CN107513580A - The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis - Google Patents
The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis Download PDFInfo
- Publication number
- CN107513580A CN107513580A CN201710988317.4A CN201710988317A CN107513580A CN 107513580 A CN107513580 A CN 107513580A CN 201710988317 A CN201710988317 A CN 201710988317A CN 107513580 A CN107513580 A CN 107513580A
- Authority
- CN
- China
- Prior art keywords
- porphyromonas gingivalis
- liquid
- gum base
- base type
- porphyromonas
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis, belong to technical field of biological.This method prepares the artificial saliva containing porphyromonas gingivalis first, and for the handling characteristics of gum base type product, machine is chewed using full analogue simulation to simulate the mastication processes of gum base type tobacco product, not only allow for the interaction of product leachable and saliva Porphyromonas gingivalis, also fully simulate adhesive attraction of the mastication processes gum base type product to bacterium, so that chewing of the whole change procedure closer to people, quantitative fluorescent PCR analysis is carried out to the porphyromonas gingivalis content of saliva in mastication processes afterwards, so as to investigate the change of the porphyromonas gingivalis content before and after using gum base type tobacco product in saliva, oral health for gum base type tobacco product provides reference.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of gum base type tobacco product is to porphyromonas gingivalis
The detection method of influence.
Background technology
Shown according to national third time oral health epidemiological investigation result, chronic periodontitis (chronic period
Ontitis, CP) illness rate reach more than 80%, and the main reason for be more than 35 years old crowd's absence of tooth.Modern medicine
Research shows that chronic periodontitis is due to that patient's oral hygiene environment is poor more, and periodontium caused by the factor such as Occlusal Trauma is gradually
The property entered is destroyed, and shows as halitosis, gum redness, bleeding initial stage, long is not treated, and tooth mobility, alveolar bone can be caused to break more
It is bad, even have tooth pulled out, it is also relate to the function of the important organs such as the heart, lung, kidney in addition.Periodontium damage with it is micro- under a variety of gums
Biology is relevant, and its Porphyromonas gingivalis (Porphyromonas gingivalis, P.gingivalis) is considered as slow
One of property most important pathogenic bacteria of periodontitis, the bacterium often detects in patients with periodontitis subgingival plaque and saliva, in periodontal health
It can also be detected in crowd.Porphyromonas gingivalis has a series of causes such as lipopolysaccharides, capsular polysaccharide, pili, gingipain
Cause of disease, can succeed attack to host cell, and escape immune system.
Gum base type smoke-free tobacco product (Tobacco chewing gum) be it is a kind of added in matrix it is a certain amount of
A kind of mouth smoke-free tobacco product made of tobacco extract, spices and some other edible additive.Gum base type tobacco
The occupation mode of product is to be put into mouth to chew, and the leachable of chewing can converge aggregation in the oral cavity with saliva, be practised according to consumption
Used difference is spued or swallowed.During product use, influence of the product to human mouth health is an important investigation
Index.Current tobacco product carries out the detection of some physical and chemical index only for product in itself, and to micro- in human mouth
Biotic influence research is less, and the research in particular for oral cavity Porphyromonas gingivalis is not related to.Therefore how to overcome existing
The problem of deficiency for having technology is current technical field of biological urgent need to resolve.
The content of the invention
The invention aims to solve the deficiencies in the prior art, there is provided a kind of gum base type tobacco product is to porphyromonas
The detection method that monad influences, this method prepares the artificial saliva containing porphyromonas gingivalis first, and is directed to gum base type
The handling characteristics of product, machine is chewed using full analogue simulation to simulate the mastication processes of gum base type tobacco product, to mastication processes
The porphyromonas gingivalis content of middle saliva carries out quantitative fluorescent PCR analysis, so as to investigate before using gum base type tobacco product
The change of the porphyromonas gingivalis content in saliva, the oral health for gum base type tobacco product provide reference afterwards.
To achieve the above object, the technical solution adopted by the present invention is as follows：
The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*104-1*106CFU/mL, obtain the artificial saliva containing porphyromonas gingivalis, numbering 1#
Liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) contains gum several times
The artificial saliva of Detection of Porphyromonas, is manually chewed, and in mastication processes, collects the saliva of different chew time points；
Step (3), the saliva for the different chew time points that step (2) is collected into and the list containing porphyromonas of step (1)
The artificial saliva of born of the same parents bacterium is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract each time point nutrient solution respectively afterwards
In porphyromonas gingivalis DNA, while extract the reference culture original DNA of porphyromonas gingivalis；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient enter as template
Row quantitative fluorescent PCR is analyzed, and draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR is carried out using the porphyromonas gingivalis DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate gum Porphyromonas in each time point nutrient solution using sterilizing distilled water according to standard curve
Bacterial content；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagatagg c-TAMRA.
It is further preferred that the specific method of step (2) is：
0.8-1.2g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, implantation step (1)
Artificial saliva 10mL containing porphyromonas gingivalis, is manually chewed；Set and chew mechanical tooth with frequency progress once in 5 seconds
Occlusion and left and right mill changing of the relative positions work up and down, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every 4
Secondary occlusion up and down and left and right, which are ground the changing of the relative positions and done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；It is complete
Liquid into rear collection solution ware, numbering is 2# liquid；
The artificial saliva 10mL containing porphyromonas gingivalis of step (1) is reinjected for the second time, is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 10 seconds frequencies once, totally 10 times, occlusion width is 10mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing porphyromonas gingivalis of step (1), is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 5 seconds frequencies once, totally 30 times, occlusion width is 8mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
It is further preferred that the specific method of step (3) is：
(3.1) co-culture：2# liquid, 3# liquid and the 4# liquid in step (2) and the 1# liquid in step (1) are taken, often
Kind of liquid is divided into 3 equal portions, is respectively put into anaerobic culture box and is co-cultured at 37 DEG C, during the culture of every kind of equal portions of liquid 3
Between be respectively tri- periods of 0h, 2h, 6h；
(3.2) nutrient solution Porphyromonas gingivalis DNA extraction：Take out the nutrient solution 1mL after the culture of step (3.1)
It is placed in sterile 1.5mL centrifuge tubes, centrifuges 10min under being afterwards 12000r/min in 4 DEG C, rotating speed, remove supernatant, add
50 μ L microorganism PCR lysates, after being well mixed, thermal denaturation 15min at 80 DEG C is placed in, is then centrifuged under 1000r/min
1min, takes supernatant, that is, obtains nutrient solution Porphyromonas gingivalis DNA；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：It is 10 to take 1mL concentration7CFU/mL gum
Detection of Porphyromonas after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganisms PCR and split in the 1.5mL centrifuge tubes of sterilizing
Liquid is solved, the thermal denaturation 15min at 80 DEG C, then 10s is centrifuged under 1000r/m, takes supernatant, that is, obtain porphyromonas gingivalis
Reference culture original DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
It is further preferred that the reference culture to porphyromonas gingivalis is needed before quantitative fluorescent PCR analysis is carried out
Original DNA, the purity of porphyromonas gingivalis DNA in each time point nutrient solution judge that decision method is：By testing sample
The OD value that DNA is determined under wavelength 260nm, 280nm respectively；When the OD value under wavelength 260nm and in wavelength 280nm
When the ratio of lower OD value is 1.7~1.9, shows that purity is qualified, quantitative fluorescent PCR analysis can be carried out, conversely, then showing pure
Spend unqualified, it is impossible to carry out quantitative fluorescent PCR analysis.
It is further preferred that the specific method of step (4) is：
The μ L of reference culture original DNA 5 of porphyromonas gingivalis are taken, adds the μ L of water 45 and fully mixes, then take turns doing 10 times
Concentration gradient dilutes, and dilutes 6 times altogether, as standard curve template；
Take each nutrient solution Porphyromonas gingivalis DNA with sterilized water by 1:Test sample is used as after the dilution of 8 volume ratios
Template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
It is further preferred that during quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes it average
Value is calculated.
Compared with prior art, its advantage is the present invention：
The present invention can simulate human mouth and chew what oral cavity Porphyromonas gingivalis during gum base type tobacco product occurred
Change, there is certain directive significance for understanding influence of the gum base type tobacco product to oral health.Using chewing simulating machine nozzle
Gum base type tobacco product is chewed, not only allows for the interaction of product leachable and saliva Porphyromonas gingivalis, it is also abundant
Adhesive attraction of the mastication processes gum base type product to bacterium is simulated, so that chewing of the whole change procedure closer to people.
Employ fluorescence quantifying PCR method to analyze saliva Porphyromonas gingivalis, microorganism can contain in real-time tracking saliva
The change of amount, it ensure that the accuracy and high efficiency of experiment.3 dynamic saliva collection points and after chewing when devising chewing simultaneously
3 co-cultivation periods, dynamic saliva collection point is to simulate chewing dynamic process of people when using tobacco product, during co-cultivation
Between section to simulate oral cavity static process of the people after chewing, make detection more fully accurate.
Brief description of the drawings
Fig. 1 is real-time fluorescence quantitative PCR detection porphyromonas gingivalis canonical plotting；
Fig. 2 is nutrient solution Porphyromonas gingivalis testing result figure.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright scope.In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art
Or carried out according to product description.Material therefor or the unreceipted production firm person of equipment, it is that can be obtained by buying
Conventional products.
Lysis Buffer for Microorganism to Direct PCR are limited purchased from precious bioengineering (Dalian)
Company, specification is referring to network address http://www.docin.com/p-317504172.html.
It is the products of ZL 201610555175.8 that the full analogue simulation that the present invention uses, which chews machine,.
PCR kit for fluorescence quantitative of the present invention is purchased from KAPA PROBE FAST qPCR.
The present invention prepares artificial saliva and prepared by the conventional method of the art.
Blank control template of the present invention is for the baseline value of calibration standard curve using sterilizing distilled water.
Gum base type tobacco product used in the embodiment of the present invention is purchased from cigarette industry responsibility Co., Ltd in Yunnan.
The reference culture of porphyromonas gingivalis used in the embodiment of the present invention is purchased from Guangdong Province's Microbiological Culture Collection
The heart.
Embodiment 1
The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*104CFU/mL, obtains the artificial saliva containing porphyromonas gingivalis, and numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) contains gum several times
The artificial saliva of Detection of Porphyromonas, is manually chewed, and in mastication processes, collects the saliva of different chew time points；Specific side
Method is：
1.2g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, implantation step (1) contains tooth
The artificial saliva 10mL of gum Detection of Porphyromonas, is manually chewed；Set and chew mechanical tooth with above and below frequency progress once in 5 seconds
Occlusion and left and right mill changing of the relative positions work, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every on 4 times
Lower occlusion and left and right, which are ground the changing of the relative positions and done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of
The liquid in solution ware is collected, numbering is 2# liquid；
The artificial saliva 10mL containing porphyromonas gingivalis of step (1) is reinjected for the second time, is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 10 seconds frequencies once, totally 10 times, occlusion width is 10mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing porphyromonas gingivalis of step (1), is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 5 seconds frequencies once, totally 30 times, occlusion width is 8mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
Step (3), the saliva for the different chew time points that step (2) is collected into and the list containing porphyromonas of step (1)
The artificial saliva of born of the same parents bacterium is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract each time point nutrient solution respectively afterwards
In porphyromonas gingivalis DNA, while extract the reference culture original DNA of porphyromonas gingivalis；Specific method is：
(3.1) co-culture：2# liquid, 3# liquid and the 4# liquid in step (2) and the 1# liquid in step (1) are taken, often
Kind of liquid is divided into 3 equal portions, is respectively put into anaerobic culture box and is co-cultured at 37 DEG C, during the culture of every kind of equal portions of liquid 3
Between be respectively tri- periods of 0h, 2h, 6h；
(3.2) nutrient solution Porphyromonas gingivalis DNA extraction：Take out the nutrient solution 1mL after the culture of step (3.1)
It is placed in sterile 1.5mL centrifuge tubes, centrifuges 10min under being afterwards 12000r/min in 4 DEG C, rotating speed, remove supernatant, add
50 μ L microorganism PCR lysates, after being well mixed, thermal denaturation 15min at 80 DEG C is placed in, is then centrifuged under 1000r/min
1min, takes supernatant, that is, obtains nutrient solution Porphyromonas gingivalis DNA；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：It is 10 to take 1mL concentration7CFU/mL gum
Detection of Porphyromonas after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganisms PCR and split in the 1.5mL centrifuge tubes of sterilizing
Liquid is solved, the thermal denaturation 15min at 80 DEG C, then 10s is centrifuged under 1000r/m, takes supernatant, that is, obtain porphyromonas gingivalis
Reference culture original DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient enter as template
Row quantitative fluorescent PCR is analyzed, and draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR is carried out using the porphyromonas gingivalis DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate gum Porphyromonas in each time point nutrient solution using sterilizing distilled water according to standard curve
Bacterial content；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagataggc-TAMRA；
The reference culture original DNA to porphyromonas gingivalis, each time point are needed before quantitative fluorescent PCR analysis is carried out
The purity of porphyromonas gingivalis DNA in nutrient solution judges that decision method is：By the DNA of testing sample respectively in wavelength
The OD value determined under 260nm, 280nm；When the OD value under wavelength 260nm and the OD value under wavelength 280nm
When ratio is 1.7~1.9, shows that purity is qualified, quantitative fluorescent PCR analysis can be carried out, conversely, then showing that purity is unqualified, no
Quantitative fluorescent PCR analysis can be carried out.
The specific method of step (4) is：
The μ L of reference culture original DNA 5 of porphyromonas gingivalis are taken, adds the μ L of water 45 and fully mixes, then take turns doing 10 times
Concentration gradient dilutes, and dilutes 6 times altogether, as standard curve template；
Take each nutrient solution Porphyromonas gingivalis DNA with sterilized water by 1:Test sample is used as after the dilution of 8 volume ratios
Template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
Embodiment 2
The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*106CFU/mL, obtains the artificial saliva containing porphyromonas gingivalis, and numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) contains gum several times
The artificial saliva of Detection of Porphyromonas, is manually chewed, and in mastication processes, collects the saliva of different chew time points；Specific side
Method is：
0.8g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, implantation step (1) contains tooth
The artificial saliva 10mL of gum Detection of Porphyromonas, is manually chewed；Set and chew mechanical tooth with above and below frequency progress once in 5 seconds
Occlusion and left and right mill changing of the relative positions work, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every on 4 times
Lower occlusion and left and right, which are ground the changing of the relative positions and done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of
The liquid in solution ware is collected, numbering is 2# liquid；
The artificial saliva 10mL containing porphyromonas gingivalis of step (1) is reinjected for the second time, is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 10 seconds frequencies once, totally 10 times, occlusion width is 10mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing porphyromonas gingivalis of step (1), is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 5 seconds frequencies once, totally 30 times, occlusion width is 8mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
Step (3), the saliva for the different chew time points that step (2) is collected into and the list containing porphyromonas of step (1)
The artificial saliva of born of the same parents bacterium is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract each time point nutrient solution respectively afterwards
In porphyromonas gingivalis DNA, while extract the reference culture original DNA of porphyromonas gingivalis；Specific method is：
(3.1) co-culture：2# liquid, 3# liquid and the 4# liquid in step (2) and the 1# liquid in step (1) are taken, often
Kind of liquid is divided into 3 equal portions, is respectively put into anaerobic culture box and is co-cultured at 37 DEG C, during the culture of every kind of equal portions of liquid 3
Between be respectively tri- periods of 0h, 2h, 6h；
(3.2) nutrient solution Porphyromonas gingivalis DNA extraction：Take out the nutrient solution 1mL after the culture of step (3.1)
It is placed in sterile 1.5mL centrifuge tubes, centrifuges 10min under being afterwards 12000r/min in 4 DEG C, rotating speed, remove supernatant, add
50 μ L microorganism PCR lysates, after being well mixed, thermal denaturation 15min at 80 DEG C is placed in, is then centrifuged under 1000r/min
1min, takes supernatant, that is, obtains nutrient solution Porphyromonas gingivalis DNA；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：It is 10 to take 1mL concentration7CFU/mL gum
Detection of Porphyromonas after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganisms PCR and split in the 1.5mL centrifuge tubes of sterilizing
Liquid is solved, the thermal denaturation 15min at 80 DEG C, then 10s is centrifuged under 1000r/m, takes supernatant, that is, obtain porphyromonas gingivalis
Reference culture original DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient enter as template
Row quantitative fluorescent PCR is analyzed, and draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR is carried out using the porphyromonas gingivalis DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate gum Porphyromonas in each time point nutrient solution using sterilizing distilled water according to standard curve
Bacterial content；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagataggc-TAMRA；
The reference culture original DNA to porphyromonas gingivalis, each time point are needed before quantitative fluorescent PCR analysis is carried out
The purity of porphyromonas gingivalis DNA in nutrient solution judges that decision method is：By the DNA of testing sample respectively in wavelength
The OD value determined under 260nm, 280nm；When the OD value under wavelength 260nm and the OD value under wavelength 280nm
When ratio is 1.7~1.9, shows that purity is qualified, quantitative fluorescent PCR analysis can be carried out, conversely, then showing that purity is unqualified, no
Quantitative fluorescent PCR analysis can be carried out.
The specific method of step (4) is：
The μ L of reference culture original DNA 5 of porphyromonas gingivalis are taken, adds the μ L of water 45 and fully mixes, then take turns doing 10 times
Concentration gradient dilutes, and dilutes 6 times altogether, as standard curve template；
Take each nutrient solution Porphyromonas gingivalis DNA with sterilized water by 1:Test sample is used as after the dilution of 8 volume ratios
Template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
During quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes its average value to be calculated.
Embodiment 3
The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*105CFU/mL, obtains the artificial saliva containing porphyromonas gingivalis, and numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) contains gum several times
The artificial saliva of Detection of Porphyromonas, is manually chewed, and in mastication processes, collects the saliva of different chew time points；Specific side
Method is：
1g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, implantation step (1) contains gum
The artificial saliva 10mL of Detection of Porphyromonas, is manually chewed；Set and chew mechanical tooth to be stung above and below frequency progress once in 5 seconds
Close and the left and right mill changing of the relative positions is made, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every about 4 times
Occlusion and left and right, which are ground the changing of the relative positions and done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of receive
Collect the liquid in solution ware, numbering is 2# liquid；
The artificial saliva 10mL containing porphyromonas gingivalis of step (1) is reinjected for the second time, is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 10 seconds frequencies once, totally 10 times, occlusion width is 10mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing porphyromonas gingivalis of step (1), is manually chewed；Set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 5 seconds frequencies once, totally 30 times, occlusion width is 8mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
Step (3), the saliva for the different chew time points that step (2) is collected into and the list containing porphyromonas of step (1)
The artificial saliva of born of the same parents bacterium is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract each time point nutrient solution respectively afterwards
In porphyromonas gingivalis DNA, while extract the reference culture original DNA of porphyromonas gingivalis；Specific method is：
(3.1) co-culture：2# liquid, 3# liquid and the 4# liquid in step (2) and the 1# liquid in step (1) are taken, often
Kind of liquid is divided into 3 equal portions, is respectively put into anaerobic culture box and is co-cultured at 37 DEG C, during the culture of every kind of equal portions of liquid 3
Between be respectively tri- periods of 0h, 2h, 6h；
(3.2) nutrient solution Porphyromonas gingivalis DNA extraction：Take out the nutrient solution 1mL after the culture of step (3.1)
It is placed in sterile 1.5mL centrifuge tubes, centrifuges 10min under being afterwards 12000r/min in 4 DEG C, rotating speed, remove supernatant, add
50 μ L microorganism PCR lysates, after being well mixed, thermal denaturation 15min at 80 DEG C is placed in, is then centrifuged under 1000r/min
1min, takes supernatant, that is, obtains nutrient solution Porphyromonas gingivalis DNA；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：It is 10 to take 1mL concentration7CFU/mL gum
Detection of Porphyromonas after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganisms PCR and split in the 1.5mL centrifuge tubes of sterilizing
Liquid is solved, the thermal denaturation 15min at 80 DEG C, then 10s is centrifuged under 1000r/m, takes supernatant, that is, obtain porphyromonas gingivalis
Reference culture original DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient enter as template
Row quantitative fluorescent PCR is analyzed, and draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR is carried out using the porphyromonas gingivalis DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate gum Porphyromonas in each time point nutrient solution using sterilizing distilled water according to standard curve
Bacterial content；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagataggc-TAMRA；
The reference culture original DNA to porphyromonas gingivalis, each time point are needed before quantitative fluorescent PCR analysis is carried out
The purity of porphyromonas gingivalis DNA in nutrient solution judges that decision method is：By the DNA of testing sample respectively in wavelength
The OD value (optical density, OD) determined under 260nm, 280nm；When the OD value under wavelength 260nm and in ripple
When the ratio (OD260 and OD280 ratios) of OD value is 1.7~1.9 under long 280nm, shows that purity is qualified, fluorescence can be carried out
Quantitative PCR analysis, conversely, then showing that purity is unqualified, it is impossible to carry out quantitative fluorescent PCR analysis.
The specific method of step (4) is：
The μ L of reference culture original DNA 5 of porphyromonas gingivalis are taken, adds the μ L of water 45 and fully mixes, then take turns doing 10 times
Concentration gradient dilutes, and dilutes 6 times altogether, as standard curve template；
Take each nutrient solution Porphyromonas gingivalis DNA with sterilized water by 1:Test sample is used as after the dilution of 8 volume ratios
Template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
During quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes its average value to be calculated.
As a result as shown in Fig. 1, table 1 and Fig. 2, table 1 is the testing result of nutrient solution Porphyromonas gingivalis content.
Wherein, standard curve is y=-3.960log (x)+36.90, r2=0.9791；Afterwards by each time point nutrient solution
Ct values be updated in standard curve, try to achieve corresponding to porphyromonas gingivalis content.
Table 1
Note：Unit is CFU/mL, M ± SD.
By table 1 and Fig. 2, it can learn that the nutrient solution Porphyromonas gingivalis content after chewing decreases, wherein 2#
Liquid reduce it is the most notable, this be probably due to gum base type tobacco product have the function that absorption porphyromonas gingivalis, and
The growth rate of bacterium also decreases in culture, and reason is probably that the material for chewing dissolution has certain suppression porphyromonas
The effect of unit cell bacteria growing.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table is as shown in table 2.
Table 2
| Primer and probe | Sequence 5 ' -3 ' |
| Forward primer | 5’-tacccatcgtcgccttggt-3’(SEQ ID NO.1) |
| Reverse primer | 5’-cggactaaaaccgcatacacttg-3’(SEQ ID NO.2) |
| Probe | FAM-atttatagctgtaagataggc-TAMRA(SEQ ID NO.3) |
Sequence table
<110>Cigarette industry Co., Ltd in Yunnan
<120>The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence ()
<400> 1
tacccatcgt cgccttggt 19
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence ()
<400> 2
cggactaaaa ccgcatacac ttg 23
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence ()
<400> 3
atttatagct gtaagatagg c 21
Claims (6)
1. the detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis, it is characterised in that including following step
Suddenly：
Step（1）, artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, porphyromonas gingivalis is existed
Amount in artificial saliva is 1*104-1*106 CFU/mL, obtains the artificial saliva containing porphyromonas gingivalis, and numbering is 1# liquid
Body；
Step（2）, gum base type tobacco product is put into chewing simulating machine, and implantation step several times（1）Contain porphyromonas
The artificial saliva of monad, is manually chewed, and in mastication processes, collects the saliva of different chew time points；
Step（3）, by step（2）The saliva for the different chew time points being collected into and step（1）Contain porphyromonas gingivalis
Artificial saliva be put into anaerobic culture box together and co-cultured at 37 DEG C；Extract respectively afterwards in each time point nutrient solution
Porphyromonas gingivalis DNA, while extract the reference culture original DNA of porphyromonas gingivalis；
Step（4）, carried out using the reference culture original DNA of 10 times of porphyromonas gingivalis of concentration gradient dilution as template glimmering
Fluorescent Quantitative PCR is analyzed, and draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the porphyromonas gingivalis DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate each time point nutrient solution Porphyromonas gingivalis using sterilizing distilled water according to standard curve
Content；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Anti-sense primer
For 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagataggc-TAMRA.
2. the detection method that gum base type tobacco product according to claim 1 influences on porphyromonas gingivalis, its feature
It is, step（2）Specific method be：
0.8-1.2g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, implantation step（1）Contain tooth
The artificial saliva 10mL of gum Detection of Porphyromonas, is manually chewed；Set and chew mechanical tooth with above and below frequency progress once in 5 seconds
Occlusion and left and right mill changing of the relative positions work, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every on 4 times
Lower occlusion and left and right, which are ground the changing of the relative positions and done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of
The liquid in solution ware is collected, numbering is 2# liquid；
Step is reinjected for the second time（1）The mL of artificial saliva 10 containing porphyromonas gingivalis, manually chewed；Nozzle is set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 10 seconds frequencies once, totally 10 times, occlusion width is 10mm；Together
When, start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, every time
The anglec of rotation for gathering rolling clamping plate is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects step（1）The mL of artificial saliva 10 containing porphyromonas gingivalis, manually chewed；Nozzle is set
Chew mechanical tooth and occlusion up and down and left and right mill changing of the relative positions work were carried out with 5 seconds frequencies once, totally 30 times, occlusion width is 8mm；Meanwhile
Start opening and closing left and right rotary components, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, gather every time
The anglec of rotation of rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
3. the detection method that gum base type tobacco product according to claim 2 influences on porphyromonas gingivalis, its feature
It is, step（3）Specific method be：
（3.1）Co-culture：Take step（2）In 2# liquid, 3# liquid and 4# liquid and step（1）In 1# liquid, every kind of liquid
Body is divided into 3 equal portions, is respectively put into anaerobic culture box and is co-cultured at 37 DEG C, the incubation time point of every kind of equal portions of liquid 3
Wei not tri- periods of 0h, 2h, 6h；
（3.2）Nutrient solution Porphyromonas gingivalis DNA extraction：Take out step（3.1）Culture after nutrient solution 1mL be placed in
In sterile 1.5mL centrifuge tubes, 10min is centrifuged under being afterwards 12000r/min in 4 DEG C, rotating speed, supernatant is removed, adds 50 μ
L microorganism PCR lysates, after being well mixed, thermal denaturation 15min at 80 DEG C is placed in, then centrifuges 1min under 1000r/min,
Supernatant is taken, that is, obtains nutrient solution Porphyromonas gingivalis DNA；
（3.3）The extraction of the reference culture original DNA of porphyromonas gingivalis：It is 10 to take 1mL concentration7CFU/mL porphyromonas
Monad after 12000 r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganisms PCR cracking in the 1.5mL centrifuge tubes of sterilizing
Liquid, the thermal denaturation 15min at 80 DEG C, then centrifuges 10 s under 1000r/m, takes supernatant, that is, obtains porphyromonas gingivalis
Reference culture original DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
4. the detection method that the gum base type tobacco product according to claim 1 or 3 influences on porphyromonas gingivalis, it is special
Sign is, the reference culture original DNA to porphyromonas gingivalis, each time point are needed before quantitative fluorescent PCR analysis is carried out
The purity of porphyromonas gingivalis DNA in nutrient solution judges that decision method is：By the DNA of testing sample respectively in wavelength
The OD value determined under 260nm, 280nm；When the OD value under wavelength 260nm and the OD value under wavelength 280nm
When ratio is 1.7 ~ 1.9, shows that purity is qualified, quantitative fluorescent PCR analysis can be carried out, conversely, then showing that purity is unqualified, it is impossible to
Carry out quantitative fluorescent PCR analysis.
5. the detection method that gum base type tobacco product according to claim 3 influences on porphyromonas gingivalis, its feature
It is, step（4）Specific method be：
The μ L of reference culture original DNA 5 of porphyromonas gingivalis are taken, adds the μ L of water 45 and fully mixes, then take turns doing 10 times of concentration
Gradient dilution, dilute 6 times altogether, as standard curve template；
Take each nutrient solution Porphyromonas gingivalis DNA with sterilized water by 1:Test sample template is used as after the dilution of 8 volume ratios；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
μ L of 200 nmol/L of sense primer 0.175,
μ L of 200 nmol/L of anti-sense primer 0.175,
μ L of 250 nmol/L of probe 0.175,
μ L of sterilized water 1.475,
μ L of template 8,
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s, 1min at 60 DEG C of annealing/extension at 95 DEG C of denaturation,
Carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point culture is calculated according to standard curve
Liquid Porphyromonas gingivalis content.
6. the detection method that gum base type tobacco product influences on porphyromonas gingivalis according to claim 1 or 5, it is special
Sign is that when quantitative fluorescent PCR analysis detects, each pattern detection in triplicate, takes its average value to be calculated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710988317.4A CN107513580A (en) | 2017-10-21 | 2017-10-21 | The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710988317.4A CN107513580A (en) | 2017-10-21 | 2017-10-21 | The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107513580A true CN107513580A (en) | 2017-12-26 |
Family
ID=60727466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710988317.4A Pending CN107513580A (en) | 2017-10-21 | 2017-10-21 | The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107513580A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507789A (en) * | 2011-11-17 | 2012-06-20 | 中国烟草总公司郑州烟草研究院 | Method for detecting nicotine release behavior of gum base type smoke-free tobacco product |
| CN102507790A (en) * | 2011-11-17 | 2012-06-20 | 中国烟草总公司郑州烟草研究院 | In-vitro simulation detection method for bioavailability of nicotine in tobacco gum base product |
| CN106053731A (en) * | 2016-07-14 | 2016-10-26 | 云南中烟工业有限责任公司 | Simulating mechanical tongue |
| CN106222234A (en) * | 2016-08-26 | 2016-12-14 | 云南中烟工业有限责任公司 | A kind of gum base type tobacco product that detects is on the method for impact outside human mouth microbial body |
-
2017
- 2017-10-21 CN CN201710988317.4A patent/CN107513580A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507789A (en) * | 2011-11-17 | 2012-06-20 | 中国烟草总公司郑州烟草研究院 | Method for detecting nicotine release behavior of gum base type smoke-free tobacco product |
| CN102507790A (en) * | 2011-11-17 | 2012-06-20 | 中国烟草总公司郑州烟草研究院 | In-vitro simulation detection method for bioavailability of nicotine in tobacco gum base product |
| CN106053731A (en) * | 2016-07-14 | 2016-10-26 | 云南中烟工业有限责任公司 | Simulating mechanical tongue |
| CN106222234A (en) * | 2016-08-26 | 2016-12-14 | 云南中烟工业有限责任公司 | A kind of gum base type tobacco product that detects is on the method for impact outside human mouth microbial body |
Non-Patent Citations (3)
| Title |
|---|
| JUHI BAGAITKAR ET AL: "Tobacco Upregulates P. gingivalis Fimbrial Proteins Which Induce TLR2 Hyposensitivity", 《PLOS ONE》 * |
| JUHI BAGAITKAR ET AL: "Tobacco-induced alterations to Porphyromonas gingivalis-host interactions", 《ENVIRON MICROBIOL.》 * |
| THIAGO CRUVINEL SILVA ET AL: "Oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children", 《BRAZ J ORAL SCI.》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kadir et al. | Prevalence of Candida species in Turkish children: relationship between dietary ıntake and carriage | |
| Eşian et al. | Salivary level of streptococcus mutans and lactobacillus spp. related to a high a risk of caries disease | |
| CN110257297A (en) | Lactobacillus paracasei PC-01 and its application for being used to promote oral health of one plant of separation | |
| CN113005055B (en) | Lactobacillus plantarum for preventing and/or treating periodontitis, culture thereof, and preparation and application thereof | |
| Ambrosio et al. | Detection and quantification of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in bacteremia induced by interdental brushing in periodontally healthy and periodontitis patients | |
| TWM542208U (en) | Oral health examination and assessment system | |
| Gies et al. | CHEMICAL STUDIES OF THE RELATIONS OF ORAL MICROORGANISMS TO DENTAL CARIES ¹ | |
| CN1840206A (en) | Model construction of human flora-associated piggy and molecular method for detecting flora in intestine tract of baby pig | |
| CN109330942A (en) | A kind of formulation of tooth-paste containing oral cavity probiotics | |
| Martao Florio et al. | Time of initial acquisition of mutans streptococci by human infants | |
| CN111518713B (en) | Lactobacillus rhamnosus capable of preventing and/or treating dental caries and application thereof | |
| Liu et al. | Typing of mutans streptococci by arbitrarily primed PCR in patients undergoing orthodontic treatment | |
| TWI757586B (en) | Plant ferment, preparation method thereof and use thereof for liver health care | |
| CN107513580A (en) | The detection method that a kind of gum base type tobacco product influences on porphyromonas gingivalis | |
| CN111826304A (en) | Streptococcus thermophilus and application thereof | |
| CN107630072A (en) | The detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis | |
| Yilmaz et al. | Adherence frequency of CANDIDA ALBICANS on nasoalveolar molding (NAM) appliances | |
| CN107513581A (en) | The detection method that a kind of gum base type tobacco product influences on streptococcus mutans | |
| CN114164157A (en) | Lactobacillus salivarius strain ZK-88 for inhibiting inflammation, relieving swelling and aching of gum, and improving oral flora balance | |
| CN107619856A (en) | A kind of detection method of buccal tobacco product to the total bacteria effect of saliva | |
| CN113046258A (en) | Lactobacillus rhamnosus capable of preventing and/or treating periodontitis and application thereof | |
| CN107699611A (en) | A kind of detection method of gum base type tobacco product to the total bacteria effect of saliva | |
| CN108771070B (en) | Compound functional beverage prepared by fermenting celery juice with eurotium cristatum, preparation method and application | |
| Al-Abdul et al. | Isolation and Identification of Bacteria from Diabetic and Non-Diabetic Patients With Periodontitis | |
| CN107523645A (en) | The detection method that a kind of buccal tobacco product influences on streptococcus mutans |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |