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Probe set and kit for detecting pathogenic gene of autoinflammatory disease

A technology of inflammatory diseases and disease-causing genes, applied in the field of biomedicine, can solve problems such as the inability to implement prenatal interventions in genetic counseling, the pain of patients and their families, and hinder the quality of the population, so as to shorten the inspection cycle, shorten the detection time, The effect of improving diagnostic efficiency

Active Publication Date: 2019-02-22
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The next-generation sequencing technology (next-generation sequencing technology) has the advantages of fast, accurate and low cost, and can detect various types of mutations in multiple genes at the same time, and has been widely used in the etiology detection of genetic defects and molecular genetic diagnosis, but so far, there have been no high-throughput capture probes or kits specifically for the detection of autoinflammatory disease-related genes, making the progress of this disease-related field seriously lagging behind, and the diagnostic rate is low. Genetic counseling and necessary prenatal intervention measures cannot be implemented, which not only brings great pain and heavy economic burden to patients and their families, but also seriously hinders the substantial improvement of the overall quality of the population in our country

Method used

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  • Probe set and kit for detecting pathogenic gene of autoinflammatory disease
  • Probe set and kit for detecting pathogenic gene of autoinflammatory disease
  • Probe set and kit for detecting pathogenic gene of autoinflammatory disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] A probe set for detecting autoinflammatory disease-causing genes, the probe set includes probes capable of specifically capturing the following 50 genes at the same time: ADAR, COPA, NLRP3, SLC29A3, HPS1, HPS6, RNASEH2C , TNFRSF1A, GUCY2C, MVK, LACC1, RNASEH2B, RNF31, PSTPIP1, MEFV, NOD2, PLCG2, NLRP1, CARD14, LPIN2, ACP5, RNASEH2A, ADGRE2, NLRP12, ADAM17, NLRC4, IL36RN, IL1RN, IFIH1, AP1S3, RBCK1, FERMT1 , SAMHD1, ADA2, USP18, HPS4, TREX1, COL7A1, SH3BP2, OTULIN, TMEM173, PSMB8, TNFAIP3, SHARPIN, DDX58, POLA1, IL10, IL10RA, IL10RB, ISG15.

[0069] The probes included in capturing and detecting each of the above genes include probes whose base sequences are shown in SEQ ID No. 1-50.

Embodiment 2

[0071] A kit for detecting autoinflammatory disease-causing genes, comprising the following components: the probe set (160ul, 150ng / ul) obtained in Example 1, enrichment buffer (208ul), hybridization buffer ( 800ul), binding buffer (3.2ml), first rinse (9ml), second rinse (45ml), NaOH solution (0.1M, 1ml), Tris-HCl buffer (1M, pH 7.5, 1.2ml) , PCR reaction solution (580ul), TE buffer solution (800ul). Wherein each buffer composition is as follows:

[0072] 1. Enrichment buffer (per 20ul):

[0073] Human cot-1 DNA (purchased from Invitrogen), 7ul; salmon sperm DNA (purchased from Invitrogen), 3ul;

[0074] Specific blocking primers as shown in SEQ ID No.51 and SEQ ID No.52, a total of 10ul, each primer concentration is 1nmol / ul:

[0075] 2. Hybridization buffer: 5 times concentration of SSPE, 5 times concentration of Denhardt solution, 5mM EDTA, 0.1% SDS;

[0076] 3. Binding buffer: 1M NaCl, 10mM Tris-HCl (pH 7.5), 1mM EDTA

[0077] 4. The first rinse solution: 1 times the...

Embodiment 3

[0118] Test kit effect test for detecting autoinflammatory disease-causing genes:

[0119] Using the kit described in Example 2 of the present invention to detect 3 samples, the results confirmed that the capture rate of the target disease-causing / susceptible gene was satisfactory, and more than 50% of the original short sequences could be compared back to the reference sequence of the target region, the target region The average amount of effective sequencing data reaches 200Mb, and the average sequencing depth of the target region is 300X, which is much higher than the general genetic disease diagnosis requirements (generally 100X).

[0120] 1. Child patient number C150710C01201, male, 4 years old, was tested using the kit of the present invention, and the test results were as follows: figure 1 Shown is a mutation in the gene TNFRSF1A. A mutation occurs at chr12-6442930*, that is, a deletion mutation c.295T>A occurs in exon3, and the disease phenotype is periodic fever, aut...

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Abstract

The invention provides a probe set and a kit for detecting a pathogenic gene of an autoinflammatory disease. The probe set comprises probes capable of simultaneously specifically capturing pathogenicgenes affecting inflammasome defects, non-inflammasome defects and autoinflammatory diseases associated with defects in an interferon pathway. The probe set and the kit for detecting the pathogenic gene of the autoinflammatory disease are capable of simultaneously detecting the pathogenic genes of 50 types of known autoinflammatory diseases; based on the probe set which is high in targeted screening, stable and reliable in gene acquisition and used for detecting the pathogenic gene of the autoinflammatory disease, and combined with a high-throughput sequencing method, a diagnosis cycle of theautoinflammatory disease can be accelerated, the diagnostic efficiency can be improved, and several new diseases can be diagnosed, especially for children with the autoinflammatory diseases, so that the effects of no missed detection, no missed diagnosis, relatively small amount of data analysis, shortened inspection cycle are achieved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a probe group and a kit for detecting autoinflammatory disease-causing genes. Background technique [0002] Autoinflammatory diseases (autoinflammatory disorders, AIDs) are a group of hereditary inflammatory diseases, characterized by fever, rash, arthralgia, arthritis, eye lesions, etc., often accompanied by elevated inflammatory proteins. Autoinflammatory diseases are a large category of primary immunodeficiency diseases. As of 2018, 50 disease-causing genes have been discovered, and new genes are constantly being discovered. [0003] The second-generation high-throughput sequencing technology (Next Generation Sequencing, NGS) is also called high-throughput sequencing technology. The core idea of ​​the second-generation sequencing technology is sequencing by synthesis (Sequencing by Synthesis), that is, by capturing the The existing technology platforms mainly ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 王薇宋红梅伍建
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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