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DNA probe pool for detecting kawasaki disease related gene and SNP locus, as well as preparation method and application thereof

A DNA probe, Kawasaki disease technology, applied in the field of molecular biology, can solve the problem of inability to accurately classify patients and provide reliable test results, disease pathogenesis cannot be accurately diagnosed, no similar detection products for Kawasaki disease, etc. problems, to achieve the effect of reducing fatality and disability rates, achieving early diagnosis and early treatment, and reducing burden

Active Publication Date: 2018-06-19
SHANGHAI CHILDRENS HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing detection methods are mainly based on cytokines and echocardiography. Cardiac echocardiography is a first-line diagnostic technique for diagnosing coronary artery lesions, but it cannot accurately diagnose the pathogenesis of the disease, and cannot provide reliable detection for accurate typing and medication of patients result
The missed detection rate of cytokines is about 67%, and the direct use of whole-genome next-generation sequencing is too costly. At the same time, a large number of useless sequencing results will make analysis very difficult
Currently, there is no similar test product for Kawasaki disease on the market

Method used

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  • DNA probe pool for detecting kawasaki disease related gene and SNP locus, as well as preparation method and application thereof
  • DNA probe pool for detecting kawasaki disease related gene and SNP locus, as well as preparation method and application thereof
  • DNA probe pool for detecting kawasaki disease related gene and SNP locus, as well as preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0055] Another aspect of the embodiments of the present invention also provides the aforementioned method for preparing a DNA probe pool for detecting genes related to Kawasaki disease and SNP sites, which includes:

[0056] Select the exon regions of genes involved in T cell receptor signaling pathway, Toll-like receptor signaling pathway, cytokine receptor signaling pathway, and TGF-β signaling pathway, and screen a total of 560 genes related to Kawasaki disease, including 466 SNP positions point;

[0057] Obtain different transcripts of each gene, map them to the HG19 human standard reference genome through position information, and select sequences that can cover all transcripts;

[0058] Make the exon part extend 15bp to the intron at the 3' and 5' ends,

[0059] Each of the oligonucleotide probe sequences has at least 50% sequence overlap, the probe sequence with a CG content of 40-60% in the target region covers 2 times, and the CG content in the target region is less ...

Embodiment example

[0094] Wherein, as a more specific implementation case of the present invention, a method for qualitative detection of Kawasaki disease gene in vitro comprises the following steps:

[0095] (1) DNA probe preparation

[0096] (1.1) DNA probe design: Kawasaki disease-related immune signaling pathways were obtained from KEGG, and a total of 560 genes including 466 SNP sites were screened through the literature.

[0097] The principles of oligonucleotide design are:

[0098] 1) Different transcripts of each gene are mapped to the HG19 human standard reference genome through position information, and sequences that can cover all transcripts are selected.

[0099] figure 1 For the exon collection method of the 6 transcripts of the PTPN22 gene, different transcripts are mapped to the hg19 reference genome, and a schematic diagram containing the sequences of all transcripts is generated; the sequence interval used to design probes includes the exons of all transcripts Exon region. ...

Embodiment 1

[0141] Screening of markers for predicting the risk of coronary artery injury induced by Kawasaki disease

[0142] Screening methods and steps such as Figure 7 Shown:

[0143] 1. Approved by the Ethics Committee, take 1mL of whole blood from 50 children with Kawasaki disease complicated with CAL and 1mL of whole blood from 50 children with Kawasaki disease without CAL, use EDTA anticoagulant tubes uniformly for blood collection, and store at 4°C for no more than 48 hours , long-term storage in liquid nitrogen.

[0144] 2. Take 200uL of whole blood and use the QiaAmp Blood Mini Kit for DNA extraction. The extraction operation is carried out in full accordance with the operation manual of the kit.

[0145] 3. Take 1ug of the extracted DNA, and use the KAPA LTP DNA Library Preparation Kit to construct the next-generation sequencing library. The library construction operation is completely carried out according to the operation manual of the kit.

[0146] 4. Group the construc...

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Abstract

The invention discloses a DNA probe pool for detecting kawasaki disease related gene and SNP locus, as well as preparation method and application thereof. The DNA probe pool comprises 558 oligonucleotide probe sequences which are shown by SEQ ID NO:3-SEQ ID NO:560. Each oligonucleotide probe sequence comprises a 21bp universal sequence at the 5' end, a 120bp sequence which can be specifically bonded with gene, and a 21bp universal sequence at the 3' end; the 21bp universal sequence at the 5' end and the 21bp universal sequence at the 3' end of the oligonucleotide probe sequence are shown by SEQ ID NO:1-SEQ ID NO:2 respectively. According to the detection method disclosed by the invention, the detection cost can be reduced, the detection precision can be improved, and an in-vitro qualitative detection means can be provided for the kawasaki disease pathogenesis, prognosis and medicinal cure effect, as well as an effective auxiliary diagnosis way can be provided for reducing coronary artery lesion and other heart diseases triggered in the late phase of kawasaki disease.

Description

technical field [0001] The present invention relates to the field of molecular biology technology, more specifically, to the field of DNA technology, in particular to a DNA probe pool for detecting genes and SNP sites related to Kawasaki disease, its preparation method, and its Use for predicting Kawasaki disease-induced coronary artery injury and IVIG drug sensitivity. Background technique [0002] Kawasaki Disease (KD), also known as mucocutaneous lymph node syndrome (Mucocutaneouslymph node syndrome), is an acute systemic inflammatory syndrome of medium and small vessels that occurs frequently in children. It was first reported by Japanese scholar Tomisaku Kawasaki in 1967. Coronary artery lesions (CALs) caused by this disease are the most common cause of acquired heart disease in children in developed countries, mainly including coronary artery dilatation, coronary aneurysm, coronary artery stenosis, coronary artery fistula formation, etc., serious Patients may develop ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2535/122
Inventor 黄敏贾佳朱丹颖宁倩倩张泓杨竞宋思瑞
Owner SHANGHAI CHILDRENS HOSPITAL
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