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Marker related to generation of congenital megacolon and application of marker

A Hirschsprung's disease, marker technology, applied in the field of genetic engineering, to achieve the effect of high throughput, simple and fast operation, and high sensitivity

Active Publication Date: 2021-09-17
SOOCHOW UNIV AFFILIATED CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In humans, hsa_circ_0000740 is located at chromosome 17 negative strand 5364258 to 5365866, and there is no record in the prior art that hsa_circ_0000740 is related to the occurrence of Hirschsprung’s disease

Method used

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  • Marker related to generation of congenital megacolon and application of marker
  • Marker related to generation of congenital megacolon and application of marker
  • Marker related to generation of congenital megacolon and application of marker

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Screening of the present embodiment, Hirschsprung's disease-related gene markers, comprising the steps of:

[0054] (1) Sample collection: Following informed consent, and under the premise of the Ethics Committee agreed to collect four cases of Hirschsprung Hirschsprung's patients and normal intestinal tissue;

[0055] (2) Preparation of RNA samples: treated with Rnase R (. Epicentre, Inc) Step (1) Total RNA was digested tissues collected, removed and circular RNA enriched linear RNA;

[0056] (3) reverse transcribed and labeled: using the random primer method (Arraystar Super RNALabeling Kit; Arraystar) in step (2) RNA was reverse transcribed and labeled cRNAs obtained;

[0057] (4) hybridization: The labeled cRNAs hybridize to Arraystar Human circRNA Array v2 (8x15K, Arraystar) on the slide after washing, using an Agilent scanner G2505C chip is scanned;

[0058] (5) Data processing: Agilent feature extraction software (11.0.1.1 Version) analysis array image obtained using...

Embodiment 2

[0060] In this embodiment, samples were extracted RNA, including the steps of:

[0061] (1) Sample: Following informed consent, and under the premise of the Ethics Committee agreed to collect 15 cases of patients with Hirschsprung megacolon tissue and normal intestinal tissue;

[0062] (2) RNA Sample Extraction:

[0063] 1) comprises a reagent TRIZOL reagent (Invitrogen life technologies), chloroform (Shanghai Chemical Reagent Co.), isopropanol (Shanghai Chemical Reagent Co., Ltd.), 100% ethanol (Shanghai Chemical Reagent Co., Ltd.), 75% ethanol (in DEPC-treated formulated water), water and free RNA enzyme-free RNA enzyme glycogen (Invitrogen lifetechnologies);

[0064] 2) Take 70mg tissue samples, 1mL of TRIZOL reagent was added, and homogenized with an electric homogenizer;

[0065] 3) The homogenate samples were incubated at 20 ℃ 5min, 1mL of each sample homogenate TRIZOL reagent was added 0.2mL of chloroform, the tube cap tightly, shaken vigorously by hand for 15 seconds after...

Embodiment 3

[0070] This embodiment will be cDNA synthesized using RNA prepared in Example 2.

[0071] Reagents: RNA inhibitors (Epicentre), SuperScript TM III Reverse Transcriptase (Invitrogen), 5 × RT buffer (Invitrogen), 2.5mM dNTP mixture (dATP, dGTP, dCTP and dTTP each 2.5mM) (HyTest Ltd) and Primer (Invitrogen Biotechnology Co., Ltd).

[0072] Instrument: clean bench (Xun Haibo Medical Equipment Factory Co., Ltd.), DK-8D type electric thermostat bath (Hysen letter Experimental Instrument Co.), and GeneAmp PCR System 9700 (Applied Biosystems).

[0073] During operation comprising the steps of:

[0074] (1) An annealing mixture:

[0075]

[0076] (2) The mixture was placed in a water bath at 65 deg.] C 5min, placed on ice for 2min, after centrifugation, the centrifuge tube in the RT reaction solution were successively added:

[0077]

[0078] (3) mixed at 37 ℃ 1min, pipette pipetting gently mixed, incubated for 50 deg.] C 60min, 70 ℃ incubated for 15min to inactivate the enzyme, the cD...

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Abstract

The invention relates to a marker related to generation of congenital megacolon and an application of the marker. The marker of the congenital megacolon comprises hsa_circ_0000740. According to the marker related to generation of the congenital megacolon and the application of the marker, a DNA diagnosis technology is utilized, a high-specificity hsa_circ_0000740 is used as the marker of the child congenital megacolon for the first time, and the adopted detection product not only can make the diagnosis of the congenital megacolon meet the requirements of strong specificity and high sensitivity, but also has the comprehensive advantages of simple and rapid operation, no intervention, high flux, low cost and the like.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, and it is related to a marker related to the occurrence of congenital giant colon. Background technique [0002] Hirschsprung Disease (HSCR) is a birth defective disease in children with intestinal nerve development. The pathological mechanism is intestinal neural crest cell migration and differentiation of intestinal neurons, which leads to sustained spasm in the lack of intestinal nerve. It is one of the common congenital intestinal diseases in children. [0003] The timely diagnosis and treatment of congenital giant colon can reduce the dangers of congenital giant colitis and achieve good prognosis. The preoperative diagnostic method is mainly a barium enema, a rectal biopsy, rectal pressure, to determine whether it is necessary to implement "giant junction radical". At present, the ruthenium is the most important diagnostic method. After the barium is natural, the expansion and stenosis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/113
CPCC12Q1/6883C12Q1/686C12Q2600/178C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 方芳黄顺根郭万亮
Owner SOOCHOW UNIV AFFILIATED CHILDRENS HOSPITAL