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Probe set and reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes

A Hirschsprung and susceptibility gene technology, applied in the field of genetic engineering and molecular genetics, can solve problems such as lagging progress, high incidence, and hindering population quality, achieving solid theoretical basis and highly targeted effects

Active Publication Date: 2016-01-20
首都儿科研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The second-generation high-throughput sequencing technology (next-generation sequencing technology) has the advantages of fast, accurate, and low cost. It 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 inheritance. However, up to now, there have been no high-throughput probes, chips or kits specifically for the detection of pathogenic / susceptible genes of Hirschsprung and related diseases, which makes the progress of this disease-related field seriously lagging behind. High levels, genetic counseling and necessary prenatal interventions 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 reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes
  • Probe set and reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes
  • Probe set and reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: Design and preparation of the probe set of the present invention

[0066] 1. Screening of pathogenic / susceptible genes

[0067]Based on the previous research on the results of whole-exome level genetic screening of Hirschsprung population with the largest sample size in the world, combined with a large number of database searches, screening of patient gene sequencing results, and comparison of symptoms in mouse disease models Work, finally identified 172 pathogenic / susceptible genes, mainly including four categories: (1), 15 HSCR and related syndrome pathogenic genes: RET, EDNRB, SEMA3D, SEMA3C, NRG1, PHOX2B, SOX10, ECE1 , EDN3, GDNF, KIAA1279, L1CAM, NRTN, TCF4, ZEB2; (2), literature and RNASeq results suggest 19 genes highly related to human enteric nervous system development: NAV2, WWOX, KIAA0368, PLEKHA1, MAPK10, LARGE, SYN3 , GRIN2B, LRRTM4, SOX2, NCRNA00158 (LINC00158), DLX2, GFRA1, ARAF, GRB10, HOXA2, PHACTR4, TLX2, ZIC2; (3), 120 mutations involve...

Embodiment 2

[0073] Embodiment 2: Composition, preparation and use of the kit of the present invention

[0074] The kit for detecting the pathogenic / susceptible genes of Hirschsprung and related syndromes described in this embodiment is to carry out the molecular inheritance of the individual under examination by detecting the mutations of the above-mentioned 172 pathogenic / susceptible genes. Kits for medical diagnosis or risk prediction.

[0075] The components contained in the kit are: the probe set obtained in Example 1 (160ul, 150ng / ul), enrichment buffer (208ul), hybridization buffer (800ul), binding buffer (3.2ml), washing Solution 1 (9ml), rinse solution 2 (45ml), NaOH solution (0.1M, 1ml), Tris-HCl buffer (1M, pH7.5, 1.2ml), PCR reaction solution (580ul), TE buffer (800ul ). Wherein each buffer composition is as follows:

[0076] (1) Enrichment buffer (per 20ul):

[0077] Human cot-1 DNA (purchased from Invitrogen), 7ul; salmon sperm DNA (purchased from Invitrogen), 3ul;

[00...

Embodiment 3

[0123] Embodiment 3: verification of the use effect of the kit of the present invention

[0124] Using the kit of the present invention to detect 72 samples, the results confirmed that the capture rate of the target pathogenic / susceptible gene was satisfactory, more than 97.7% of the original short sequences could be compared back to the reference sequence of the target region, and the average effective The amount of sequencing data reaches 272.4Mb, and the average sequencing depth of the target region is 263X (see Table 1), which is much higher than the general genetic disease diagnosis requirements (generally 20X).

[0125] Table 1 The quality of sequencing data for pathogenic / susceptible genes

[0126]

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Abstract

The invention provides a probe set and a reagent kit used for detecting pathopoiesia / susceptibility genes of congenital megacolon and relative syndromes. At most 172 pathopoiesia / susceptibility genes related to diseases can be detected at the same time, and reference can be provided for affected individual molecular genetics diagnosis, mass survey of congenital megacolon high-incidence areas, screening of high-risk affected groups in congenital megacolon family members and corresponding genetic counseling or prenatal intervention.

Description

field of invention [0001] The invention belongs to the field of genetic engineering and molecular genetics, and specifically relates to a probe group and a kit for personalized capture and sequencing of pathogenic / susceptible genes of Hirschsprung and related syndromes. Background technique [0002] Hirschsprungdisease (HSCR), also known as intestinal aganglionosis, is the most common abnormality of the digestive tract in children that causes functional intestinal obstruction, and is a typical congenital abnormality of the enteric nervous system. The main clinical manifestations are delayed or no meconium excretion, followed by abdominal distension and intestinal obstruction. my country is a country with a high incidence rate. At present, the incidence rate of live births in my country is about 1 / 3,000, and males are significantly higher than females (about 4:1), that is, nearly 5,500 newborns will have this kind of birth defect every year, second only to congenital anorect...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q2600/166
Inventor 姜茜伍建李颀林朋龚勋张震肖萍苏琳李龙
Owner 首都儿科研究所
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