CN101833613A - A kind of oral microbial community database and its application - Google Patents

Abstract

The invention discloses an oral microbial community database. The oral saliva from persons under different healthy conditions is used as a sample source of the database, a sample saliva microbial community and the genome information thereof are acquired by carrying out DNA extraction, PCR (Polymerase Chain Reaction) amplification and high-throughput sequencing on the sample saliva, and the data is updated and maintained by a computer. The database can be used for the further analysis on the structural components of the oral microbial community of various persons, the risk assessment and prediction on the etiological factors of disease microorganisms and the assessment on human health status, and the screening of biomarkers and prognosis judgment indicators of disease diagnosis.

Description

A kind of oral microbial community database and its application

technical field

The present invention relates to the field of human symbiotic flora and public health. Specifically, through a new generation of high-throughput sequencing technology, the species and genome information related to the oral symbiotic flora represented by the saliva microbial flora of the human oral system are obtained. An Oral Microbiome Database Established in a State Population and Its Application to Disease Risk Assessment, Prediction, and Diagnosis.

Background technique

As an important part of human health, oral health affects the quality of life to a great extent, so the prevention of oral diseases is very important. Oral diseases, such as dental caries, periodontal disease, oral mucosal disease, oral cancer, oral manifestations of HIV, malaria, and oral and maxillofacial trauma, are important public health problems. The impact of oral disease on individuals and communities cannot be ignored due to excruciating pain, impaired function, and reduced quality of life. The World Health Organization has been committed to arousing people's widespread attention to oral health around the world, calling on and guiding governments, all walks of life and individuals to take effective measures to prevent the occurrence of oral diseases, aiming to improve the oral health of all mankind.

How to improve the level of oral health is not only to provide remedial treatment after the disease occurs, but more importantly, to find the cause of the disease, carry out disease prevention and control work, screen disease-sensitive groups, and carry out effective early prevention and early prevention. Diagnosis and early treatment. More and more studies have made people gradually realize that the mutual transformation of human diseases and health depends not only on the genetic genes carried by human chromosomes, but also on the interaction between a large number of symbiotic bacteria in the human body and with the human body itself. Scholars such as Peterson (Peterson, J., S. Garges, et al. (2009). "The NIH Human Microbiome Project." Genome Res 19 (12): 2317-2323), the symbiotic flora carried by human individuals The number is far more than ten times the number of cells that make up the human body itself. The birth of the symbiotic flora genome theory extends the meaning of "human" to the microbial community carried by human individuals. It is these symbiotic flora that endow human beings independent of themselves. Complex individual traits acquired by evolution. Such a "human" can be defined as a symbiotic combination of human cells and microbial cells, carrying two sets of genomes, one set is the human genome inherited from parents from a vertical relationship, which encodes about 25,000 genes; A large number of symbiotic flora that parasitize the human body later, as the second genome of the human body, are the main carriers of acquired endowments. The sum of their genetic information is called the "microbiome" or "metagenome". The number of encoded genes is around 1 million. The two sets of genomes coordinate and harmonize with each other, maintaining the health of the human body. In order to comprehensively analyze the human microbiome and reveal the relationship between human health and disease states and the human microbiome, the National Institutes of Health (NIH) announced on December 19, 2007 the official launch of a new genetic project - human symbiotic flora Genome Project (HMP), to characterize microorganisms in 5 areas of the human body known to host microbial communities, including the digestive tract, oral cavity, skin, nasal cavity, and female genitourinary system, to determine whether there is a common core among individuals The existence of human microbiome, so as to deepen the understanding of whether the change of human microbiome is related to human health status, and build the connection between the existence and change of microorganisms in specific areas of the human body and specific diseases.

Contents of the invention

The purpose of the present invention is to provide a database of oral microbial flora, which is the first to analyze the oral system microbial flora. This database uses 454 and Solexa next-generation high-throughput sequencing technology to conduct 16S rRNA and whole-genome sequencing of oral saliva microorganisms from different populations and different health states. Through supercomputer operations, it can be used for human disease risk prediction and Diagnostic Oral Microbiome Database. The database covers species information at different levels from bacteria, fungi and virus phylum to genus on the biological phylogenetic tree, and covers almost all genome-wide functional genes with detailed annotations. The computer storage data volume is as high as 10 ⁵ G above. This result has brought people's understanding of oral bacterial species to an unprecedented depth, providing more candidate genes and entry points for disease research, prediction and diagnosis.

Specifically, the oral microbial community database provided by the present invention is based on the oral saliva of people with different health status as the database sample source, through DNA extraction, polymerase chain reaction amplification and high-throughput sequencing, to obtain the sample saliva microbial community and The genome information is updated and maintained by computer.

The oral microbial community database, wherein, the crowd includes: adults, children, elderly people, and people with special disease sources; the health status of the crowd includes: complete health, dental caries, periodontal disease, pulp disease, halitosis, refractory root apex Inflammation, diabetes, patients after tumor radiotherapy and chemotherapy, AIDS, tuberculosis and viral hepatitis.

The oral microflora database, wherein the characteristics of the saliva microflora include oral bacteria, fungi and viruses.

The oral microbial community database, wherein the oral microbial genome information is obtained using 454 and Solexa high-throughput sequencers.

The oral microbial community database, wherein the genome information covers the 16S and 18S rDNA sequences for each oral microbial ribosome and the metagenomic sequences of all microorganisms.

The database constructed by the invention can be applied in risk assessment, prediction and auxiliary diagnosis of clinical oral diseases, AIDS, tuberculosis, viral hepatitis, obesity and diabetes.

Detailed ways

The oral microbial community database provided by the present invention is obtained by collecting saliva samples from various sources of diseases (such as dental caries, periodontal disease, refractory apicitis, etc.) and people in different health states, and constructing 16S rDNA and 18S rDNA tags The sequence library and the whole genome library, with the help of high-throughput sequencing, construct the database information of the saliva microbial community for each sample, which mainly includes the following steps (the following is an example for caries-sensitive groups):

1) Collect saliva from different populations, then lyse the cells with lysis buffer (50mM Tris, pH8.0, 50mM EDTA, 50mM sucrose, 100mM NaCl, 1% SDS), and extract the whole genome of microorganisms in saliva by the known high-salt method DNA (gDNA).

2) For the 16S rDNA tag sequence library, by synthesizing a sample-specifically labeled PCR probe with 6 bases, using a known polymerase chain reaction (PCR) method to amplify the 16S hypervariable region fragment of the sample DNA, and then After the PCR products were purified, a library was built and sequenced on a 454 machine.

3) By comparing the RDP database (The Ribosomal Database Project, http://rdp.cme.msu.edu/, which contains constantly updated ribosome-related data covering the bacterial and archaeal domains, it contains about 1358,426 items Sequence information: as of January 2010), while using a relatively mature joint analysis process (mainly including commercial software MOTHUR, ULUST, CD-HIT, RDP-CLASSIFIER, etc.), with the help of supercomputers (mainly Suguang Tianchao TC2600 blade server, Composed of Tiankuo A950r-F rack server and Tiankuo A620r-FX rack server, with a total computing capacity of 3.6 trillion times per second) to obtain species diversity and abundance information of samples.

4) For the whole genome (or metagenomic) database, directly purify the PCR product and build the library, and use Solexa to perform sequencing on the machine through specific markers. By comparing KEGG (KyotoEncyclopedia of Genes and Genomes, http://www.genome.jp/kegg/), CAZyme (Carbohydrate-Active enZYmes Database, http://www.cazy.org/) and other large-scale genome-wide and Metabolic pathway database for reconstruction of related metabolic pathways and annotation and analysis of functional genes.

5) On the basis of the above research, construct a saliva microbial community database, which contains all microbial flora (including the diversity and abundance of each flora) and their corresponding 16S rDNA and whole genome information in oral samples from different populations.

According to the above steps, those skilled in the art can realize.

The present invention builds a database of saliva flora for specific diseases and healthy people based on an in-depth understanding of oral flora. The database can be used to explore the pathogenic factors of disease microorganisms, and to explore possible biological screening markers and prognosis indicators for diseases. , which is conducive to discovering the hidden relationship between the internal and local flora of the disease.

Examples are given below for further description.

Example 1

1. Extraction of saliva DNA by high-salt method

1) Add 30 μl of proteinase K and 150 μl of 10% SDS to the saliva-buffer, and incubate overnight in a water bath at 53°C.

2) Add 400 μl of 5M NaCl and incubate on ice for 10 minutes, centrifuge at 13,000 rpm for 10 minutes at high speed, and take the supernatant.

3) Add 800 μl of isopropanol, incubate at room temperature for 10 minutes, and centrifuge at 13,000 rpm for 15 minutes at high speed.

4) Discard the supernatant, wash the precipitate three times with 500 μl of 70% ethanol, and dissolve in 30 μl of EB solution.

2. Construction of 16S rDNA tag sequence library

1) Amplify the V4-V5 hypervariable region with uniquely labeled primers

50μl PCR system includes:

10xbuffer 5μl

dNTP 4 μl

DNA polymerase 0.25μl

H ₂ O 36.75 μl

Primer pair 1μl*2

template 2μl

PCR reaction conditions:

94℃, 5min

72℃, 10min

2) Cut the gel to recover and purify the PCR products, and mix different samples in equimolar numbers

3) Build the library according to the 454FLX Titanium sequencing standard procedure (including blunt end filling, adapter ligation, small piece end removal, DNA library fixation, gap filling and single-stranded DNA library separation)

4) Perform on-machine sequencing according to the 454FLX Titanium sequencing standard (including emulsified PCR amplification, bead recovery and enrichment, and sequence primer linking and on-machine sequencing)

3. For whole-genome (or metagenomic) databases, after purification of the sample DNA, build the library directly according to the Solexa GAⅡ standard library building process (including nitrogen interruption, end fill-up, adding adapters at both ends, and cutting the gel to recover 300-400bp fragments) PCR amplification), the constructed DNA library was planted in the Flow cell on the ClusterStation, amplified by bridge PCR and hybridized with sequencing primers, and sequenced on SolexaGAII.

4. Data Analysis

16S rDNA sequence:

1) Quality screening of raw data (preliminary screening based on the quality evaluation value obtained from sequencing, length, ambiguous bases, and possible chimeras)

2) Perform OTU analysis (Operational Taxonomy Unit) on the screened sequences, and count the bacterial species diversity information.

3) Perform species identification on the screened sequences, study the species and abundance of bacteria at each taxonomic level, and look for statistical differences in the abundance of bacteria at different taxonomic levels from bacterial phylum to bacterial genus in disease samples and healthy samples; For samples from disease and healthy sources, conduct Unifrac (http://bmf2.colorado.edu/unifrac/index.psp) analysis based on the phylogenetic information of the entire microbial community, compare the differences in community structure, and monitor diseases based on the above basis and screening indicators.

Whole genome (or metagenomic) data:

1) Preliminary screening of metagenomic data (remove sequences containing ambiguous bases), and remove human genome source sequences by comparing with human reference genomes

2) Compare with metabolic databases including KEGG, CAZyme and other metabolic databases, annotate the sequence, reconstruct corresponding metabolic pathways and compare functional gene differences.

5. Due to the complex bacterial populations covered by oral microorganisms, the genome information is also huge. At present, the database has been updated, maintained and background managed by supercomputers. The access to the database information is based on the user name and password of the payment system to log in. The login website is www.microbialdb.org.

The above is a further description of the present invention in conjunction with specific implementation examples. It should be understood by those skilled in the art that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention also has various Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the claims and their equivalents.

Claims (6)

1. oral microbial community database, be to be the database sample source with different health status crowd's saliva of buccal cavity, extraction by DNA, polymerase chain reaction (PCR) amplification and high-flux sequence, obtain sample saliva microflora and genomic information thereof, carry out the renewal and the maintenance of data by computing machine.

2. oral microbial community database according to claim 1, wherein, the crowd comprises: adult, children, aged crowd and special disease source crowd; The population health state comprises: fully healthy, dental caries are sick, patient, acquired immune deficiency syndrome (AIDS), tuberculosis and virus hepatitis after the periodontosis, dental pulp disease, halitosis, intractable apical pericementitis, diabetes, tumor radiotherapy chemotherapy.

3. oral microbial community database according to claim 1, wherein, saliva microflora feature comprises oral bacteria, fungi and virus.

4. oral microbial community database according to claim 1, wherein employing 454 of oral microorganism genomic information and Solexa high-flux sequence instrument obtain.

5. oral microbial community database according to claim 4, genomic information are wherein contained the grand genome sequence at the ribosomal 16S of each oral microorganism, 18SrDNA sequence and all microorganisms.

6. the constructed application of database in risk assessment, prediction and the auxiliary diagnosis of clinical mouth disease, acquired immune deficiency syndrome (AIDS), tuberculosis, virus hepatitis, obesity and diabetes of claim 1.