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A dna library for detecting pathogenic genes of idiopathic pulmonary fibrosis and its application

A DNA library and pulmonary fibrosis technology, applied in the field of DNA library, can solve the problems of indistinguishability, low throughput, and lack of specificity in inspection

Active Publication Date: 2019-09-10
汪道文
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both bronchoscopic lung biopsy and bronchoalveolar lavage cell analysis will bring pain to patients, and patients with poor basic conditions may not even tolerate it
Imaging and serological tests can help in the diagnosis of idiopathic pulmonary fibrosis, but these tests lack specificity and cannot completely distinguish idiopathic pulmonary fibrosis from other diseases such as similar connective tissue diseases
At the same time, the traditional gene detection method based on Sanger sequencing has the disadvantage of low throughput, and only one amplification region can be detected in one reaction, which cannot meet the requirements of multi-gene detection area and multi-sample detection timeliness

Method used

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  • A dna library for detecting pathogenic genes of idiopathic pulmonary fibrosis and its application
  • A dna library for detecting pathogenic genes of idiopathic pulmonary fibrosis and its application
  • A dna library for detecting pathogenic genes of idiopathic pulmonary fibrosis and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] 1. Reagents used in the method:

[0076] Ion AmpliSeq TM Library Kit 2.0, Ion PI TM Hi-Q TM OT2 200Kit, Ion PI TM Hi-Q TM Sequencing 200Kit, Ion Xpress Barcode Adapters 1-16Kit, Ion PI TM Chip v3

[0077] 2. Specimen collection and storage

[0078] (1) Specimen collection: The specimen is the peripheral blood of the patient. Blood is 5ml of venous blood taken routinely, treated with EDTA anticoagulant.

[0079] (2) Storage: It can be detected immediately, stored at 4°C for one week, and stored at -80°C for more than one week.

[0080] 3. Detection steps and result analysis:

[0081] (1) Extraction of genomic DNA of specimens: DNA extraction of specimens was carried out according to the operation instructions of the blood DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0082] (2) Super multiplex PCR amplification and library building of the target detection region: the whole exons of 92 genes involved in the present invention ar...

Embodiment 2

[0118] The primer pool of the present invention contains 81 idiopathic pulmonary fibrosis pathogenic genes newly discovered by the inventor, as shown in Table 1 above. The newly discovered 81 idiopathic pulmonary fibrosis pathogenic genes come from the inventor's research accumulation and family investigation over the years. The inventors first searched the known pathogenic genes of idiopathic pulmonary fibrosis through the KEGG signaling pathway and the homologous gene library to find the homologous genes of the known pathogenic genes and the key genes on the same pathogenic pathway. Then, targeted high-throughput sequencing of candidate disease-causing genes was carried out in the large-scale case sample library of Chinese yellow-race patients accumulated over the years, and bioinformatics analysis was carried out to screen and find disease-causing mutations. Cases with pathogenic mutations were screened for follow-up and further family analysis, and the entire family of the...

Embodiment 3

[0120] Using the method provided by the present invention and using 92 gene libraries, the gene detection was carried out on 200 patients with idiopathic pulmonary fibrosis but whose etiology was not clear. Analysis of 85 cases of idiopathic pulmonary fibrosis carrying gene mutations found that 68 patients carried two or more mutations, the average age of onset was 58.7 years, and only one mutation was carried by patients There were 17 cases with an average age of onset of 63.1 years. Moreover, patients with only one mutation have significantly better clinical symptoms and pulmonary diffusion function than those with two or more mutations.

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Abstract

The invention discloses a DNA library for detecting idiopathic pulmonary fibrosis pathogenic genes with a targeted high-throughput semiconductor sequencing technique and an application of the DNA library. Specifically, a primer pool is designed according to 92 idiopathic pulmonary fibrosis pathogenic genes, super-multiplet PCR amplification is performed on sample genome DNA, an amplification product is sequenced with the high-throughput semiconductor sequencing technique, pathogenic mutations are searched, and a theoretical basis of genetics and molecular biology is provided for clinical diagnosis. The DNA library has the characteristics of being accurate, rapid, flexible and low in cost. Involved 92 gene detection regions can be used for detecting various types of idiopathic pulmonary fibrosis related to known genes and have great significance and clinical value in diagnosis and differential diagnosis of the idiopathic pulmonary fibrosis.

Description

technical field [0001] The invention relates to a DNA library for detecting and diagnosing pathogenic genes of idiopathic pulmonary fibrosis through targeted high-throughput semiconductor sequencing technology and its application. Specifically, according to the pathogenic gene of idiopathic pulmonary fibrosis, supermultiplex PCR primers that can cover the exons and adjacent regions of the above genes are designed, and supermultiplex PCR amplification is performed on the sample genomic DNA. Sequencing technology performs sequencing to find pathogenic mutations, clarify the genetic etiology of idiopathic pulmonary fibrosis, and provide a theoretical basis for genetics and molecular biology for clinical diagnosis. It belongs to the gene detection technology in the clinical detection technology in the field of biomedicine. Background technique [0002] Idiopathic pulmonary fibrosis (IPF) is an interstitial pneumonia of unknown cause, confined to the lungs, progressive fibrosis i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/6883C40B40/08
Inventor 汪道文熊维宁李宗哲邓燕晗刘娟
Owner 汪道文
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