Oral metagenome data analysis method based on high-flux sequencing

A metagenomic and data analysis technology, applied in the field of bioinformatics, can solve the problem of inability to accurately explore the pathogenesis of primary Sjögren's syndrome, and achieve the effect of comprehensive identification data

Pending Publication Date: 2019-08-27
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, there are few comprehensive and comprehensive analyzes of the oral microbial flora of patients with primary Sjögren's syndrome, and even less use of metagenomics methods for analysis, so it is impossible to accurately explore the pathogenesis of primary Sjögren's syndrome

Method used

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  • Oral metagenome data analysis method based on high-flux sequencing
  • Oral metagenome data analysis method based on high-flux sequencing
  • Oral metagenome data analysis method based on high-flux sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1. Selection of primary Sjögren's syndrome group and control group

[0036] There was no significant difference in the average age between the primary Sjogren's syndrome group and the normal control group. The collected clinical cases were all female patients. In order to avoid the result error caused by gender selection, all the healthy volunteers were female. Compared with healthy controls, all samples of patients with primary Sjögren's syndrome met two or more of the three diagnostic criteria, and systemic diseases such as hepatitis, conjunctivitis, and diabetes were excluded. See Table 1 for details.

[0037]

[0038] The definition of primary Sjogren's syndrome in this study is in line with the new diagnostic criteria for SS proposed by the American College of Rheumatology (ACR) in 2012 based on the cohort data study of Sjögren's Syndrome International Clinical Collaboration Alliance (SICCA). In the experiment, patients with primary Sjögren's syndrome...

Embodiment 2

[0039] Example 2. Sample Collection

[0040] 30 minutes before collecting the saliva sample, advise to wash away the debris in the mouth with drinking water, and do not eat, drink, smoke or chew gum until the sample is collected. Use a saliva DNA sample collection tube to collect 1ml of the subject’s saliva according to the sampling instructions of the collector, mix it with saliva preservation solution 1:1, and store it at room temperature (the collector has prepared sealed tubes, protective solutions, etc., and does not need low-temperature storage , can be stored at room temperature, the protection of DNA is good, the DNA integrity is good, and there is little degradation).

Embodiment 3

[0041] Example 3. DNA extraction and PCR amplification

[0042] Take 250 mg of oral saliva samples at the end of the sample box respectively, and use The oral DNA Kit was used to extract the sample DNA, using Mobio Power DNA Clean-UpKit is used for DNA purification, and the purified genomic DNA is detected by 1% agarose gel electrophoresis. The design of the primers is specifically synthesized according to the region to be determined in the DNA. The primers are used to amplify the V4-V5 variable regions of 16SrRNA. The sequences of the primers are as follows:

[0043] 515F: 5'-barcode-GTGCCAGCMGCCGCGG)-3';

[0044] 907R: 5'-CCGTCAATTCMTTTRAGTTT-3';

[0045] PCR uses TransGenAP221-02: TransStart FastpfuDNA polymerase; 20μL reaction system: 5×FastPfu Buffer 4μL, DNA template 10ng, FastPfu Polymerase 0.4μL, dNTPs (2.5mM) 2μL, upstream and downstream primers (5μM) 0.4μL each;

[0046] Adopt ABI The 9700-type gene thermal cycler performs PCR amplification, and the amplifica...

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Abstract

The invention relates to an oral metagenome data analysis method. According to the data analysis method of the invention, an oral microflora of a sjogren syndrome patient is analyzed based on a high-flux sequencing facility; furthermore saliva microbe diversity of the primary sjogren syndrome patient is analyzed based on cluster analysis, diversity index analysis, evolution branch analysis and abundance analysis; the composition difference of the microflora in the oral cavity of the primary sjogren syndrome patient in China is evaluated; the substantial different flora is screened and a treatment guidance is supplied.

Description

technical field [0001] This application relates to the field of bioinformatics, in particular, to a method for analyzing oral metagenomic data based on high-throughput sequencing. Background technique [0002] Oral microbial flora belongs to the category of metagenomics, and its imbalance can cause many oral diseases, such as gingivitis, periodontitis, caries, oral cancer, orthodontic infection, implant infection, etc. At present, the research on oral microbial community mainly includes traditional separation and purification methods, physiological and biochemical methods, and DNA-based molecular biology methods. These methods need to screen out and identify oral microorganisms according to their growth characteristics. For most of the non-culturable microorganisms, humans cannot simulate their natural growth state and the relationship between species and the environment. Therefore, for many non-culturable microorganisms in the oral cavity, their physiological and biochemica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B20/30G16B50/00G16B40/00
CPCG16B20/30G16B50/00G16B40/00
Inventor 周爽王旻蔡叶
Owner CHINA PHARM UNIV
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