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Screening and application of feces gene marker

A gene marker and gene technology, applied in the field of biomedicine, can solve the problems of PD diagnosis with low sensitivity and specificity, poor specificity, and inconsistent research results.

Active Publication Date: 2019-04-19
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. Clinical symptoms of PD: Motor symptoms are the main basis for the diagnosis of PD at present. However, typical motor symptoms such as bradykinesia, muscle rigidity and resting tremor only appear when a large number of dopaminergic neurons are lost, and cannot be used as markers for early diagnosis
Non-motor symptoms such as hyposmia, REM sleep behavior disorder, and constipation can occur much earlier than motor symptoms, but the sensitivity and specificity for the diagnosis of PD are not high
[0005] 2. Neuroimaging: currently known imaging methods for PD diagnosis include positron emission tomography (PET), single-photon emission computed tomography (single-photonemission computed tomography, SPECT), magnetic resonance imaging (magnetic resonance imaging, MRI), etc., are difficult to popularize due to high price, poor practicality or poor specificity
But findings so far from blood and saliva samples are not quite the same
[0007] Therefore, a biomarker with high sensitivity and specificity that can be widely used in clinical PD diagnosis has not been found so far.

Method used

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  • Screening and application of feces gene marker
  • Screening and application of feces gene marker
  • Screening and application of feces gene marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 65 eligible PD patients and their healthy spouses were screened from 120 sporadic PD patients. The inclusion criteria of PD patients include: (1) patients with clinically confirmed primary Parkinson's disease, and the diagnostic criteria refer to the diagnostic criteria of Parkinson's disease in the British Brain Bank; (2) exclude atypical and secondary Parkinson's syndrome; ( 3) No history of stroke, dementia, or any serious nervous system disease; (4) No malignant disease, such as tumor, heart failure, etc.; (5) No other chronic diseases affecting intestinal flora: including diabetes, liver disease, etc. sclerosis, cardiovascular disease, etc.; (6) no autoimmunity, bleeding disease; (7) no serious intestinal disease, including irritable bowel syndrome, etc. Healthy spouses were required to have no medical history. Subjects taking antibiotics within 3 months of stool sample collection were excluded from this study. Finally, 40 PD patients (PD group, including 21 fema...

Embodiment 2

[0161] Fluorescent real-time quantitative PCR validation of gene markers

[0162] The 25 gene sequences screened by shotgun metagenomics sequencing were designed for Real-timePCR primers, and the primers were optimized and screened according to the product fragments, Tm values ​​and the population distribution of the target gene with primer 5 software. The specific primers are shown in Table 2 shown.

[0163] Table 2 Design of primers corresponding to target genes

[0164]

[0165]

[0166] 2. Standard preparation

[0167] Plasmid construction containing the gene of interest. Synthesize the target gene sequence. After synthesis, select the appropriate restriction site according to the characteristics of the gene sequence, connect it to the prokaryotic pET-28a(+) vector, and ensure the correct sequence of the plasmid through first-generation sequencing. The schematic diagram of plasmid construction is as follows Figure 4 shown.

[0168] 3. Real-time fluorescence qu...

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Abstract

The invention relates to the field of biomedicine, and particularly to screening and application of a feces gene marker based on high-flux metagenome sequencing analysis. According to the method, through extracting feces DNA for performing macro genome sequencing and ROC curve analysis by means of a shotgun method, the gene marker is screened, and furthermore the gene marker is verified through fluorescent real-time quantified PCR for obtaining consistency. According to the method, metagenome high-flux shotgun sequencing technical measurement is firstly performed in feces of Chinese PD patients, and a screening method for the feces gene marker is established; various different genes in the feces of the PD patient are found; 25 feces gene markers are screened as PD diagnosis markers for auxiliary diagnosis of the PD patient; and the method has important academic meaning and application prospect for enriching and further understanding the pathological mechanism of PD.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to the screening and application of feces gene markers based on high-throughput metagenomics sequencing analysis. Background technique [0002] Parkinson's disease (PD) is a common slowly progressive neurodegenerative disease in middle-aged and elderly people, which seriously affects the quality of life of patients. The main clinical features are motor symptoms, including resting tremor, muscle rigidity, bradykinesia, and abnormal posture and gait. With the acceleration of my country's population aging process, the incidence of PD is increasing day by day, and the number of cases is increasing year by year. At present, there are more than 2 million PD patients in my country. The main pathological changes of PD are abnormal aggregation of α-synuclein (a-syn), leading to the selective loss of dopaminergic neurons in the substantia nigra compacta of the midbrain and the formation of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B20/20G16B40/00
Inventor 肖勤钱逸维陈生弟杨晓东徐绍卿
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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