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Primers for detecting plurality of newborn inherited metabolic disease causing genes and kit

A technology for genetic metabolic diseases and neonates, applied in the field of genetic detection of neonatal genetic metabolic diseases, can solve the problems of low throughput and small genetic metabolic disease screening, achieve early diagnosis and early treatment, and reduce fatalities rate and disability rate, effect of reducing burden

Active Publication Date: 2015-12-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of these diagnostic methods for IMD screening has limitations: (1) Generally, the throughput is low, and generally only one to dozens of species can be detected at a time, which cannot meet the year-by-year increase in the number of detectable diseases of IMD, and requires genetic metabolic (2) The detection methods of different IMDs are different, and the samples are diverse. For example, some diseases detect protein levels, while others detect gene levels. Some diseases require urine samples, while others require blood samples. sample
Therefore, it is not suitable for large-scale screening of inherited metabolic diseases, and there is an urgent need for a high-throughput detection technology for various diseases of IMD.

Method used

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  • Primers for detecting plurality of newborn inherited metabolic disease causing genes and kit
  • Primers for detecting plurality of newborn inherited metabolic disease causing genes and kit
  • Primers for detecting plurality of newborn inherited metabolic disease causing genes and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: In this example, six genes including non-syndromic deafness-related pathogenic genes GJB2, GJB3, SLC26A4, 12SrRNA, COCH and DFNA5 were selected.

[0068] 1. Sample preparation (acquisition of sequencing library)

[0069] The steps of performing two rounds of PCR amplification on the exon and exon-intron binding region of the gene to obtain the sequencing library are as follows:

[0070] 1. Acquisition of disease-causing gene information.

[0071] Search for non-syndromic deafness on the OMIM (Online Mendelian Inheritance in Man) website or NCBI's OMIM database to obtain hotspot pathogenic genes related to it and obtain information on related mutation sites.

[0072] 2. Search the sequence and position information of the exons and introns of each relevant pathogenic gene in NCBI.

[0073] 3. Multiplex PCR primer design and synthesis

[0074] Corresponding PCR primers were designed for the exons of the above genes. According to the aforementioned design prin...

Embodiment 2

[0121] Embodiment 2: Referring to the method of Example 1, for a certain glucose-6-phosphate dehydrogenase deficiency patient, the primers were designed using the method of the present invention, and the first round of primer sequences are as follows in Table 2:

[0122] Table 2, G6PD gene first-round PCR primer sequence list

[0123]

[0124]

[0125] After the first round of PCR was completed, high-throughput sequencing was carried out after the second round of PCR (see Example 1 for specific steps and methods), and the results obtained were as follows:

[0126] The high-throughput sequencing results of patients with G6PD deficiency are:

[0127]

[0128] The c.678C>G mutation site of the G6PD gene is verified with the gold standard Sanger method (with normal people as a control), and it is found that it is completely consistent with the mutation type detected by the present invention, such as Figure 5 Shown:

[0129] Figure 5 It is the Sanger sequencing result...

example 3

[0133] Example 3: with reference to the method of Example 1, to a newborn of a certain Gitelman syndrome patient, utilize the method of the present invention to related disease-causing gene SLC12A3 gene design primer (the primer of first-round PCR is as table 3) carries out PCR amplification high Through-put sequencing was used to determine whether the patient's son suffered from Gitelman syndrome, and the following results were obtained:

[0134] Table 3, SLC12A3 gene first-round PCR primer sequence list

[0135]

[0136]

[0137] The results of high-throughput sequencing of newborns of patients with Gitelman syndrome are:

[0138]

[0139] The C.176_179delACAAA deletion mutation site of the SLC12A3 gene was verified by the gold standard Sanger method, and it was found to be completely consistent with the mutation type detected in the present invention. After the ACAAA sequence was deleted, a frameshift mutation was caused, and it was heterozygous at the same time, r...

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Abstract

The invention discloses a design way of primers for in-vitro qualitative detection of newborn inherited metabolic disease genes. The design way comprises the following steps: (1) acquiring relevant disease-causing gene information of inherited diseases: acquiring the relevant disease-causing gene information, including gene location information and mutation site information of a plurality of inherited diseases through an OMIM (Online Mendelian Inheritance in Man) database, and acquiring exon and intron information of relevant genes from an NCBI (National Center for Biotechnology Information) database according to the obtained gene information; (2) designing primers specific to the relevant genes: designing corresponding specific primers F1 and R1 specific to an exon and an exon-intron combination area of each gene. The invention also discloses the first-round PCR (Polymerase Chain Reaction) primer sequence list of syndromic deafness, a first-round PCR primer list of G6PD genes, and a first-round PCR primer sequence list of SLC12A3 genes. The primers can be used for detecting relevant genes of 22 types of common newborn inherited diseases.

Description

technical field [0001] The invention relates to the fields of molecular biology and medical detection, in particular to a method for detecting genetic metabolic diseases in newborns that can be used on a next-generation sequencing platform. Background technique [0002] Inherited metabolic diseases (Inherited Metabolic Diseases, IMD), also known as Inborn Errors of Metabolism (IEM), are caused by mutations in the coding genes of certain enzymes, carrier proteins, membranes or receptors necessary to maintain the normal metabolism of the body, causing A type of disease in which the function of the encoded product changes, resulting in corresponding laboratory abnormalities and clinical symptoms. It involves abnormalities in the metabolism of amino acids, organic acids, fatty acids, urea cycle, carbohydrates, lysosomes, and steroids. Most of these diseases are single-gene genetic diseases, and a few are mitochondrial genetic diseases. After the onset of such diseases, they of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 谭海芹余文菁洪旭涛宓娅娜
Owner ZHEJIANG UNIV
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