CN106636423A - Analysis method for diversity of oral cavity flora and disease-associated flora marker - Google Patents
Analysis method for diversity of oral cavity flora and disease-associated flora marker Download PDFInfo
- Publication number
- CN106636423A CN106636423A CN201710019638.3A CN201710019638A CN106636423A CN 106636423 A CN106636423 A CN 106636423A CN 201710019638 A CN201710019638 A CN 201710019638A CN 106636423 A CN106636423 A CN 106636423A
- Authority
- CN
- China
- Prior art keywords
- pcr
- primer
- sample
- flora
- primer pair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an analysis method for the diversity of oral cavity flora based on a second-generation high throughput sequencing technology and a disease-associated flora marker. The method comprises the following steps of (1) extracting a DNA of a to-be-detected saliva sample; (2) preparing a PCR reaction system, and carrying out PCR amplified reaction of V1-V2 regions of bacteria 16S rDNA, wherein the PCR reaction system comprises a PCR amplification template, a universal primer pair and the like; (3) carrying out detection of a target segment of a PCR product and purification of the PCR product; (4) carrying out second-generation high throughput sequencing on the purified PCR product and detecting the species of microorganisms in the saliva sample; (5) sequencing by using an Ion Torrent PGM sequencing system; and (6) carrying out bioinformatics analysis by using a FLASH, QIIME, usearch61. The diversity and the difference of the oral cavity flora of a patient are detected through the second-generation high throughput sequencing technology, so that the analysis method has the advantages that the composition and the functions of the flora can be more deeply analyzed in more detail by using the new-generation high throughput technology in comparison with a traditional flora detection method.
Description
Technical field
The present invention relates to field of biological technology detection, specifically, the invention provides a kind of be based on two generation high-flux sequences
The multifarious technology of oral cavity flora and method in the detection Disease body of technology.
Background technology
In oral cavity hundreds of millions of microorganisms and its metabolite human energy metabolism, absorption of nutrient ingredients, it is congenital and
The aspects such as acquired immunity, stomach and oral cavity function are played an important role, once commensalism symbiosis between host and oral microorganism
Stable state be broken, various human diseases will be induced.Have now realized that oral microorganism and human health and disease
Substantial connection, only from mankind itself's angle goes out to send research human diseases far from enough, it has to be taken into account to common with mankind's commensalism
The effect of raw oral microorganism.
Be applied at present human oral cavity microorganism diversity and differentiation research method it is more, mainly have traditional detection
The methods such as method, structure gene library method, genetic fingerprint profile technique, molecular hybridization and DNA sequencing technology.It is wherein traditional
Microorganism detection method is with various medium culture separate microorganisms, and to be tried by Grain stain, biochemistry and serology
The method such as test to determine microbe species, micro organism quantity is determined by methods such as doubling dilution, colony countings.But due to passing
In system microbial culture technique, the enrichment or decay of bacterial strain is inevitable, and original Tiny ecosystem structure is changed, and this can cause to grind
Study carefully result and there is relatively large deviation.
In sum, apply and develop the detection method for being directed to the diversity and otherness of Disease oral cavity flora, energy
The technical support and theoretical foundation of the correlation being enough better understood by for people between oral microorganism and disease.
The content of the invention
The purpose of the present invention is to provide a kind of diversity and the detection of otherness for patients with gastric cancer oral cavity flora
Method.
It is also another object of the present invention to provide a kind of by designing specific primer (two sections of artificial synthesized oligonucleotides
Sequence a, primer is complementary with a DNA profiling chain of genes of interest one end, and another primer is another with the genes of interest other end
One DNA profiling chain is complementary), it is adaptable to the diversity of multiple types Disease oral cavity flora and the detection method of otherness.
A further object of the present invention is the mutual pass being better understood by for people between oral microorganism and disease
The technical support and theoretical foundation of system.
1. in the present invention, the primer pair (upstream and downstream primer is referred to as pair of primers) is not limited to by the sequence institute
Show two single strand dnas composition primer pair, as long as can specific amplification inspection in need it is microbe-derived
Primer pair.
The invention provides the specific primer pair of a kind of diversity of detection patients with gastric cancer oral cavity flora and otherness, its
Be characterised by, the primer pair be can specific amplification inspection in need microbe-derived primer pair.The primer
To the primer pair being made up of two single strand dna sequences.The primer pair
Upstream primer is：5’-AGAGTTTGATCMTGGCTCAG-3’；
Downstream primer is：5’-GCTGCCTCCCGTAGGAGT-3’.
Described primer pair, for biotechnology and detection field, by two generation high throughput sequencing technologies, precisely detects stomach
The diversity and otherness of cancer patient's oral cavity flora.
2. the present invention also provides a kind of method for judging the diversity and otherness of oral cavity flora in Disease body to be measured,
It is characterized in that comprise the following steps,
(1) saliva sample to be measured is provided；
(2) from the saliva sample, its sample DNA is extracted；
(3) PCR reaction systems are configured, enters performing PCR reaction；
(4) for PCR product carries out the detection of target fragment；
(5) for the PCR primer of purposeful fragment carries out PCR primer purifying；
(6) PCR primer, as detection object, is carried out micro- in saliva sample with two generation high throughput sequencing technologies with step 5
The detection of biological species；
(7) for two generation sequencing results carry out data analysis and process.
3. the extracting method of the collection of saliva sample of the present invention and saliva sample DNA is：
(1) acquisition method of the saliva sample is specially：
1) time be preferably selected in the 9 of the morning:00-11:Between 00, and should not take food within an hour before sampling；With
Clear water is gargled；
2) after gargling 5 minutes, in 30 minutes collect 5ml salivas in the sampling pipe of a 50ml (if conditions permit,
In sampling process, it is ensured that adopt foster pipe and be placed on ice), remind patient to cough mucus；
3) after the completion of sampling, 4 DEG C, 2600g is centrifuged 15min；Supernatant is abandoned, the lid of sampling pipe is tightened, carry out information note
Record, including the person's of being sampled name, admission number and sampling time.
(2) the saliva DNA extraction method is specially：
With the saliva sample of centrifuged deposit, the extraction that sample DNA is carried out with improved CTAB extraction process is selected.
1) lid is opened, the precipitation in sampling pipe is transferred in 1.5 milliliters of Ep pipes with liquid-transfering gun, 12000 rotating speeds, from
The heart 5 minutes.
2) 2 × CTAB stirring concussions for adding 300 microgram, 65 DEG C of preheatings are suspension.
3) 95 DEG C of heating water baths 8 minutes after EP pipes winding sealed membrane.
4) imitative (the removing layer) mixed liquor of 300 microlitres of phenol, vibration is added to mix, 10000 rotating speeds are centrifuged 5 minutes；
5) by supernatant 200 microlitres move in the sterile centrifugation tube of new 1.5 milliliter, add the molten of 3 times of volumes (600 microlitres)
Glue, vibration is mixed, and at twice (every time about 400 microlitres) move into siliceous post, stands 2 minutes, and 12000 rotating speeds are centrifuged 30 seconds；
6) liquid is abandoned, adds about 400 microlitres of cleaning solution, 12000 rotating speeds to be centrifuged in siliceous post 30 seconds；
7) previous step is repeated；
8) waste liquid in recovery tube is abandoned, blank pipe centrifugation, 12000 rotating speeds are centrifuged 2 minutes；
9) siliceous post is moved into 1.5 milliliters of new sterile centrifugation tubes, adds 60 microlitres of TE eluents, (TE eluents need to be carried
Front 65 DEG C of water-baths), 12000 rotating speeds are centrifuged 30 seconds；
10) the 60 microlitres of liquid collected in centrifuge tube, as DNA masterplates, in -20 DEG C of Refrigerator stores.
4. the judgement of DNA content of the present invention is specially：
(1) DNA to extracting carries out NanoDrop2000 spectrophotometers and detects its purity, finds 1.6 ＜ OD260/
280 ＜ 2.0, the equal nanograms of ＞ 10 of extraction DNA concentration/microlitre.
(2) pcr amplification reaction is substance PCR reactions, and reaction system is included：
10 × buffer, forward primer and reverse primer, DNA profiling, dNTP, Taq enzyme, sterile purified water.The DNA of extraction
For template, with described primer, fluorescent quantitative PCR is carried out.
(3) reaction system of 50 microlitres of the PCR reactions described in includes：
| Reagent | Usage amount | Final concentration |
| Taq enzyme solution | 0.8 microlitre | |
| Upstream primer (10 micro- rub) | 0.5 microlitre | 0.1 micro- rubs |
| Downstream primer (10 micro- rub) | 0.5 microlitre | 0.1 micro- rubs |
| 10 × buffer solution | 5 microlitres | |
| DNA profiling | 2 microlitres | |
| DNTP solution | 4 microlitres | |
| Sterile purified water | 37.2 microlitres | |
| Cumulative volume | 15 microlitres |
Reaction system is total to：50 microlitres.
(4) the pcr amplification reaction condition described in is：
(5) target fragment of the PCR reactions described in carries out detecting that the method for using is agarose gel electrophoresis method for detecting.
(6) described in PCR primer purifying specific implementation step be：
1) during PCR reactant liquors to be moved to the centrifuge tube of clean 1.5 milliliters, add 240 microlitres Buffer B3 (from-
20 degree of refrigerators take out), fully mix.
2) mixed liquor is all moved into adsorption column, after standing 2 minutes, 9000 × g is centrifuged 30 seconds, and filter liquor is added again
In adsorption column, pillar is crossed again, adsorption column is put into same collecting pipe.
3) 500 microlitres of Wash Solution are added in adsorption column, 9000 × g is centrifuged 30 seconds, in discarding collecting pipe
Liquid, adsorption column is put into same centrifuge tube, and is repeated once.
4) suction attached column and collecting pipe are put into centrifuge, 9000 × g is centrifuged 1 minute.
5) 30 microlitres of TE are added in adsorbed film central authorities, is stored at room temperature 2 minutes, 9000 × g is centrifuged 1 minute.By the DNA for obtaining
It is placed in -20 degree to preserve.
5. the basic step of two generations sequencing of the present invention is：
(1) sequencing library builds
1) 79 microlitres of DNA profiling (totally 100 nanogram) the mix reagent composition in the pipe of 1.5 milliliters of low absorption is taken, fully
It is incubated at room temperature after mixing 20 minutes.
2) useReagent purifying the DNA of filling-in, with the magnetic bead of 1.8 times of volumes to sample
Purified in this.
3) P and A joints, the mix reagent in the pipe of 0.2 milliliter of low absorption are linked in the end reparation of DNA and fragment
Composition, enters again performing PCR reaction after fully mixing.
4) useReagent purifying the DNA of filling-in, with the magnetic bead of 1.0 times of volumes to sample
Purified in this.
5) the quantitative library concentration of quantifying PCR method, with the method for quantitative fluorescent PCR for the library for building carry out it is fixed
Amount, and library is diluted to by 100 picomoles according to quantitative result.
(2) prepared by template
1) instruments of Ion one Touch 2 prepare, and after instrument power source is opened, are initialized.In initialized process
In, note the volume of waste liquid collected in waste collection pipe.
2) add Ion One Touch Oil and Ion PGM OT2 Recorvery Solution, add these examinations
Should sufficiently by the reverse mixing of reagent before agent.
3) amplification reaction solution prepares, and according to Laboratory Manual, amplification reaction solution is sequentially added in reaction vessel, and runs
Mix 3 times.
4) Ion one Touch 2 are run, the kit class for selecting this sequencing corresponding is should be noted that when instrument is run
Type：Ion PGM Template OT2 400 Kit.
(3) template enrichment
1) the Sphere Particles of Ion PGM Template OT2 400 are reclaimed, according to what is noted in operating instruction
Problem, ISP templates are reclaimed.
2) Melt-off solution is configured, takes 1.5ml centrifuge tubes, configured by operating instruction.
3) Dynabeads MyOne Streptavidin C1 Beads are prepared, by Dynabeads MyOne
After Streptavidin C1 Beads fully shakings, 13 microlitres are drawn in 1.5 milliliters of centrifuge tubes, be placed on magnetic frame, by wine
After essence is taken out, Dynabeads MyOne Streptavidin C1 Beads Wash Solution 130 μ l, Ran Houchong are added
Concussion is divided to mix.
4) prepare template and be enriched with eight platoons, sequentially add the reagent in 8 holes according to operating instruction.
5) ES is run, eight platoons is put into groove, 10 microlitres of neutralization buffer is then added in collecting pipe.
6) after end of run, collecting pipe is removed from instrument immediately, then mixes the abundant pressure-vaccum of reagent.
(4) machine sequencing on
1) open after PGM sequenators, PGM washings are carried out first.The deionized water of 250 milliliters of 18M Ω is added in W1,
Then washing button is pressed.
2) initialization of PGM is carried out, according to operating instruction, corresponding reagent will be added in W1, W2, W3, it is clean by one
Chip initialized.When being initialized, it should be ensured that the types of agents of selection matches with this sequencing.
3) prepare dNTP solution, 20 microlitres of dNTP solution is added in the centrifuge tube of each 50ml, then change new
Sipper, screws.
4) program setting is sequenced, it is then used according to this sequencing with the server corresponding to computer to access sequenator
Reagent, consumptive material, and the result required by subsequent analysis, carry out the setting of sequencing program.
5) inspection of chip, takes a brand-new chip, then does chip detection, if passed through, illustrates that chip is not asked
Topic, can carry out subsequent experimental procedure.
6) loading template prepares, and Control Ion Sphere Particles is added in all of ISP, as right
According to.Sequencing primer is added, enters performing PCR reaction, connect the two.In the enzyme for adding 3 microlitres, 5 minutes are then stored at room temperature
.
7) chip is processed, and the chip for finishing chip detection is removed from sequenator, and the liquid of the inside is suctioned out.
8) chip loading, is slowly screwed in 30 microlitres of sample in chip with pipettor, is then carried out on rejection tablet machine
Sample holes are towards inner-outer-inner three rejection tablets.
9) sequencing program is run, the good chip of loading is placed on sequenator, operating display sets before selection
Sequencing program, is sequenced.
6. primer of the present invention is in field of biological technology detection, there is provided one kind is based on two generation high throughput sequencing technologies,
The method of the diversity and otherness of detection Disease oral cavity flora.In the above-mentioned methods, the sample to be tested can be cancer of the stomach
Patient and the saliva sample of normal person.Such as cancer of the stomach, ephrosis.
On the basis of two generation high-flux sequences, devising can expand the universal primer of multiple-microorganism to the present invention, build
The method of diversity and otherness that Disease oral cavity flora is detected based on two generation high throughput sequencing technologies is found.With routine
Method is compared, and the method for the present invention is to have carried out V1- to the DNA that the oral cavity flora in patient's body is extracted by round pcr
The amplification in V2 regions, is studied the diversity and otherness of the oral cavity flora in patient's body, and is utilized
Iontorrent platforms are sequenced, and sequencing just can be completed to the sample of 100 or so within only two days, greatly save the time.
Description of the drawings
Fig. 1 is relative abundance histogram of the difference species in each sample in patients with gastric cancer group and healthy population；
Fig. 2 is the flora biological marker of the variance analysis of different ecotone microorganisms in patients with gastric cancer group and healthy population
Thing figure；
Fig. 3 is the PCoA figures of different ecotone microbiologic populations difference in CKD and healthy population；
Fig. 4 is the flora biomarker of different ecotone microbiologic populations difference in CKD and healthy population
Figure；
Specific embodiment
In embodiments of the invention 1, the sample to be tested is systemic disease patient's saliva sample, specially normal person
Saliva sample 32, the saliva sample of patients with gastric cancer 17.Disease sample 49 is surveyed altogether.
Using said method, the extraction of DNA is carried out to the oral cavity flora in different patient's bodies, and the DNA of extraction is carried out
PCR reacts, and to the product of PCR reactions row agarose gel electrophoresis are entered, and then carries out PCR primer purifying, library is built, in preparation
Machine is sequenced, and follows the steps below successively：
1. saliva sample sampling method
The sampling of saliva sample is carried out in strict accordance with test operation flow process.
2. the extracting method of saliva DNA
The extraction of saliva DNA is carried out with CTAB methods.
The judgement of 3.DNA contents
DNA to extracting carries out the spectrophotometers of NanoDrop 2000 and detects its purity, finds 1.6 ＜ OD260/
OD280 ＜ 2.0, the equal nanograms of ＞ 10 of extraction DNA total amounts/microlitre.
4. the selection of the specific primer of bacterium
According to existing primer, the Tm values of primer, melting temperature during PCR response procedures is designed.
5.PCR amplification reaction systems
Enter the configuration of performing PCR reaction system in strict accordance with reagent proportioning.
6.PCR amplified reaction programs
Enter the setting of line program in strict accordance with pcr amplification reaction program.
The specific detection of 7.PCR products
Detect target fragment whether in 355bp or so using agarose gel electrophoresis.
The purifying of the PCR primer after the specific amplification in 8.V1-V2 areas
In strict accordance with test operation flow process, the purifying of PCR primer is carried out.
9. it is quantitative accurate DNA concentration to be carried out using QUBIT
The sample of 60-90 or so is carried out into the accurate quantification of DNA concentration, then according to the Cmin for measuring, by institute
Some samples carry out the dilution of same concentration.
10. sequencing library builds
PCR product after purification is added in EP pipes according to experimental implementation flow process, the connection of joint is carried out, end is repaiied
It is multiple etc..
It is prepared by 11. templates
After instrument washing prepared by template, the preparation of template is carried out.
12. templates are enriched with
After the machine that template is enriched with is opened, first run in advance once.Then, by collecting pipe, neutralization buffer is added,
Operation machine.
Machine sequencing on 13.
By the template after the completion of enrichment, carry out adding joint, add after sequencing reaction primer, carry out chip loading, then set
Sequencing reaction program, carries out machine sequencing.
14. carry out bioinformatic analysis using QIIME.
(1) initial data pretreatment
Initial data obtains high-quality Reads sequences through going joint, low quality filtration etc. to process, and is then combined with just
Reversely Reads, obtains 16SrDNA V1-V2 areas amplification subsequence.Merge positive direction reads using FLASH softwares, the software can
Fast and accurately to detect the Overlap sequences of forward and reverse reads, and sequence output after merging, its accuracy is 99%
More than.
(2) merging data distribution of lengths and statistic of attribute
Data carry out charge analysis using FastQC softwares after merging, the software from initial data count on the quality of data,
The information such as sequence length, GC preferences, redundancy show in the form of a web page, can intuitively find out the charge information of data.
(3) preparation of configuration file
Contain in configuration file mixing amplicon each sample important information, such as sample names, sequence label, draw
Thing sequence, sample process, pickup area, acquisition time etc..
(4) separating sample sequence
According to configuration file sequence label information separating sample, each sample essential information is counted.
(5) chimera is removed
Chimera sequence is removed using denovo modes, chimera checks that software is usearch61.
(6) screening operation taxon (Operational Taxonomic Units, OTUs)
All detached high quality samples sequences are done into cluster analysis, cluster can gather the sequence of certain homology for one
Class (acquiescence homology is 97%), each OTU represents a species.
(7) taxology composition analysis
Using RDP graders and Greengenes reference data sets.
(8) diversity analysis (alpha diversity analysis) in sample
Mainly sparse tracing analysis.
(9) diversity analysis (beta diversity analysis) between sample
The distance matrix that data are produced can be by principal coordinate analysis (Principal Coordinate Analysis, PCoA)
Visualization.
(10) LEfSe analyses
The relative abundance histogram of LDA score value histograms, microbiologic population's difference classification chart and difference characteristic.
The saliva sample 17 that disease sample is patients with gastric cancer, the saliva sample of normal person 32 are surveyed altogether in this experiment.Altogether
Survey disease sample 49.After Quality Control and analysis are carried out to sequencing result with DAS, group between different samples is drawn
Fall the difference (see Fig. 1) of microorganism, and the analysis of different classifications sample microbiologic population difference is carried out to result, so as to find
Corresponding biomarker (see Fig. 2).
In embodiments of the invention 2, the sample to be tested is saliva sample, the specially saliva sample 330 of normal person
Example, the saliva sample of CKD patient 166.
Using said method, the extraction of DNA is carried out to the oral cavity flora in different patient's bodies, and the DNA of extraction is carried out
PCR reacts, and to the product of PCR reactions row agarose gel electrophoresis are entered, and then carries out PCR primer purifying, library is built, in preparation
Machine is sequenced, and follows the steps below successively：
1. saliva sample sampling method
The sampling of saliva sample is carried out in strict accordance with test operation flow process.
2. the extracting method of saliva DNA
The extraction of saliva DNA is carried out with CTAB methods.
The judgement of 3.DNA contents
DNA to extracting carries out the spectrophotometers of NanoDrop 2000 and detects its purity, finds 1.6 ＜ OD260/
OD280 ＜ 2.0, the equal nanograms of ＞ 10 of extraction DNA total amounts/microlitre.
4. the selection of the specific primer of bacterium
According to existing primer, the Tm values of primer, melting temperature during PCR response procedures is designed.
5.PCR amplification reaction systems
Enter the configuration of performing PCR reaction system in strict accordance with reagent proportioning.
Pcr amplification reaction program
Enter the setting of line program in strict accordance with pcr amplification reaction program.
The specific detection of 7.PCR products
Detect target fragment whether in 355bp or so using agarose gel electrophoresis.
The purifying of the PCR primer after the specific amplification in 8.V1-V2 areas
In strict accordance with test operation flow process, the purifying of PCR primer is carried out.
9. it is quantitative accurate DNA concentration to be carried out using QUBIT
The sample of 60-90 or so is carried out into the accurate quantification of DNA concentration, then according to the Cmin for measuring, by institute
Some samples carry out the dilution of same concentration.
10. sequencing library builds
PCR product after purification is added in EP pipes according to experimental implementation flow process, the connection of joint is carried out, end is repaiied
It is multiple etc..
It is prepared by 11. templates
After instrument washing prepared by template, the preparation of template is carried out.
12. templates are enriched with
After the machine that template is enriched with is opened, first run in advance once.Then, by collecting pipe, neutralization buffer is added,
Operation machine.
Machine sequencing on 13.
By the template after the completion of enrichment, carry out adding joint, add after sequencing reaction primer, carry out chip loading, then set
Sequencing reaction program, carries out machine sequencing.
14. carry out bioinformatic analysis using QIIME.
(1) initial data pretreatment
Initial data obtains high-quality Reads sequences through going joint, low quality filtration etc. to process, and is then combined with just
Reversely Reads, obtains 16SrDNA V1-V2 areas amplification subsequence.Merge positive direction reads using FLASH softwares, the software can
Fast and accurately to detect the Overlap sequences of forward and reverse reads, and sequence output after merging, its accuracy is 99%
More than.
(2) merging data distribution of lengths and statistic of attribute
Data carry out charge analysis using FastQC softwares after merging, the software from initial data count on the quality of data,
The information such as sequence length, GC preferences, redundancy show in the form of a web page, can intuitively find out the charge information of data.
(3) preparation of configuration file
Contain in configuration file mixing amplicon each sample important information, such as sample names, sequence label, draw
Thing sequence, sample process, pickup area, acquisition time etc..
(4) separating sample sequence
According to configuration file sequence label information separating sample, each sample essential information is counted.
(5) chimera is removed
Chimera sequence is removed using denovo modes, chimera checks that software is usearch61.
(6) screening operation taxon (Operational Taxonomic Units, OTUs)
All detached high quality samples sequences are done into cluster analysis, cluster can gather the sequence of certain homology for one
Class (acquiescence homology is 97%), each OTU represents a species.
(7) taxology composition analysis
Using RDP graders and Greengenes reference data sets.
(8) diversity analysis (alpha diversity analysis) in sample
Mainly sparse tracing analysis.
(9) diversity analysis (beta diversity analysis) between sample
The distance matrix that data are produced can be by principal coordinate analysis (Principal Coordinate Analysis, PCoA)
Visualization.
(10) LEfSe analyses
The relative abundance histogram of LDA score value histograms, microbiologic population's difference classification chart and difference characteristic.
The common test sample sheet 496 of the implementation case, wherein 330, normal person's sample, CKD 166.With data point
Analysis software is carried out after Quality Control and analysis to sequencing result, draws the difference (see Fig. 3) of group microorganism between different samples, and
And the analysis of different classifications sample microbiologic population difference is carried out to result, so as to find corresponding biomarker (see figure
4)。
Claims (8)
1. a kind of for the 16S rDNA V1-V2 areas specific primer pair for detecting microbe species in testing sample, its feature exists
In, the primer pair be can specific amplification inspection in need microbe-derived primer pair.
2. primer pair according to claim 1, it is characterised in that the primer pair is by two single strand dna sequence groups
Into primer pair.
3. primer pair according to claim 1, it is characterised in that the primer pair
Upstream primer is：5’-AGAGTTTGATCMTGGCTCAG-3’；
Downstream primer is：5’-GCTGCCTCCCGTAGGAGT-3’.
4. the primer pair in claim 1-3 described in any claim, it is characterised in that the primer pair is used for biotechnology
Detection field, by two generation high throughput sequencing technologies, precisely detects the species of microorganism in the sample of Disease oral cavity.
5. a kind of oral cavity flora method for analyzing diversity, it is characterised in that comprise the following steps,
(1) buccal sample to be measured is provided；
(2) from the buccal sample, its sample DNA is extracted；
(3) PCR reaction systems are configured, enters performing PCR reaction；
(4) for PCR product carries out the detection of target fragment；
(5) for the PCR primer of purposeful fragment carries out PCR primer purifying；
(6) with PCR primer in step (5) as detection object, the micro- life in the sample of oral cavity is carried out with two generation high throughput sequencing technologies
The detection of species；
(7) for two generation sequencing results carry out data analysis and process.
6. method according to claim 5, it is characterised in that the pcr amplification reaction in described step (3) is substance
PCR reacts, and reaction system is included：
10 × buffer, forward primer and reverse primer, DNA profiling, dNTP, Taq enzyme, sterile purified water.
7. method according to claim 6, it is characterised in that the reaction system is 50 microlitres of reaction systems, comprising：
0.8 microlitre of Taq enzyme, each 0.5 microlitre of forward primer and reverse primer, 2 microlitres of DNA profilings, 5 microlitres of 10 × buffer
Solution, 4 microlitres of dNTP solution, 37.2 microlitres of sterile purified water.
8. the method according to any claim in claim 5-7, it is characterised in that described pcr amplification reaction bar
Part is：
95 DEG C preheat 10 minutes；
6 PCR reaction cycles：92 DEG C 45 seconds, 50 DEG C 30 seconds, 72 DEG C 30 seconds；
30 PCR reaction cycles：92 DEG C 45 seconds, 68 DEG C 30 seconds, 72 DEG C 30 seconds；
72 DEG C extend 9 minutes；
4 DEG C of insulations are preserved.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710019638.3A CN106636423A (en) | 2017-01-12 | 2017-01-12 | Analysis method for diversity of oral cavity flora and disease-associated flora marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710019638.3A CN106636423A (en) | 2017-01-12 | 2017-01-12 | Analysis method for diversity of oral cavity flora and disease-associated flora marker |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106636423A true CN106636423A (en) | 2017-05-10 |
Family
ID=58843775
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710019638.3A Pending CN106636423A (en) | 2017-01-12 | 2017-01-12 | Analysis method for diversity of oral cavity flora and disease-associated flora marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636423A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058564A (en) * | 2017-05-18 | 2017-08-18 | 济南市儿童医院（山东大学齐鲁儿童医院） | Analysis method for diversity of intestinal flora of autism of children and specific primer pair |
| CN110176275A (en) * | 2019-05-22 | 2019-08-27 | 中国药科大学 | The macro genomic data analysis method in oral cavity based on high-flux sequence |
| CN110914451A (en) * | 2017-07-19 | 2020-03-24 | 知识股份有限公司 | Method and apparatus for real-time determination of disease status based on nucleic acids |
| CN111394485A (en) * | 2020-03-30 | 2020-07-10 | 云南中烟工业有限责任公司 | Primer pair, kit and method for detecting whether people smoke or not based on salivary microorganisms |
| CN111763751A (en) * | 2019-12-19 | 2020-10-13 | 上海力拜生物科技有限公司 | Tuberculosis saliva detection biomarker and application thereof |
| JP2021010343A (en) * | 2019-07-08 | 2021-02-04 | 三菱ケミカル株式会社 | Health condition prediction method by oral cavity bacteria |
| CN114107508A (en) * | 2021-12-08 | 2022-03-01 | 中国人民解放军总医院第五医学中心 | Application of salivary microbial marker in diagnosis and differentiation of hepatitis B hepatocellular carcinoma and liver cirrhosis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140186318A1 (en) * | 2012-09-14 | 2014-07-03 | Case Western Reserve University | Probiotic controlling fungi and uses thereof |
-
2017
- 2017-01-12 CN CN201710019638.3A patent/CN106636423A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140186318A1 (en) * | 2012-09-14 | 2014-07-03 | Case Western Reserve University | Probiotic controlling fungi and uses thereof |
Non-Patent Citations (5)
| Title |
|---|
| FABRICE A,ET AL: "Exploring Microbial Diversity Using 16S rRNA High-Throughput Methods", 《JOURNAL OF COMPUTER SCIENCE & SYSTEMS BIOLOGY》 * |
| JUSTIN KUCZYNSKI,ET AL: "Using QIIME to analyze 16S rRNA gene sequences from Microbial Communities", 《CURR PROTOC MICROBIOL》 * |
| MUKHERJEE PK，ET AL: "Oral Mycobiome Analysis of HIV-Infected Patients: Identification ofPichia as an Antagonist of Opportunistic Fungi", 《PLOS PATHOG》 * |
| 夏忠奎: "基于454与Ion Torrent平台的16S rRNA测序数据的比较分析与研究", 《中国优秀硕士学位论文全文数据库》 * |
| 张军毅等: "基于分子标记的宏基因组16S rRNA基因高变区选择策略", 《应用生态学报》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058564A (en) * | 2017-05-18 | 2017-08-18 | 济南市儿童医院（山东大学齐鲁儿童医院） | Analysis method for diversity of intestinal flora of autism of children and specific primer pair |
| CN110914451A (en) * | 2017-07-19 | 2020-03-24 | 知识股份有限公司 | Method and apparatus for real-time determination of disease status based on nucleic acids |
| CN110176275A (en) * | 2019-05-22 | 2019-08-27 | 中国药科大学 | The macro genomic data analysis method in oral cavity based on high-flux sequence |
| JP2021010343A (en) * | 2019-07-08 | 2021-02-04 | 三菱ケミカル株式会社 | Health condition prediction method by oral cavity bacteria |
| CN111763751A (en) * | 2019-12-19 | 2020-10-13 | 上海力拜生物科技有限公司 | Tuberculosis saliva detection biomarker and application thereof |
| CN111394485A (en) * | 2020-03-30 | 2020-07-10 | 云南中烟工业有限责任公司 | Primer pair, kit and method for detecting whether people smoke or not based on salivary microorganisms |
| CN111394485B (en) * | 2020-03-30 | 2023-09-22 | 云南中烟工业有限责任公司 | Primer pair, kit and method for detecting whether people smoke based on saliva microorganisms |
| CN114107508A (en) * | 2021-12-08 | 2022-03-01 | 中国人民解放军总医院第五医学中心 | Application of salivary microbial marker in diagnosis and differentiation of hepatitis B hepatocellular carcinoma and liver cirrhosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106636423A (en) | Analysis method for diversity of oral cavity flora and disease-associated flora marker | |
| CN111662958B (en) | Construction method of library based on nanopore sequencing platform, method for identifying microorganisms and application | |
| CN112501268B (en) | Nanopore sequencing-based primer group and kit for rapidly identifying respiratory microorganisms and application of primer group and kit | |
| CN107058490A (en) | The analysis method of enteron aisle and oral cavity flora diversity and otherness | |
| CN107312839A (en) | A kind of material, kit and method detected for systemic infection | |
| CN110387438A (en) | Multi-primers, kit and method for enterovirus high-flux sequence | |
| CN106845155A (en) | A kind of device for detecting internal series-connection repetition | |
| CN107058564A (en) | Analysis method for diversity of intestinal flora of autism of children and specific primer pair | |
| CN110343754A (en) | A method of it is quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism | |
| CN108588246A (en) | A kind of primer, probe, method and the kit of detection lower respiratory tract bacterium specific gene | |
| JPWO2018168986A1 (en) | Genetic test method and genetic test kit | |
| CN105296642A (en) | Isothermal amplification detection kit for pork derived component nucleic acid and detection method | |
| CN103773884B (en) | For detecting the primer sets of Chlamydia pneumoniae 98KDa MOMP gene and probe and application thereof | |
| CN102146467A (en) | Reagent for detecting Yersinia pestis and method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis | |
| CN112626215B (en) | AML prognosis related gene expression detection kit | |
| CN112592965B (en) | E.coli host DNA residue detection kit adopting TaqMan probe method | |
| CN115725784A (en) | Kit and method for detecting pathogens related to respiratory tract infection | |
| CN115011695A (en) | Multiple cancer species identification marker based on free circular DNA gene, kit and application | |
| WO2021039777A1 (en) | Method for examining rheumatoid arthritis | |
| WO2022174117A1 (en) | Metagenomic next-generation sequencing of microbial cell-free nucleic acids in subjects with lyme disease | |
| CN109825561B (en) | Application of Prevotella in identifying and/or distinguishing individuals of different ethnic groups | |
| CN207512174U (en) | A kind of detection of nucleic acids card | |
| CN103060224B (en) | Space enterococcus faecium LCT-EF258 strain | |
| CN109652573A (en) | For Salmonella typhimurtum or the site VNTR, detection primer group and the determination method of its single-phase bacterium mutation parting detection | |
| WO2023040997A1 (en) | Single gene test method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170510 |