CN106446599A - Method for screening oral pathogenic biomarkers of infant caries - Google Patents
Method for screening oral pathogenic biomarkers of infant caries Download PDFInfo
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Abstract
The invention relates to the field of biomarkers, and specifically provides a method for screening oral pathogenic biomarkers of infant caries. The method comprises the following steps of: acquiring an oral sample of an infant individual; converting DNA information of the sample into sample microorganism information; carrying out wilcoxon rank sum inspection by taking information of all the oral microorganisms of the above sample as input variables and utilizing a model established on the basis of health and disease states; and if the verification result is that p is less than 0.05, determining that an oral pathogenic biomarker is screened. The method provided by the invention is wide in application; a series of biomarkers providing functions not only can be used as detection objects, but also can be used as potential therapeutic targets; the method is not only suitable for large-scale population assessment, but also suitable for realizing long-term monitoring for an individual; and an application form of the method not only can detect the oral state of the infant individuals at the moment, but also can be used as a method for predicting and assessing the future caries states of the individuals.
Description
Technical field
The present invention relates to microbial biomarker field, specifically provide a kind of method of the oral cavity pathogenic organism label of screening infant dental caries.
Background technology
Dental caries be occur tooth chronic infectious disease it is considered to be the most common infectious disease of the mankind, be also incidence rate highest one class disease in Pediatric Oral Emergency.NIH (National Institutes of Health; NIH) propose the concept of " caries (early childhood caries; ECC) ", refer to that the dental caries of the one or many deciduous teeth that the child less than 71 months occurs are bad, because of dental caries mistake tooth or the infant because of dental caries filling.The longitudinal survey result that World Health Organization's crowd oral health national to 186 was carried out up to 20 years shows：Apply the enforcement of the control strategies such as fluorine, fissure blockade, diet control by local, though various countries' household's DMFT index has integrally declined, in most of country, dental caries still affect the school age population of 60%-90%.And China's third time oral health epidemiological investigation PRELIMINARY RESULTS also shows:The prevalence of 5 years old Primary Caries diseases is 66.0%, and the caries prevalence rate of 12 years old dental caries dental caries is 28.9%.
Dental caries are fallen ill for irreversible process, directly result in the defect of dental tissue or even whole absence of tooth, a series of infection and pain can be caused, also child's masticatory function can be reduced, increase permanent tooth caries risk, impact face appearance and pronunciation correctness, or even impact mouth jaw is developed, even cause malocclusion, have a strong impact on the life quality of life of patient.Because dental caries have, age of onset is early, and fast-growth, autonomous symptom is inconspicuous, the disease damage feature such as extensively, only passes through clinic mechanotherapy means and is processed, the defect of pathological changes tissue of tooth is inevitable during usual clinical definite.But repair the purpose that cavity can not be fully achieved treatment and control dental caries merely, and inappropriate dental treatment can increase the danger suffering from dental caries on the contrary.Therefore, the risk assessment for dental caries or even preventative strategies can are all the current Important Problems studied.
The risk factor of dental caries is complex, and under the multifactor collective effect such as microorganism, heredity, environment, induction tissue of tooth demineralization and remineralization level are unbalance, lead to the generation of chronic destructive disease, wherein microorganism is key link.Past is studied mainly for Streptococcus mutans (Streptococcus mutans), S.mutans is as the research of main " cariogenic bacteria ", because of its stronger function of producing acidity, acid resistance and extracellular synthesis glucosan, determine a large amount of impact dental caries pathogenetic mutans streptococcus strains, find that codified one class that changes of S.mutans gene participates in the albumen that biomembrane develops formation, its polymorphism can improve biomembranous virulence, increase dental caries and cause danger.But with more deep to the research of Streptococcus mutans 26S Proteasome Structure and Function both at home and abroad, more and more presence studied the cariogenic characteristics of support and non-fully only rely upon Streptococcus mutans, also have other oral cavity bacteriums to be considered closely related with the generating process of dental caries simultaneously.
Additionally, the appraisal procedure of the infant risk dental caries commonly used at present mainly includes：Oral microorganism counting method (as Streptococcus mutans and saliva lactic acid bacterial count method), saliva chemistry detects (such as, the buffer capacity of saliva and pH value detection), situation (during host's preliminary investigation, dental caries situation is suffered from oral cavity) is gone on dental caries basis, individual hygienic habit (bacterial plaque situation as can be seen), life-form structure, diet (as sugar intake) and/or social economy's level etc..However, there are a lot of limitation in these prediction and evaluation method：Including these indexs of (i) major part relatively subjective, be easily introduced that larger personal error, different detection are more difficult to compare and repeat.As being to judge it is more difficult to or else compare with tester based on visual observation and tester are personal when detecting individual primary oral condition.For another example when hygienic habit, the glucide absorption personal information such as frequency and fluorine source generally gather, equal detected person or its guardian offer are it is more difficult to accurate quantitative analysis are assessed.(ii) the other diagnostic detection of current chair, saliva nature examination and/or iconography detect for most of infant, and still more difficult cooperation and acceptance, cause its doctor poor from property.(iii) collection (as questionnaire form) of individual information is relative with microbial enumeration method expends larger human and material resources and time.(iv) individual dental caries experience is still currently the only generally acknowledged evaluation index, so generation of its unpredictable new dental caries.And major part sensitive group has no dental caries experience record in early stage.V () existing method is higher for screening adult's accuracy, but more limited for infant applicability.
In sum it would be highly desirable to screening and identifying other potential microbial biomarkers related in dental caries generation, and its detection method to it.
Content of the invention
For weak point above-mentioned present in prior art, present invention aim at a kind of method of the oral cavity pathogenic organism label of screening infant dental caries.
For achieving the above object, the technical solution adopted in the present invention is：
A kind of method of the oral cavity pathogenic organism label of screening infant dental caries, take infant individuality oral cavity sample, the DNA information of sample is converted into sample microbial information, and using the information of the whole oral microorganism of above-mentioned sample as input variable, the model set up using health and morbid state, by rank test, inspection correction p<0.05 is oral cavity pathogenic organism label；Described oral cavity pathogenic organism label is the fertile Pseudomonas (Prevotella) of Prey of dental plaque and/or saliva sample、The Ya Puleiwo bacterium that dwells (Prevotella denticola) in dental plaque in the level of kind、Speckle Prey in dental plaque in the level of kind irrigates bacterium (Prevotella maculosa)、True mouth Prey in dental plaque in the level of kind irrigates bacterium (Prevotella veroralis)、Prey in saliva in the level of kind irrigates bacterium DO039 (Prevotella DO039)、Xi Shi Prey in saliva in the level of kind irrigates bacterium (Prevotella histicola)、Pale Prey in saliva in the level of kind irrigates bacterium (Prevotella pallens)、Saliva Prey in saliva in the level of kind irrigates bacterium (Prevotella salivae)、True mouth Prey in saliva in the level of kind irrigates one or more in bacterium (Prevotella veroralis).
The structure of the model of described health and morbid state foundation：
1) using the one or more oral cavity pathogenic organism label obtaining and its abundance as input variable；
2) utilize random forest method, the individual health of corresponding for input variable infant and dental caries information are carried out two classification, builds the mathematical model based on specific oral cavity pathogenic organism markers tests oral status.
Further screening technique：
1) obtain the DNA of infant individuality oral cavity sample；Obtain 16s RNA or the full-length genome information of DNA information；
2) using bioinformatics method, DNA information is converted to oral cavity pathogenic organism label information；
3) by the abundance messages of the individual pathogenic organism label of above-mentioned biomarker information acquisition infant；
4) utilize random forest method, using the abundance of individual dental caries mark of correlation thing as input variable, using the dental caries detection model of foundation, sample is carried out with two classification analysises wilcoxon rank sum inspection correction p<0.05, that is, obtain and be oral cavity pathogenic organism label.
Described infant individuality oral cavity sample is dental plaque or saliva on infant gum.
Mentioned microorganism label can react the individual health/dental caries state now of infant, and the following dental caries of individuality a situation arises.
The present invention has advantages below and beneficial effect：
1. the object collection of the present invention and process simple, no invasive, low cost；
2. the assessment of the present invention is objective, automatization, it is possible to provide exact numerical；
3. the model of the present invention is set up and to optimize easily operated, data processing efficient；
4. the present invention is widely used：It provides a series of microbial biomarkers of function to can be used as detection object, simultaneously also can be used as potential therapy target；Its application is applicable not only to large-scale crowd assessment, also can realize long term monitoring for individuality；Its application form not only can detect infant individuality now oral status, also can be used as the method for forecast assessment dental caries state of individual future.
Brief description
Fig. 1 implements the oral microbial community architectural feature figure providing for the present invention；
Fig. 2 implements the oral microorganism composition figure filtering out time correlation by random forest homing method providing for the present invention；
Fig. 3 implements the pathogenic bacterium (Prey irrigates Pseudomonas (Prevotella)) of the dental caries correlation screened by random forest homing method providing and its to the result figure distinguishing health and dental caries for the present invention；
The model that Fig. 4 implements to provide for the present invention is applied to predict relative healths sample result figure.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment the present invention is described in further detail.
The present invention to screen the microbial biomarker related with identification dental caries by the use of oral plaque and saliva microbiologic population and its to apply as embodiment, including following content：
(1) collect Pediatric Oral Emergency health status clinical information (table 1)：
The oral health of the Guangzhou south Chinese and English full-time child of kindergarten is tracked investigating, every half a year checks once, continue 1 year three times inspections, it is spaced afterwards 1 year and checked again, child's dmfs (dental caries according to investigation records, lose, dental filling number) index, select the child with following three class oral health variation characteristics to include this subject study according to this research purpose：1. healthy group (H2H group):Oral cavity dental caries situation remains 17 children of health；2. dental caries group, including dental caries generation group (H2C group):Oral cavity dental caries situation experience newly sends out 21 children of process from health to dental caries, and dental caries progression group (C2C group):Dental caries situation experience in oral cavity is from having suffered from 12 children to dental caries evolution for the dental caries.Inclusion criteria includes：About 4 years old age, the whole eruption of 20 deciduous teeth, exclusion standard includes：There is the oral disease such as whole body system disease and periodontal, halitosis, take antibiotic within three months.Obtain volunteer guardian with regard to the matters such as whole experiment flow items details and the announcement of later data to agree to, and sign Informed Consent Form.On the gum being taken during the examination of mouth choosing all selected children, dental plaque and saliva sample amount to 284.
Investigation method:Combined in the way of spy is examined by inspection by two tooth body dental pulp specialists and checked that examination apparatus autoclave sterilization removes soft dirt by cotton swab if necessary.Unified understanding, method and standard before checking, the Kappa value of standard consistency inspection is all higher than 0.92.Using World Health Organization (WHO)《Oral health surveys basic skills》(1997) diagnostic criteria to dental caries.Hat dental caries diagnostic criteria：The nest ditch point gap of tooth or shiny surface have under obvious cavity or obvious enamel and destroy or clearly can visit and soften hole bottom or the disease damage of hole wall is designated as dental caries, include charges or the fissure blockade person that simultaneously has dental caries.Following performance is had to lack and during other positive symptom, do not list dental caries recording interval in：1. white or Chalk mottle point；2. visit coloring or the rough spot examining no softening；3. glaze particle gap or the coloring of nest ditch, but destruction of moving under water under no obvious enamel；4. severe dental fluorosis are arrived in, glossy, matter is hard, have dolly dimple；5. according to distribution or medical history, observe and cause disease damage dental caries because of abrasion in conjunction with palpation, inspection.
The sample clinical data that table 1 present example provides
(2) children's salivary and supragingival plaque sample are collected：
Sample previous hour experimenter to avoid taking food and drink water, every sub-sampling all in the morning 9:100-12:00, during sampling, child keeps gently facing upward head, eye closing, upright seat.Collect child's nonirritant saliva about 3-5ml in 50ml sterile centrifugation tube, and every 1ml is sub-packed in 1.5ml centrifuge tube；Reuse the bacterial plaque 1 minute that aseptic toothbrush gathers on whole eruption deciduous teeth gums, the bacterial plaque adhering on toothbrush is transferred to the 50ml centrifuge tube filling 10ml distilled water, avoids during sampling touching other sites of oral cavity such as mucosa.Sample is numbered respectively and is placed in -80 DEG C of preservation DNA to be extracted.
(3) extracting genome DNA and PCR amplification 16S rRNA genetic fragment
Using high salt DNA extraction method.Centrifuge tube 13, the 000rpm/min centrifugation 15min respectively of bacterial plaque and saliva will be filled, and abandon supernatant, be separately added into 1ml lysate, in cracking liquid mixture, add 30 μ L E.C. 3.4.21.64s and 150 μ L 10%SDS, incubated overnight is shaken in 53 DEG C of water-baths.400 μ L 5M NaCl are added to cultivate 10min on ice, 13,000rpm/min centrifugation 10min.Add isopyknic saturation phenol solution, be mixed into emulsion form to aqueous phase phenol, with 13,000rpm/min centrifugation 15min, draw the sticky aqueous phase in upper strata and manage to new, repeat phenol and extract once.Plus isopyknic chloroform isoamyl alcohol mixed liquor (24：1), rotate and mix, with 13,000rpm/min centrifugation 15min, take upper strata sticky aqueous phase transfer.Add 800 μ L isopropanols, incubated at room temperature 1min, with 13,000rpm/min centrifugation 15min.Abandon supernatant, 70% ethanol is washed twice, is dissolved in 50 μ L TE solution after being dried.
Using Qubit ultramicron spectrophotometer quantitation DNA concentration, electrophoresis detection DNA integrity.DNA after extraction is stored in -20 DEG C.About 15ng DNA is used for building 16S amplification library.
For obtaining relatively accurate germline development information, choose V1-V3 hypervariable region (Escherichia coli positions 5-534) in 16S rRNA fragment and expand target fragment as PCR.Determine PCR forward primer (5 '-NNNNNNN-TGGAGAGTTTGATCCTGGCTCAG-3 ') and downstream primer (5 '-NNNNNNN-TACCGCGGCTGCTGGCAC-3 '), NNNNNNN is IDtag, it is seven bases of the random combine designing for the different sample source of difference, it is separately added into 5 ' ends of upstream and downstream primer, complete multiple samples using this Multi-example parallel signature technology and be sequenced on sequenator simultaneously.
Each sample carries out three PCR amplifications, PCR reaction system (25 μ L) comprises the Gotag Hotstart polymerase of 12.5 μ L, each 1 μ L upstream and downstream primer (concentration 5pM), 1 μ L genomic DNA (5ng μ L-1), 9.5 μ L PCR rank sterilized water, are reacted in Thermocycler PCR system.Reaction condition is set as：95 DEG C of denaturations 2min, 94 DEG C of degeneration 30s, anneal 56 DEG C of 25s, 72 DEG C of extension 25s, totally 25 circulations, last 72 DEG C of extension 5min.All carry out gel electrophoresiss (1.2%Q agarose after PCR primer mixing, 5V cm-1,40min), confirm expanding effect, agarose gel is placed under uviol lamp, extract the DNA band of about 500bp length, carry out reclaiming according to the operating process that Qiagen MiniElute test kit provides, purification purpose fragment DNA, washed with 20 μ L.
(4) 454GS FLX Titanium sequencing
Main flow is as follows：1. library preparation, quantitative using Agilent BioAnalyzer 2100 biological analyser and PicoGreen ultramicron spectrophotometer joint, different samples are built three parts of DNA library altogether with after equimolar mixing, is connected modification with specific linkers, degenerative treatments reclaim single stranded DNA；2. emulsifying PCR, DNA library is fixed on magnetic bead, through expanding emulsifying, forms oil water mixture, each DNA segment carries out independent parallel amplification in microreactor, produces millions of meter identical copies.Break emulsified state, recovery purifying is incorporated into the DNA fragmentation on magnetic bead；3. sequencing reaction, the magnetic bead carrying DNA is mixed with other reactants, put into and be placed in PTP plate in 454 GS FLX Titanium machines, the interpolation of each nucleotide complementary with template strand can produce fluorescence signal and be captured by CCD camera, be gradually completing sequencing；4. data collection, carries out base parsing by system information instrument to sequencing reaction data.
(5) the high flux data conversion of acquisition is become specific microbiologic population data
Sequence quality controls：454 high-quality sequence analysis flow processs are based primarily upon MOTHUR platform, set quality control specifications, and standard compliant sequence fragment is considered high-quality sequence, is retained.1. at least one end primer can be matched it is allowed to editing distance (insertion, delete, disappearance, the base quantity of mispairing) be less than 2；2. sequence length is more than 150bp；3. the base reading frame of one 50bp is set, starts base one by one from first base of every sequence and be moved rearwards by, often move a base, calculate once the mass fraction average of this reading inframe, this performance figure average need to be more than 35；4. do not contain fuzzy base；5. sequence label mispairing quantity is allowed to be less than 1.After tentatively filtering, need further sequence to be carried out the examination of mistake that is sequenced, including the step such as " preclustering " and the lookup of chimera (Chimera) sequence.Select UCHIME program looks and delete these sequences.
Germline development information analysiss based on 16S data base：Carry out horizontal bacterial strains information from door to kind using MOTHUR sorting technique for Human Oral Cavity core microorganism 16S data base (CORE) to incorporate into, count the sequence number of each sample each species in each categorization levels respectively, and calculate its ratio with the sequence number of this population of samples acquisition, thus obtaining the relative abundance of each species of each class.
(6) impact (Fig. 1) that different factors are distributed for oral cavity flora：
Structure of community computational methods based on Jie Sen-aromatic (Jensen-Shannon) matrix：It also can investigate the difference of abundance in sample bacterium kind level in addition to the evolutionary distance of sample room.Antibacterial wealth of species distribution in sample can be regarded as the probability distribution of species, it is possible to use the Mutual information entropy (Jensen-Shannon divergence, JSD) of this probability distribution of sample room is measuring the difference of the microorganism group of sample room.The computing formula apart from D (a, b) of sample room is as follows：
PaAnd PbAbundance distribution in representative sample a and sample b respectively.JSD (X, Y) defines the Mutual information entropy between different probability distribution X and Y (Jensen-Shannon divergence) in two samples.
KLD is the Kullback-Leibler dispersion between X and Y, and specific computational methods are as follows：
Non-supervisory principal coordinate analysis：Jensen-Shannon matrix is carried out principal coordinate analysis (PCoA:Principal Coordinates Analysis) to show oral microbial community architectural feature between different samples，Each species information is considered as the independent variable not associated mutually by PCoA，It is analyzed with the matrix of sample × variable relative abundance，With on the premise of not considering Environmental Factors，Without prejudice、The inherent flora result of overall observation sample，Find one or more potential variable (principal coordinates，Principal coordinate，PC) with farthest compared with low dimensional best explain in sample variation，Each principal coordinate represents explainable overall structure degree of variation under this dimension，Thus reach Data Dimensionality Reduction processing and the purpose to sample sequence，The score (Score) of wherein sample is the linear combination of species score.
Displacement multi-variate statistical analyses result display oral microbial community has obvious age characteristicss, these age characteristicss are relevant with ontogeny Maturity and health status, support to differentiate caries and healthy sample according to oral microbial community, prompting can set up diagnosis and prediction oral cavity caries model (Fig. 1)：
Although 1. in each ecological site, time/age factor is the most important factor determining Flora distribution.
2. but in each ecological site, other key factors affecting Flora distribution according to its importance rank order are：Health/morbid state, sample packet, individual heterogeneity.
3. in different grouping (include H2H, H2C, C2C group), in healthy group, time factor affects maximum on its flora, and time factor receives morbid state impact and suppressed to flora influence in dental caries group.
Result above is pointed out：Oral cavity flora can react host buccal health status, the particularly generation of dental caries as the medium of dental caries detection.
(7) the screening microbial biomarker related with identification dental caries and its application (Fig. 3, Fig. 4)
Depending in machine learning, random forest method is a model comprising multiple decision trees, and the classification of its output is the mode by the classification of indivedual tree outputs, this model is widely used in excavating the incidence relation between target variable and numerous explanatory variable.The method not only can set up classification or regression model, can determine that the variable distinguishing particular state or label simultaneously, and can judge the size of its separating capacity by its importance values.In this example, random forest method utilizes the randomForest software kit of R to realize, and sets up 5000 trees, other are default setting.Using the 2/3 of input data as training dataset, using the 1/3 of input data as test data set, carry out 100 experiments at random to reduce error.
Random forest machine learning (Random Forests, RF) it is a kind of machine learning based on classifier algorithm, proposed by LeoBreiman, by bootstrap resampling technique, have from training set (data set) n and repeat with putting back to randomly draw k new training sample (train set) set of sample generation, then k classification tree is generated according to self-service sample set and form random forest, depending on the classification results of new data press the how many fraction being formed of classification tree ballot, error in classification depends on the classification capacity of every one tree and the dependency between them.The possible very little of the classification capacity of single tree, but after randomly generating substantial amounts of decision tree, a test sample can select most probable classification by the classification results of every one tree after statistics., by collecting, to a large amount of classification trees, the precision of prediction that improve model, because it does not exist, overfitting, precision of prediction are high, and this model is widely used in excavating the incidence relation between target variable and numerous explanatory variable for it.
Except setting up detection model and prediction, random forest method can be used for the importance of evaluation variable, and feature selection goes to divide each node using random method, then compares the error producing under different situations.Intuitively evaluation criterion is that this variable is more important, and the impact to forecast result is also bigger.The Assessment of Important of Random Forest model explanatory variable is using similar standard：The value of a certain for all inspection specimens explanatory variable is upset at random, using former Random Forest model, test samples is forecast again, the outer error of fitting increase of bag is more, and this explanatory variable is more important.The outer error of fitting incrementss of bag can be used for quantitative assessment explanatory variable importance.This patent adopts ten times of cross validations (Ten-Fold Cross Validation) to evaluate and builds the minimum number including variable needed for model.Repeat 100 times at random, using average as the estimation to algorithm accuracy.Cross validation (Cross-Validation, CV) it is a kind of statistical analysis technique for verifying classifier performance, it is mainly used in modelling evaluation, obtaining reliable and stable model, under certain meaning, initial data (dataset) is grouped, a part is as training set (train set), another part is as checking collection (validation set), with training set, grader is trained first, recycle checking collection to test the model that obtains of training, by each error in classification as classification of assessment device performance index.And data set is divided into very by ten times of cross validations, in turn will wherein 9 parts as training data, as test data, tested for 1 part.Test every time all can draw corresponding error in classification, and 10 result averages are as the estimation to arithmetic accuracy.
First, screen and reject time correlation microorganism：For reducing the impact that time factor screens for dental caries related microorganisms label, screen using the oral microbial community antibacterial kind data in H2H group as input variable first, using each sample corresponding actual monthly age as sample information, it is revert to discrete output variable (monthly age of prediction), the preliminary mathematical model setting up detection child's individuality biological age.Variable is returned importance ranking according to it to the age, will reduce with variable and random forest regression model to distinguish the set of variables cooperation that do not significantly change of age ability be final age related microorganisms label (Fig. 2).Wherein，Include the general Salmonella of Rockwell (Prevotella loescheii) from the label of dental plaque，Denitrification Kingella (Kingella denitrificans)，Leptothrix BU064 (Leptotrichia BU064)，Multiform Fusobacterium nucleatum subspecies (Fusobacterium nucleatum subsp.polymorphum)，The outstanding bacterium 602D02 (Bergeyella 602D02) of uncle，Oral cavity core bar bacterium (Cardiobacterium valvarum)，Streptococcus mitises/streptococcus pneumoniae/baby streptococcus/Streptococcus oralis (Streptococcus mitis/Streptococcus pneumonia/Streptococcus infantis/Streptococcus oralis)，Yellow Neisseria/neisseria mucosa/neisseria pharyngis (Neisseria flava/Neisseria mucosa/Neisseria pharyngis)，Very thin Campylobacter spp (Campylobacter gracilis)，Golden yellow Neisseria (Neisseria flavescens)，Include Detection of Porphyromonas CW034 (Porphyromonas CW034) from 15 labels of saliva，Lattice step on streptococcus (Streptococcus gordonii)，Veillonella atypica/different Veillonella/veillonella parvula (Veillonella atypical/Veillonella dispar/Veillonella parvula),Oral digestion streptococcus (Peptostreptococcus stomatis)，Secondary Streptococcus sanguiss/Streptococcus oralis (Streptococcus parasanguinis/Streptococcus oralis)，Cilium bacterium BU064 (Leptotrichia BU064)，(Porphyromonas catoniae)，TM7 oral cavity taxon 352 (TM7 oral taxon 352)，General Salmonella oral cavity taxon 299 (Prevotella oral taxon 299)，Produce black general Salmonella (Prevotella melaninogenica)，Ditch Eubacterium/small and weak Eubacterium (Eubacterium sulci/Eubacterium infirmum)，The outstanding bacterium 602D02 (Bergeyella602D02) of uncle，Golden yellow Neisseria (Neisseria flavescens)，Neisseria meningitidis/neisseria polysaccharea (Neisseria meningitides/Neisseria polysaccharea)，Severe foster particle chain bacterium (Granulicatella elegans).
Second, random forest method using rote learning, using in dental caries generation group and dental caries progression group, host buccal state is for dental caries and definitely healthy sample as input variable, corresponding with the output variable that health and dental caries two are classified by it, sets up disaggregated model (Fig. 3).Additionally, the related label of age factor is all rejected from modeling variable.Based on what Prey irrigated Pseudomonas (Prevotella), result is visible to set up that health distinguished by model and the model of ability of preventing or cure a disease is close with the model using full bacterium, point out the fertile Pseudomonas of Prey can point out infant dental caries and health status.
3rd，Based on above-mentioned random forest finding model，By all variables according to it to model importance ranking，Screen and identify the microorganism that important function is had on kind of level to differentiation health and dental caries，Prevotella denticola (dwell Ya Puleiwo bacterium) wherein in dental plaque、Prevotella maculosa (speckle Prey irrigates bacterium)、Prevotella DO039 (DO039 Prey irrigates bacterium) in Prevotella veroralis (true mouth Prey irrigates bacterium) and saliva、Prevotella histicola (Xi Shi Prey irrigates bacterium)、Prevotella pallens (pale Prey irrigates bacterium)、Prevotella salivae (saliva Prey irrigates bacterium)、Prevotella veroralis (true mouth Prey irrigates bacterium) is relatively strong (Fig. 3) to category of model contribution ability.
4th, diagnostic application：Irrigate abundance expression in each sample for the bacterial strain using being screened 8 kinds of Preies all independently as input variable, using the health of sample and morbid state as classified variable, it is utilized respectively random forest method and sets up model, the accuracy rate of each model is all about in 70% (Fig. 3).
5th, prediction application：Above-mentioned set up model is applied to the prediction of relative healths sample, wherein derive from each 24 of the sample of dental plaque and saliva relative healths, it is found that sample major part is all categorized as dental caries sample, as 81% in dental plaque, (17) sample is predicted as dental caries sample (Fig. 4), prompting pathogenic bacterium may change prior to clinical symptoms, can be used for predicting the generation of dental caries.Meriting attention is, in two groups of samples that these are predicted as health or dental caries, Streptococcus mutans no difference of science of statistics (P=0.002) in two groups, and Prey is irrigated Pseudomonas and is had significant difference (P<0.05, Fig. 4), explanation Prey irrigates important function in occurring for the Pseudomonas for dental caries again, and it can point out the generation of caries.
Of the present invention can be found in Breiman L (2001) Random forests.Mach Learn 45 based on the regression analysis of random forest:5 32.) and (Knights D, Costello EK, Knight R.Supervised classification of human microbiota.FEMS Microbiol Rev.2011Mar；35(2):343-59.doi:10.1111/j.1574-6976.2010.00251.x.Epub 2010Oct 7.Review.PubMed PMID:21039646..
Certainly, described above is not limitation of the present invention, and the present invention is also not limited to the example above, those skilled in the art, in the practical range of the present invention, the change made, remodeling, adds or replaces, all should belong to protection scope of the present invention.
Claims (4)
1. a kind of screening infant dental caries oral cavity pathogenic organism label method it is characterised in that：
Take infant individuality oral cavity sample, the DNA information of sample is converted into sample microbial information, and with
The information of the whole oral microorganism of above-mentioned sample, as input variable, is set up using health and morbid state
Model, by rank test, inspection correction p<0.05 is oral cavity pathogenic organism label；Institute
State the fertile Pseudomonas of Prey that oral cavity pathogenic organism label is dental plaque and/or saliva sample
(Prevotella), the Ya Puleiwo bacterium (Prevotella of dwelling in dental plaque in kind level
Denticola), the speckle Prey in dental plaque in kind level irrigate bacterium (Prevotella maculosa),
Saliva in the fertile bacterium (Prevotella veroralis) of true mouth Prey in dental plaque in the level of kind, kind level
Xi Shi Prey in saliva in the fertile bacterium DO039 (Prevotella DO039) of Prey in liquid, kind level
Pale Prey in saliva on fertile bacterium (Prevotella histicola), kind level irrigates bacterium
Saliva Prey in saliva on (Prevotella pallens), kind level irrigates bacterium (Prevotella
Salivae), the true mouth Prey in saliva in kind level is irrigated one in bacterium (Prevotella veroralis)
Plant or several.
2. the side of the oral cavity pathogenic organism label of screening infant dental caries as described in claim 1
Method it is characterised in that：The structure of the model of described health and morbid state foundation：
1) the one or more oral cavity pathogenic organism label obtaining and its abundance are become as input
Amount；
2) utilize random forest method, by the individual health of corresponding for input variable infant and dental caries letter
Breath carries out two classification, builds the mathematics based on specific oral cavity pathogenic organism markers tests oral status
Model.
3. the oral cavity pathogenic organism label of the screening infant dental caries as described in claim 1 or 2
Method it is characterised in that：Screening technique：
1) obtain the DNA of infant individuality oral cavity sample；
2) using bioinformatics method, DNA information is converted to oral cavity pathogenic organism label letter
Breath；
3) rich by the individual pathogenic organism label of above-mentioned biomarker information acquisition infant
Degree information；
4) utilize random forest method, the abundance of individual dental caries mark of correlation thing is become as input
Amount, carries out two classification analysises wilcoxon rank sum using the dental caries detection model of foundation to sample
Inspection correction p<0.05, that is, obtain and be oral cavity pathogenic organism label.
4. the side of the oral cavity pathogenic organism label of detection infant dental caries as described in claim 1
Method it is characterised in that：Described infant individuality oral cavity sample is dental plaque or saliva on infant gum.
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