylketnuria) gene detection kit
  • PKU (Phenylketnuria) gene detection kit
  • PKU (Phenylketnuria) gene detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] One, the information of 7 polymorphic sites on the PAH gene targeted by the present invention is as follows:

[0046]The PAH gene polymorphism sites selected by the present invention include: R111X(C→T), Y204C(A→G), R243Q(G→A), R252Q(G→A), R261Q(G→A), Y356X (C→A), R413P (G→C). The arrangement of each point is as follows figure 1 shown.

[0047] 2. The main components of the detection kit:

[0048] 1. Amplification primers for 7 polymorphic sites (R111X, Y204C, R243Q, R252Q, R261Q, Y356X, R413P):

[0049]

[0050] Among the above primers, F is an upstream primer, and R is a downstream primer. R243Q, R252Q, and R261Q are adjacent polymorphic sites, and a pair of amplification primers can amplify a DNA fragment containing these three polymorphic sites.

[0051] 2. Probes targeting polymorphic sites:

[0052]

[0053]

[0054] Among the above probes, F represents the common upstream common probe, W represents the wild-type downstream probe, and M represents t...

Embodiment 2

[0082] 1. Establishment of standard atlas

[0083] Use the kit and method of implementation 1 to detect 7 sites in the wild-type samples of the known PKU gene, and the detection map is shown in Figure 5 . Use the method of implementation 3 to detect 7 sites of known PKU gene mutation samples, and the detection map is shown in Figure 6 .

[0084] 2. Practical application examples

Embodiment 3

[0087] In order to improve the specificity of the ligase connection, the present invention artificially introduces a mismatch at the third base at the 5' end of the probe to increase the specificity of the probe connection. The principle of introducing mismatches is: if the 5' end is a medium mismatch (A-A, C-C, G-G, T-T), then a moderate mismatch should be introduced; if the 5' end is a strong mismatch (G-A, T-C), a weak mismatch should be introduced (G-T, A-C); if the 5' end is a weak mismatch (G-T, A-C), then a strong mismatch (G-A, T-C) will be introduced.

[0088] The present invention artificially introduces a mismatch at the 5' end of the probe, so that the connection specificity is greatly improved. Taking the R111X site as an example, no mismatch is introduced at the 5' end of the probe ( Figure 11 In a) and introducing a mismatch ( Figure 11 The difference in b).

[0089] In the process of using multiple connection detection technology to detect SNPs, the specif...

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Abstract

The invention discloses a PKU (Phenylketnuria) gene detection kit. According to the kit, a multiple ligase detection reaction (MLDR) technology is adopted, specific probes and label probes on seven polymorphic sites on PKU determining genes (PAH) are adopted, the genotypes of seven common polymorphic sites on the PAH genes in Chinese population are detected, the genotypes of four or five detected samples can be simultaneously and accurately judged by utilizing a multiple fluorescent capillary electrophoresis detection technology, and the phenylketonuria can be diagnosed and screened. The kit disclosed by the invention can be used for rapidly and efficiently detecting the genotypes of seven polymorphic sites on the PAH genes and is a rapid, simple, convenient, economical and high-efficiency PKU gene screening and diagnosing kit.

Description

technical field [0001] The invention belongs to the technical field of biological in vitro diagnosis, and in particular relates to a kit for detecting the genotype of the determining gene (PAH) of phenylketonuria (abbreviated as PKU). Background technique [0002] Phenylketonuria (PKU for short) is a hereditary disease caused by abnormal amino acid metabolism. Due to gene mutation, the activity of phenylalanine hydroxylase in the body is reduced, which leads to the blockage of phenylalanine metabolism. The concentration of phenylalanine increases, and abnormal metabolites such as phenylpyruvate, phenylacetic acid, and phenyllactic acid increase significantly in the urine. PKU is an autosomal recessive genetic disease. The incidence of PKU in my country has obvious regional differences, showing a law of high in the north and low in the south. Generally, the incidence in southern provinces is about 1:1-30,000, while the incidence in northern provinces is mostly in 1: 6000-9000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2521/501C12Q2565/125
Inventor 郑卫国王於建胡琦杜蔚安孟祥和郭育林葛海鹏高静王邦超
Owner GUANGDONG HUAMEI ZHONGYUAN BIOLOGICAL SCI & TECH
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