CN108120679A - A kind of kit for predicting whether children are susceptible to dental caries - Google Patents

Abstract

The present invention relates to a kind of kits that can predict whether children are susceptible to dental caries, it includes maltose, α methyl D galactosides, α amarogentins, D, L α phosphoglycerols, D trehaloses, 3 methyl D glucose, turanose, glycyl L proline, L glutamine and glycyl L glutamine, 10 kinds of carbon sources altogether, for predicting risk that children suffer from saprodontia.The kit can be used in medical institutions or even in family, and just risk can be estimated before child morbidity, it is thus possible to be prevented in advance, be substantially reduced society for the resource input by dental caries.

Description

A kind of kit for predicting whether children are susceptible to dental caries

Technical field

The present invention relates to the predictions of technical field of oral medicine more particularly to children caries risk.

Background technology

Underage child dental caries (ECC) were referred to as feeding bottle dental caries or fed dental caries, in the past in terms of being considered as paediatrics and public health Global problem.This is a kind of chronic disease for betiding children, and 2-5 Sui when is most preferably sent out, but is often ignored by people. Dental caries treatment is usually more difficult, and cost is also higher.Treatment and the qualified hospital for carrying out general anesthesia treatment are to control under general anesthesia It is required to treat ECC.

Previous research is all conceived to the Phylogenetic diversity of bacteria on taxology.Although these researchs are to we provide many passes The important evidence of relation between oral microorganism and bacterial plaque, but which has limited us for being associated on microbial coenoecology Understanding depth.These researchs also more is only limitted to theoretic analysis, and there is no be converted directly into result among practical application.

Analysis for microorganism phenotype, such as metabolism, can more in depth understand biological community structure.First, Metabolic process is to determine the key point of the virulence property of oral cavity pathogenic microorganisms.In addition, internal collaboration Nutrition and Metabolism can increase Bacterial community in diversity, to maintain microbiologic population stability it is significant.

With sole carbon source utilization (SCSU) pattern of Biolog (Biolog Inc., Hayward, CA, USA) analysis samples It is a kind of the method for non-autotrophic microbe to be differentiated based on function.Have several holes on one block of plate, per hole include tetrazolium violet and The different proprietary somatomedins (single carbon source) of minimum breathe the reaction solution of reduction tetrazolium violet to refer to microbial cell Show the metabolic condition of bacterium.This analytical technology has the characteristics that high sensitivity, resolving power are strong, need not be separately cultured, microorganism Selective to the utilization of carbon source, each microorganism is different to the Utilization ability of different carbon source, therefore the measure of a variety of SCSU can be with Tested single microorganism or the metabolic characteristics fingerprint of microbiologic population are obtained, to differentiate different microorganisms (group), therefore should Technology is often used among the measure of environmental microorganisms such as edaphon detection, Eco-environmental Reconstruction assessment.

Zhang in 2014 et al. is delivered on PLOS ONE magazines determines patients with periodontitis bacterium on using Biolog technologies The paper of spot microbiologic population wherein collecting subject's bacterial plaque, analyzes 95 kinds of sole carbon source utilization patterns, obtains that tooth can be distinguished 14 kinds of single carbon sources of all scorching groups and healthy control group.

But the metabolic characteristic on microorganism in ECC patient and healthy person mouth does not have more really there is no correlative study report Have and actual product or method can be applied to based on the studies above.Although dental caries are that bacterial plaque relies on disease with periodontitis, But the detection model and corresponding conclusion for relying solely on existing periodontitis can not be helpful to obtaining the relevant information of dental caries, Reason includes：The pathogenic bacteria of periodontitis and dental caries simultaneously differ；The mutual antagonism of the pathogenic bacteria of periodontitis and dental caries, it is difficult to coexist； Periodontitis and both dental caries are different for the metabolic characteristics fingerprint of different carbon source；Processing for the two detection sample also can be It is different；Compared with healthy person, the time of significant difference or the other parameters of correlation model in experiment and its optimal are presented in carbon source Value also all can be different.

In view of dental caries are one of highest mouth diseases of mankind's illness rate, the 4th national oral health epidemiological investigation It has been shown that, China's situation of dental caries illness at this stage is also very pessimistic, therefore, for underage child dental caries, not only needs diseased mechanism, cause Cause of disease element is probed into, while is also highly desirable to a kind of product that can be to risk into accommodating prediction, to help the whole society Active prevention.

The content of the invention

In view of the above problems, it can predict whether children are susceptible to dental caries it is an object of the invention to provide one kind Kit.It can be used in medical institutions or even in family, and just risk can be carried out before child morbidity It estimates, it is thus possible to prevent in advance, substantially reduce society for the resource input by dental caries.

Specifically, predict whether susceptible to the dental caries kit of children include maltose, Alpha-Methyl-D- galactosides, α- Amarogentin, D, L- alpha-phosphates glycerine, D- trehaloses, 3- methyl-D-glucoses, turanose, glycyl-L-PROLINE, L- paddy Glutamine and glycyl-L-glutamine amount to 10 kinds of carbon sources, for predicting risk that infant suffers from saprodontia.

Kit generally platy structure, plate is equipped with to place the hole of the carbon source and color developing agent, wherein different Different carbon sources is placed in hole, color developing agent is equipped in each hole.Wherein described color developing agent is preferably tetrazolium violet.

Kit can also include hole and/or the internal hole for sky equipped with water and color developing agent, as reference.

In order to further easy to use, kit can also include phosphoric acid normal saline buffer solution.The phosphoric acid physiology salt Water buffer solution is preferably the 0.01mol/L phosphate buffers that pH value is 7.2-7.4.

Kit global design is easy to use, and can accurately predict the risk that children suffer from dental caries.

Description of the drawings

Fig. 1：Disease group (- ● -) that 24,48,72,96 are cultivated on Biolog Anaerobic culturel plates is small with control group (-- --) Average color change (average well color development, AWCD) constantly.* P ＜ 0.05

Fig. 2：Disease group (- ● -) with the different carbon source metabolic patterns of control group (-- --).* P ＜ 0.05**P ＜ 0.01

Fig. 3：Based at 24 hours between disease group (P) and control group (C) difference carbon source cluster analysis schematic diagram.

Fig. 4：10 difference carbon sources during cultivating 24 hours lead disease group (▲) and control group (■) as variable The schematic diagram of constituent analysis.

Fig. 5:Reagent cartridge configuration schematic diagram.

Specific embodiment

The invention will be further described below.

(1) experimental subjects

In a manner that computer generates random number from January 1st, 2014 to August 31 days during in Medical School of Peking University second Subject is raised in the children that the outpatient service of Pediatric Dentistry of clinic is checked or is treated.All pediatric subject parents are studying It endorsed Written informed consent before beginning.Inclusion criteria is：48~71 months ages；Without infectious, congenital periodontosis or Dental abscess；It is collected in plaque in the previous moon without using antibiotic, painting fluorine treatment or extraction.

In terms of saprodontia is diagnosed, the diagnostic criteria of a staff has carried out school with reference to the experienced colleague of another one Standard has then carried out all dental examinations.Inspection mouth mirror and dental explorer examine method by visual examination and spy and diagnose saprodontia. Tooth is cleaned with cotton balls, but does not clap X-ray.Caries of primary teeth, which loses, based on the World Health Organization (1997) Caries diagnosis criterion evaluation mends The tooth number that dental caries occur in index (dmft), i.e. a human mouth, the tooth number lost by dental caries with because dental caries do filling treatment The sum of tooth number.The infant for having the saprodontia of 5 or more is in SECC groups or is called disease group.No dental caries (CF) children are divided In control group.The tooth that filling is repaired in the past is by there is dental caries statistics.All subjects receive dentistry during in August, 2015-October It checks, the children of control group remain no dental caries state.

Generally speaking, this research incorporates 20 children, wherein 10 have severe ECC (SECC), in addition 10 conducts pair According to control.

(2) collection and processing of sample

It is required that subject is not early eating more foods after the meal and cannot brush teeth.About it is early after meal 2 it is small when, it is desirable that it is tested Person is gargled with clear water.To each children, plaque is sampled from the complete enamel of eight deciduous molars.Sample is received with sterile excavator spoon Collection, and be immediately placed on containing in 1ml hypo solution Eppendorf pipes.Sample is stored in ice bag under the conditions of 0 DEG C, It is interior when 2 is small after collection to be sent to laboratory.

(3) Biolog is analyzed

Sample suspensions are vortexed in 11mLpH7.2-7.4 phosphate buffers of 0.01mol/L and shake abundant mixing 60s, Sample is seeded on the Biolog plates (AN) of anaerobism, 100 μ L are loaded per hole.The microwell plate of Biolog includes 95 independent lists One carbon source and a water are as blank control.Contain tetrazolium violet in each hole, color change caused by reduction reaction is thin to indicate Bacterium is metabolized.The initial optical density (ODS) of bacterial suspension is measured before inoculation.Microwell plate is under conditions of 5% carbon dioxide, 37 DEG C Culture 4 days, Biolog tablets are placed in anaerobism bag.590nm OD values (OD590nm) with Biolog Micro Station with And related software measures and analysis.

(4) data analysis

For correcting background, we introduce modified OD values, i.e. experimental port subtracts control hole OD.If difference is negative, Revised OD values are zero.The overall metabolic activity of microbiologic population is represented as average color change in Biolog plates (AWCD), it is the average of correction OD value of the single carbon source per hole.Our standardization correction OD values are the correction in each hole OD values divided by each plate AWCD, therefore can avoid as caused by microbiologic population's manual sampling difference in bacterial plaque suspension just Beginning OD value changes.

Disease group and the difference application independent samples t test of the AWCD of control group plank after cultivating four days are compared Compared with.P ＜ 0.05 or p ＜ 0.01 are statistically significant.The utilization power of two groups of carbon source is determined, OD is corrected based on standardization Value determines which kind of carbon source has the difference that utilizes between two groups using independent samples t test.Between two groups of disease group and control group The definite of relation on having distinction carbon source uses cluster analysis and principal component analysis (PCA).

(5) result

The demography and Clinical symptoms of sample are shown in Table 1.

1 demography of table and Clinical symptoms

The initial absorbance value of disease group is higher than control group, and with the extension of incubation time, Biolog culture plates pass through aobvious Colour response reflects the variation of plaque metabolism well, prompts the change of Bacterial community indirectly.Specifically, 48 before culture Hour, average color change (average well color development, the AWCD) difference of disease group and control group without Statistical significance.However, after when culture 72,96 is small, the AWCD values of disease group flora are significantly higher than control group (P ＜ 0.05), Refer to Figure of description 1.Wherein, it has been found that two kinds of typical carbon metabolism patterns, referring to Figure of description 2, top two A utilized for control group is better than the carbon source that disease group group utilizes, and lower section utilizes for disease group is better than the carbon source that control group utilizes.

Specific to single carbon source, originally researched and analysed cultivate 24,48,72,96 it is small when disease group on totally four time points With the control group carbon source that utilization power has differences between the two, 10,23,9,7 carbon sources for having significant difference are found that respectively. Due to 24 it is small when Time in Vitro it is most short, thus it is speculated that truth in the closest mouth of the metabolism of flora at this time, therefore, this research 10 difference carbon sources for choosing the time point discovery carry out subsequent analysis, refer to table 2.The difference carbon source of other time point refers to table 3。

2 disease group of table and difference carbon source of the control group when 24 is small

3 disease group of table and difference carbon source of the control group when 48,72 and 96 are small

For 10 difference carbon sources that culture 24 obtains when small, this research gathers the sample of disease group and control group Alanysis (Figure of description 3) and principal component analysis (Figure of description 4), with clear and definite carbon metabolism pattern and whether it is diseased between Association.

(6) cluster analysis

Cluster analysis is a kind of analysis of exploration, and during classification, people do not provide the mark of a classification in advance Standard, cluster analysis can automatically be classified from sample data.Its principle is according to gap (the i.e. sample between individual The distance of the data of characteristic index), sample in small distance is classified as one kind, makes different classes of interindividual variation larger, Individual difference between same category is smaller.This research uses the ward (sum of squares of deviations method) in hierarchical clustering method, uses Software be R softwares.Figure of description 3 is the thermal map (heatmap) made after cluster analysis.

The small figure in the upper left corner is legend, and color lump shows the gradual change of gray scale reduction, represents each sample to each carbon source from left to right Producing level gradually increase.Because each utilization power corresponds to an above-mentioned correction absorbance, that is, share 20 × 10 =200 values, i.e. 200 color lumps, the color lump quantity according to each gray scale, it may be determined that go out the grayish counting line in small figure Item.

The transverse axis of big figure is 10 species diversity carbon sources in Fig. 3, and in the longitudinal axis, P1-10 is disease group sample, and C1-10 is control group Sample.The meaning of color lump is identical with legend.It can be seen that the corresponding low gray scale color lump of disease group sample is more than control group, illustrate disease The utilization of carbon source degree of disease group is better than control group；It can furthermore be seen that the low gray scale color lump of disease group is more in the row of the left side 7, the right 3 The low gray scale color lump of row control group is more.

For the effect of clearer displaying cluster, the process of cluster is represented in Fig. 3 with line, it is first that feature is similar Sample gathers for one kind, the group of formation again it is similar to other feature it is small birds of the same feather flock together for major class, cluster upwards successively, until all samples Originally gather for one kind.The result of bidirectional clustering is shown in Fig. 3.Big figure left side line is according to sample clustering as a result, can see It can be divided into two major classes to sample, and this coincide just with disease group and control group, it was demonstrated that suffer from dental caries children and healthy children in profit With the ability aspect of this 10 kinds of carbon sources there are larger difference, it is to have effect for whether differentiation suffers from dental caries also to illustrate this 10 kinds of carbon sources 's.Big figure top line is to be clustered according to different carbon source as a result, wherein maltose list belongs to another into one kind, other 9 kinds of carbon sources It is a kind of.And in the class where other 9 kinds, 6 kinds of carbohydrates distinguish again with 3 kinds of amino acid and peptides, illustrate that 20 samples exist It is had differences in terms of using carbohydrate and amino acid and peptides.

(7) principal component analysis (PCA)

Figure of description 4 is principal component analysis schematic diagram.When principal component analysis is that sample measurement dimension is more, such as originally grind For 10 species diversity carbon sources in studying carefully, by dimensionality reduction, with the index of several synthesis, such as principal component 1 (PC1), principal component 2 (PC2) etc., Come to sample, such as totally 20 samples are assessed for the disease group and control group of this research.This research and utilization R softwares complete it is main into Analysis.

Specifically, Figure of description 4 is a bi-plot (biplot), is being distinguished between each carbon source of Main Analysis The ability of disease group and control group.Transverse axis is principal component 1, and the longitudinal axis is principal component 2, and the percentage in bracket represents main composition variance The percentage of former variable variance is accounted for, i.e., it can represent the how many variation of entire data distribution, and the two accounts for 23.24% He respectively 17.21%.This research is primarily upon PC1.10 arrows are sent from the center of figure (0,0), represent 10 carbon sources, the direction of line segment There is certain representative meaning with length：

● the carbon source that 7 arrows to the left represent belongs to carbohydrate, because it is directed toward the negative direction of principal axis of transverse axis, illustrates these variables It is negatively correlated with PC1；

● the carbon source that 3 arrows to the right represent belongs to amino acid and peptides, these variables are positively correlated with PC1；

● the length of arrow can be regarded as separating capacity of the carbon source to disease group and control group, and arrow is longer, it by certain The ability that its direction region is assigned in sample area is stronger.

Scatterplot representative sample on figure, triangle are disease groups, and rectangle is control group.It can be seen that two groups of be in figure substantially two Side, disease group is substantially in left one side of something, and substantially in right one side of something, two groups can better be distinguished control group by PC1；By arrow and Sample, which is combined, to be seen, carbohydrate is stronger for the separating capacity of disease group, and amino acid and peptides are to the separating capacity of control group It is relatively strong.The analysis result of this and our tables 2 is also identical, i.e., disease group and is compareed to the utilization of 7 kinds of carbohydrates more than control group Group is better than disease group (statistically significant) using the ability of 3 kinds of amino acid and peptides.

With reference to above analysis as it can be seen that can efficiently distinguish dental caries patient and non-dental caries person using this 10 kinds of single carbon sources.In low age It in children, is seen by result, it is centainly susceptible to saprodontia to suffer from the infant that saprodontia is very more in dental caries, especially mouth, and has and illness It is susceptible that the similar subject of person's bacterial plaque falls within dental caries.Therefore, also can efficiently to distinguish dental caries using this 10 kinds of single carbon sources susceptible Person and the non-susceptible person of dental caries.

(8) product

More than Experiment Result is based on, the present invention proposes a kind of kit for predicting whether infant is susceptible to dental caries, kit Include maltose, Alpha-Methyl-D- galactosides, α-amarogentin, D, L- alpha-phosphates glycerine, D- trehaloses, 3- methyl Ds-Portugal Grape sugar, turanose, glycyl-L-PROLINE, L-Glutamine and glycyl-L-glutamine amount to 10 kinds of carbon sources, for pre- Survey the risk that infant suffers from saprodontia.

In addition to above 10 kinds of carbon sources, kit further includes tetrazolium violet and water, can also delay equipped with phosphoric acid physiological saline Fliud flushing.Referring to Figure of description 5,12 holes are equipped on plate altogether for kit generally platy structure.In wherein 10 holes, such as 1-10 holes are respectively provided with 10 kinds of carbon sources and tetrazolium violet, and water and tetrazolium violet are housed in 1 hole, and 1 hole is sky.

The specific method tested using the product tester is：Tester cannot eat more foods after the meal early And it cannot brush teeth.About it is early after meal 2 it is small when, it is desirable that tester is gargled with clear water.It is taken from the complete enamel of eight deciduous molars Sample plaque.Sample is collected with sterile excavator spoon, and addition fills the 0.01mol/L phosphate buffers of 1.5mL pH7.2-7.4 In centrifuge tube, sample suspensions wherein, are vortexed and shake abundant mixing 60s.Then the bacterium solution of mixing is seeded under anaerobic Kit contains in the hole of carbon source, and 100 μ L are loaded per hole.Hole comprising water is as blank control.Kit after inoculation is put In anaerobism bag, cultivated 1 day under conditions of 37 DEG C.Measure 590nm OD values (OD590nm), cluster analysis, principal component analysis, and It is compared with the corresponding data of patient in our databases, healthy person, finally draws neurological susceptibility of the subject for dental caries.

It is recited above simply to illustrate a kind of embodiment for the kit for predicting whether infant is susceptible to dental caries of the present invention, Due to being easy to carry out several modifications and change on this basis for the those of ordinary skill in same technique field, this Specification is not intended to a kind of kit for predicting whether infant is susceptible to dental caries of the present invention being confined to shown and described tool In body structure or compositing range, thus it is every it is possible that the corresponding modification being utilized and equivalent, belong to the present invention and applied The scope of the claims.

Claims (6)

1. a kind of kit for predicting whether children are susceptible to dental caries, it is characterised in that including maltose, Alpha-Methyl-D- galactolipins Glycosides, α-amarogentin, D, L- alpha-phosphates glycerine, D- trehaloses, 3- methyl-D-glucoses, turanose, glycyl-L-PROLINE, L-Glutamine and glycyl-L-glutamine amount to 10 kinds of carbon sources, for predicting risk that children suffer from saprodontia.

2. kit as described in claim 1, generally platy structure, plate is equipped with to place the carbon source and colour developing Wherein placing the different carbon sources in different holes, color developing agent is equipped in each hole for the hole of agent.

3. kit as claimed in claim 2, wherein the color developing agent is tetrazolium violet.

4. kit as claimed in claim 2, further includes the hole equipped with water and color developing agent and/or internal be used as empty hole is joined According to.

5. the kit as described in claim 1-4 any one, further includes phosphoric acid normal saline buffer solution.

6. kit as claimed in claim 5, the phosphoric acid normal saline buffer solution is the 0.01mol/ that pH value is 7.2-7.4 L phosphate buffers.