CN107513581A - The detection method that a kind of gum base type tobacco product influences on streptococcus mutans - Google Patents
The detection method that a kind of gum base type tobacco product influences on streptococcus mutans Download PDFInfo
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Abstract
The present invention relates to the detection method that a kind of gum base type tobacco product influences on streptococcus mutans, belong to technical field of biological.This method prepares the artificial saliva containing streptococcus mutans 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 streptococcus mutans in saliva, 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 streptococcus mutans content of saliva in mastication processes afterwards, so as to investigate the change of the streptococcus mutans 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 influences on streptococcus mutans
Detection method.
Background technology
Dental caries are classified as one of epidemic infection disease for seriously endangering human health by the World Health Organization (WHO), are
Under many factors collective effect such as bacterium, the chronic of hard tooth tissue generation, progressive, destructive disease.Cariogenic factor
Including：Bacterium, food, host and action time etc..Oral cavity bacterium species is a lot of, wherein mainly anaerobic bacteria and amphimicrobian
Bacterium, including streptococcus, lactobacillus and actinomyces etc..Recall rate is high in the oral cavity for streptococcus mutans, is main close to 90%
The cariogenic bacteria wanted, it by stick, produce it is sour and acidproof etc. cause dental hard tissue to destroy, so as to cause the formation of carious tooth.
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 streptococcus mutans in oral cavity is not related to.Therefore existing skill how is overcome
The problem of deficiency of art 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 deforming hammer
The detection method that bacterium influences, this method prepares the artificial saliva containing streptococcus mutans first, and makes for gum base type product
With feature, machine is chewed using full analogue simulation to simulate the mastication processes of gum base type tobacco product, to saliva in mastication processes
Streptococcus mutans content carries out quantitative fluorescent PCR analysis, so as to investigate the change before and after using gum base type tobacco product in saliva
The change of shape hammer bacterial content, the oral health for gum base type tobacco product provide reference.
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 streptococcus mutans, comprises the following steps：
Step (1), artificial saliva is prepared, and streptococcus mutans is added into artificial saliva, make streptococcus mutans artificial
Amount in saliva is 1*104-1*106CFU/mL, obtains the artificial saliva containing streptococcus mutans, and numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and is deformed containing for implantation step (1) several times
Streptococcic artificial saliva, is manually chewed, and in mastication processes, collects the saliva of different chew time points；
Step (3), the salivas of the different chew time points that step (2) is collected into contain streptococcus mutans with step (1)
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
Streptococcus mutans DNA, while extract the reference culture original DNA of streptococcus mutans；
Step (4), the reference culture original DNA that 10 times of streptococcus mutans is diluted using concentration gradient are glimmering as template progress
Fluorescent Quantitative PCR is analyzed, and draws streptococcus mutans quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the streptococcus mutans DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate streptococcus mutans content in each time point nutrient solution using sterilizing distilled water according to standard curve；
Wherein, sense primer used in quantitative fluorescent PCR reaction is 5 '-gcctacagctcagagatgctattct-
3’；Anti-sense primer is 5 '-gccatacaccactcatgaattga-3 '；Probe is FAM-
tggaaatgacggtcgccgttatgaa-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 streptococcus mutans, 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 streptococcus mutans of step (1) is reinjected for the second time, is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 10 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10mm；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 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing streptococcus mutans of step (1), is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 5 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8mm；Meanwhile open
Dynamic 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 turn over every time
The anglec of rotation for 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) in nutrient solution streptococcus mutans DNA extraction：The nutrient solution 1mL after the culture of step (3.1) is taken out to 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 1min is centrifuged under 1000r/min, takes
Supernatant, that is, obtain streptococcus mutans DNA in nutrient solution；
(3.3) extraction of the reference culture original DNA of streptococcus mutans：It is 10 to take 1mL concentration7CFU/mL deformation hammer
Bacterium after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganism PCR lysates in the 1.5mL centrifuge tubes of sterilizing,
Thermal denaturation 15min at 80 DEG C, then centrifuges 10s under 1000r/m, takes supernatant, that is, obtains the reference culture of streptococcus mutans
Original DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
It is further preferred that need the reference culture to streptococcus mutans original before quantitative fluorescent PCR analysis is carried out
DNA, the purity of streptococcus mutans DNA in each time point nutrient solution judge that decision method is：The DNA of testing sample is distinguished
The OD value determined under wavelength 260nm, 280nm；When under wavelength 260nm OD value with wavelength 280nm light it is close
When the ratio of angle value is 1.7~1.9, shows that purity is qualified, quantitative fluorescent PCR analysis can be carried out, conversely, then showing that purity does not conform to
Lattice, 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 streptococcus mutans 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；
Streptococcus mutans DNA and sterilized water are taken in each nutrient solution 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：
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；
Streptococcus mutans quantitative fluorescent PCR standard curve is drawn afterwards, and each time point culture is calculated according to standard curve
Streptococcus mutans content in liquid.
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 the change that streptococcus mutans occurs in oral cavity during gum base type tobacco product,
There is certain directive significance for understanding influence of the gum base type tobacco product to oral health.Matrix is chewed using chewing simulating machine
Type tobacco product, the interaction of product leachable and streptococcus mutans in saliva is not only allowed for, also fully simulates chewing
Process gum base type product is to the adhesive attraction of bacterium, so that chewing of the whole change procedure closer to people.Employ fluorescence
Quantifying PCR method is analyzed streptococcus mutans in saliva, can in real-time tracking saliva content of microorganisms change, ensure that
The accuracy and high efficiency of experiment.3 dynamic saliva collection points and 3 co-cultivation times after chewing when devising chewing simultaneously
Section, dynamic saliva collection point co-culture the period to simulate people to simulate chewing dynamic process of people when using tobacco product
Oral cavity static process after chewing, makes detection more fully accurate.
Brief description of the drawings
Fig. 1 is real-time fluorescence quantitative PCR detection streptococcus mutans canonical plotting；
Fig. 2 is streptococcus mutans testing result figure in nutrient solution.
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 streptococcus mutans used in the embodiment of the present invention is purchased from Guangdong Province's Culture Collection.
Embodiment 1
The detection method that a kind of gum base type tobacco product influences on streptococcus mutans, comprises the following steps：
Step (1), artificial saliva is prepared, and streptococcus mutans is added into artificial saliva, make streptococcus mutans artificial
Amount in saliva is 1*104CFU/mL, obtains the artificial saliva containing streptococcus mutans, and numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and is deformed containing for implantation step (1) several times
Streptococcic artificial saliva, is manually chewed, and in mastication processes, collects the saliva of different chew time points；Specific method is：
1.2g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, containing for implantation step (1) becomes
The streptococcic artificial saliva 10mL of shape, is manually chewed；Set and chew mechanical tooth with frequency progress occlusion up and down once in 5 seconds
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, stung every about 4 times
Close and the left and right mill changing of the relative positions is done and once gathers tumbling action, the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect
Liquid in solution ware, numbering are 2# liquid；
The artificial saliva 10mL containing streptococcus mutans of step (1) is reinjected for the second time, is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 10 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10mm；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 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing streptococcus mutans of step (1), is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 5 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8mm；Meanwhile open
Dynamic 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 turn over every time
The anglec of rotation for rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
Step (3), the salivas of the different chew time points that step (2) is collected into contain streptococcus mutans with step (1)
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
Streptococcus mutans DNA, while extract the reference culture original DNA of streptococcus mutans；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) in nutrient solution streptococcus mutans DNA extraction：The nutrient solution 1mL after the culture of step (3.1) is taken out to 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 1min is centrifuged under 1000r/min, takes
Supernatant, that is, obtain streptococcus mutans DNA in nutrient solution；
(3.3) extraction of the reference culture original DNA of streptococcus mutans：It is 10 to take 1mL concentration7CFU/mL deformation hammer
Bacterium after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganism PCR lysates in the 1.5mL centrifuge tubes of sterilizing,
Thermal denaturation 15min at 80 DEG C, then centrifuges 10s under 1000r/m, takes supernatant, that is, obtains the reference culture of streptococcus mutans
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 streptococcus mutans is diluted using concentration gradient are glimmering as template progress
Fluorescent Quantitative PCR is analyzed, and draws streptococcus mutans quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the streptococcus mutans DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate streptococcus mutans content in each time point nutrient solution using sterilizing distilled water according to standard curve；
Wherein, sense primer used in quantitative fluorescent PCR reaction is 5 '-gcctacagctcagagatgctattct-
3’；Anti-sense primer is 5 '-gccatacaccactcatgaattga-3 '；Probe is FAM-tggaaatgacggtcgccgttatga
a-TAMRA。
The reference culture original DNA to streptococcus mutans, the culture of each time point are needed before quantitative fluorescent PCR analysis is carried out
The purity of streptococcus mutans DNA in liquid judges that decision method is：By the DNA of testing sample respectively in wavelength 260nm, 280nm
The OD value of lower measure；When OD value and the OD value under wavelength 280nm under wavelength 260nm ratio for 1.7~
When 1.9, show 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 fluorescence and determine
Measure PCR analyses.
The specific method of step (4) is：
The μ L of reference culture original DNA 5 of streptococcus mutans 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；
Streptococcus mutans DNA and sterilized water are taken in each nutrient solution 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：
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；
Streptococcus mutans quantitative fluorescent PCR standard curve is drawn afterwards, and each time point culture is calculated according to standard curve
Streptococcus mutans content in liquid.
Embodiment 2
The detection method that a kind of gum base type tobacco product influences on streptococcus mutans, comprises the following steps：
Step (1), artificial saliva is prepared, and streptococcus mutans is added into artificial saliva, make streptococcus mutans artificial
Amount in saliva is 1*106CFU/mL, obtains the artificial saliva containing streptococcus mutans, and numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and is deformed containing for implantation step (1) several times
Streptococcic artificial saliva, is manually chewed, and in mastication processes, collects the saliva of different chew time points；Specific method is：
0.8g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, containing for implantation step (1) becomes
The streptococcic artificial saliva 10mL of shape, is manually chewed；Set and chew mechanical tooth with frequency progress occlusion up and down once in 5 seconds
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, stung every about 4 times
Close and the left and right mill changing of the relative positions is done and once gathers tumbling action, the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect
Liquid in solution ware, numbering are 2# liquid；
The artificial saliva 10mL containing streptococcus mutans of step (1) is reinjected for the second time, is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 10 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10mm；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 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing streptococcus mutans of step (1), is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 5 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8mm；Meanwhile open
Dynamic 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 turn over every time
The anglec of rotation for rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
Step (3), the salivas of the different chew time points that step (2) is collected into contain streptococcus mutans with step (1)
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
Streptococcus mutans DNA, while extract the reference culture original DNA of streptococcus mutans；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) in nutrient solution streptococcus mutans DNA extraction：The nutrient solution 1mL after the culture of step (3.1) is taken out to 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 1min is centrifuged under 1000r/min, takes
Supernatant, that is, obtain streptococcus mutans DNA in nutrient solution；
(3.3) extraction of the reference culture original DNA of streptococcus mutans：It is 10 to take 1mL concentration7CFU/mL deformation hammer
Bacterium after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganism PCR lysates in the 1.5mL centrifuge tubes of sterilizing,
Thermal denaturation 15min at 80 DEG C, then centrifuges 10s under 1000r/m, takes supernatant, that is, obtains the reference culture of streptococcus mutans
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 streptococcus mutans is diluted using concentration gradient are glimmering as template progress
Fluorescent Quantitative PCR is analyzed, and draws streptococcus mutans quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the streptococcus mutans DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate streptococcus mutans content in each time point nutrient solution using sterilizing distilled water according to standard curve；
Wherein, sense primer used in quantitative fluorescent PCR reaction is 5 '-gcctacagctcagagatgctattct-
3’；Anti-sense primer is 5 '-gccatacaccactcatgaattga-3 '；Probe is FAM-tggaaatgacggtcgccgttatga
a-TAMRA。
The reference culture original DNA to streptococcus mutans, the culture of each time point are needed before quantitative fluorescent PCR analysis is carried out
The purity of streptococcus mutans DNA in liquid judges that decision method is：By the DN A of testing sample respectively wavelength 260nm,
The OD value determined under 280nm；When the ratio of OD value and the OD value under wavelength 280nm under wavelength 260nm is
When 1.7~1.9, show 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 is analyzed.
The specific method of step (4) is：
The μ L of reference culture original DNA 5 of streptococcus mutans 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；
Streptococcus mutans DNA and sterilized water are taken in each nutrient solution 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：
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；
Streptococcus mutans quantitative fluorescent PCR standard curve is drawn afterwards, and each time point culture is calculated according to standard curve
Streptococcus mutans content in liquid.
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 streptococcus mutans, comprises the following steps：
Step (1), artificial saliva is prepared, and streptococcus mutans is added into artificial saliva, make streptococcus mutans artificial
Amount in saliva is 1*105CFU/mL, obtains the artificial saliva containing streptococcus mutans, and numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and is deformed containing for implantation step (1) several times
Streptococcic artificial saliva, is manually chewed, and in mastication processes, collects the saliva of different chew time points；Specific method is：
1g gum base type tobacco products are put into the solution ware that full analogue simulation chews machine, implantation step (1) contains deformation
Streptococcic artificial saliva 10mL, is manually chewed；Set chew mechanical tooth with 5 seconds frequencies once carry out occlusion up and down and
The left and right mill changing of the relative positions is made, and totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every about 4 times occlusions
And the left and right mill changing of the relative positions is done and once gathers tumbling action, the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect it is molten
Liquid in liquid ware, numbering are 2# liquid；
The artificial saliva 10mL containing streptococcus mutans of step (1) is reinjected for the second time, is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 10 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10mm；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 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the artificial saliva 10mL containing streptococcus mutans of step (1), is manually chewed；Set and chew
Mechanical tooth carries out occlusion up and down with 5 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8mm；Meanwhile open
Dynamic 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 turn over every time
The anglec of rotation for rolling clamping plate is 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
Step (3), the salivas of the different chew time points that step (2) is collected into contain streptococcus mutans with step (1)
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
Streptococcus mutans DNA, while extract the reference culture original DNA of streptococcus mutans；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) in nutrient solution streptococcus mutans DNA extraction：The nutrient solution 1mL after the culture of step (3.1) is taken out to 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 1min is centrifuged under 1000r/min, takes
Supernatant, that is, obtain streptococcus mutans DNA in nutrient solution；
(3.3) extraction of the reference culture original DNA of streptococcus mutans：It is 10 to take 1mL concentration7CFU/mL deformation hammer
Bacterium after 12000r/m centrifugations 5min, abandons supernatant, adds 50 μ L microorganism PCR lysates in the 1.5mL centrifuge tubes of sterilizing,
Thermal denaturation 15min at 80 DEG C, then centrifuges 10s under 1000r/m, takes supernatant, that is, obtains the reference culture of streptococcus mutans
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 streptococcus mutans is diluted using concentration gradient are glimmering as template progress
Fluorescent Quantitative PCR is analyzed, and draws streptococcus mutans quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the streptococcus mutans DNA in each time point nutrient solution as template respectively
Analysis, blank control template calculate streptococcus mutans content in each time point nutrient solution using sterilizing distilled water according to standard curve；
Wherein, sense primer used in quantitative fluorescent PCR reaction is 5 '-gcctacagctcagagatgctattct-
3’；Anti-sense primer is 5 '-gccatacaccactcatgaattga-3 '；Probe is FAM-tggaaatgacggtcgccgttatga
a-TAMRA。
The reference culture original DNA to streptococcus mutans, the culture of each time point are needed before quantitative fluorescent PCR analysis is carried out
The purity of streptococcus mutans DNA in liquid judges that decision method is：By the DNA of testing sample respectively in wavelength 260nm, 280nm
The OD value (optical density, OD) of lower measure；When the OD value under wavelength 260nm and the light under wavelength 280nm
When the ratio (OD260 and OD280 ratios) of density value is 1.7~1.9, shows that purity is qualified, quantitative fluorescent PCR point can be carried out
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 streptococcus mutans 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；
Streptococcus mutans DNA and sterilized water are taken in each nutrient solution 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：
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；
Streptococcus mutans quantitative fluorescent PCR standard curve is drawn afterwards, and each time point culture is calculated according to standard curve
Streptococcus mutans content in liquid.
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 streptococcus mutans content in nutrient solution.
Wherein, standard curve is y=-4.215log (x)+42.39, r2=0.9964；Afterwards by each time point nutrient solution
Ct values be updated in standard curve, try to achieve corresponding to streptococcus mutans content.
Table 1
Note：Unit is CFU/mL, M ± SD.
By table 1 and Fig. 2, it can learn that streptococcus mutans content decreases in the nutrient solution after chewing, wherein 2# liquid
Reduce it is the most notable, this be probably due to gum base type tobacco product have the function that adsorb streptococcus mutans, and in culture carefully
The growth rate of bacterium also decreases, and reason is probably that there is the material for chewing dissolution certain suppression streptococcus mutans to grow
Effect.
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’-gcctacagctcagagatgctattct-3’(SEQ ID NO.1) |
| Reverse primer | 5’-gccatacaccactcatgaattga-3’(SEQ ID NO.2) |
| Probe | FAM-tggaaatgacggtcgccgttatgaa-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 streptococcus mutans
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence ()
<400> 1
gcctacagct cagagatgct attct 25
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence ()
<400> 2
gccatacacc actcatgaat tga 23
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence ()
<400> 3
tggaaatgac ggtcgccgtt atgaa 25
Claims (6)
1. the detection method that a kind of gum base type tobacco product influences on streptococcus mutans, it is characterised in that comprise the following steps：
Step（1）, artificial saliva is prepared, and streptococcus mutans is added into artificial saliva, make streptococcus mutans in artificial saliva
In amount be 1*104-1*106 CFU/mL, obtains the artificial saliva containing streptococcus mutans, and numbering is 1# liquid；
Step（2）, gum base type tobacco product is put into chewing simulating machine, and implantation step several times（1）Containing deformation hammer
The artificial saliva of bacterium, 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）The people containing streptococcus mutans
Work saliva is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract the deformation in each time point nutrient solution respectively afterwards
Streptococcus DNA, while extract the reference culture original DNA of streptococcus mutans；
Step（4）, fluorescence is carried out as template using the reference culture original DNA of 10 times of streptococcus mutans of concentration gradient dilution and determined
PCR analyses are measured, draw streptococcus mutans quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR analysis is carried out using the streptococcus mutans DNA in each time point nutrient solution as template respectively, it is empty
White contrast template calculates streptococcus mutans content in each time point nutrient solution using sterilizing distilled water according to standard curve；
Wherein, sense primer used in quantitative fluorescent PCR reaction is 5 '-gcctacagctcagagatgctattct-3 '；Under
Trip primer is 5 '-gccatacaccactcatgaattga-3 '；Probe is FAM-tggaaatgacggtcgccgttatgaa-
TAMRA。
2. the detection method that gum base type tobacco product according to claim 1 influences on streptococcus mutans, it is characterised in that
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）Containing become
The streptococcic artificial saliva 10mL of shape, is manually chewed；Set and chew mechanical tooth with frequency progress occlusion up and down once in 5 seconds
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, stung every about 4 times
Close and the left and right mill changing of the relative positions is done and once gathers tumbling action, the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect
Liquid in solution ware, numbering are 2# liquid；
Step is reinjected for the second time（1）The mL of artificial saliva 10 containing streptococcus mutans, manually chewed；Chewing machine is set
Tool tooth carries out occlusion up and down with 10 seconds frequencies once and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10mm；Meanwhile open
Dynamic 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 turn over every time
The anglec of rotation for 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 streptococcus mutans, manually chewed；Chewing machine is set
Tool tooth carries out occlusion up and down with 5 seconds frequencies once and the left and right mill changing of the relative positions is made, and 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 rolling every time
The anglec of rotation of 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 streptococcus mutans, it is characterised in that
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）Streptococcus mutans DNA extraction in nutrient solution：Take out step（3.1）Culture after nutrient solution 1mL be placed in it is sterile
In 1.5mL centrifuge tubes, 10min is centrifuged under being afterwards 12000r/min in 4 DEG C, rotating speed, supernatant is removed, it is micro- to add 50 μ L
Biological PCR lysate, after being well mixed, thermal denaturation 15min at 80 DEG C is placed in, then 1min is centrifuged under 1000r/min, takes
Clear liquid, that is, obtain streptococcus mutans DNA in nutrient solution；
（3.3）The extraction of the reference culture original DNA of streptococcus mutans：It is 10 to take 1mL concentration7CFU/mL streptococcus mutans in
In the 1.5mL centrifuge tubes of sterilizing, after 12000 r/m centrifugations 5min, supernatant is abandoned, 50 μ L microorganism PCR lysates are added, 80
Thermal denaturation 15min at DEG C, 10 s are then centrifuged under 1000r/m, take supernatant, that is, the reference culture for obtaining streptococcus mutans is former
Beginning 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 streptococcus mutans, its feature exist
In in the reference culture original DNA to streptococcus mutans, each time point nutrient solution is needed before carrying out quantitative fluorescent PCR analysis
Streptococcus mutans DNA purity judge that decision method is：The DNA of testing sample is surveyed under wavelength 260nm, 280nm respectively
Fixed OD value；When the ratio of OD value and the OD value under wavelength 280nm under wavelength 260nm is 1.7 ~ 1.9,
Show 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 streptococcus mutans, it is characterised in that
Step（4）Specific method be：
The μ L of reference culture original DNA 5 of streptococcus mutans are taken, adds the μ L of water 45 and fully mixes, then take turns doing 10 times of concentration gradients
Dilution, dilute 6 times altogether, as standard curve template；
Streptococcus mutans DNA and sterilized water are taken in each nutrient solution 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；
Streptococcus mutans quantitative fluorescent PCR standard curve is drawn afterwards, and is calculated according to standard curve in each time point nutrient solution
Streptococcus mutans content.
6. the detection method that gum base type tobacco product influences on streptococcus mutans according to claim 1 or 5, its feature exists
In when quantitative fluorescent PCR analysis detects, each pattern detection in triplicate, takes its average value to be calculated.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US20170211131A1 (en) * | 2015-11-02 | 2017-07-27 | General Biologicals Corporation | Method for evaluating oral health |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170211131A1 (en) * | 2015-11-02 | 2017-07-27 | General Biologicals Corporation | Method for evaluating oral health |
| 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 |
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| MING-YUN LI等: "Role of sortase in Streptococcus mutans under the effect of nicotine", 《INTERNATIONAL JOURNAL OF ORAL SCIENCE》 * |
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