CN107699611A - A kind of detection method of gum base type tobacco product to the total bacteria effect of saliva - Google Patents
A kind of detection method of gum base type tobacco product to the total bacteria effect of saliva Download PDFInfo
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Abstract
The present invention relates to a kind of gum base type tobacco product to the detection method of the total bacteria effect of saliva, belong to technical field of biological.This method gathers the saliva of volunteer 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 product leachable and the interaction of bacterium 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 total bacterial content in saliva in mastication processes afterwards, so as to investigate the change of total bacterial 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 the total bacteria effect of saliva
Detection method.
Background technology
Nasidze etc. is had found by analyzing 120 healthy population saliva samples of global 12 countries and regions
204 bacterial gene category, wherein 39 are the oral cavity Pseudomonas being never described in the past, 64 are unknown kind.The research
Find simultaneously, oral bacteria group has individual specificity, but diversity is hardly influenceed by geographical configuration.It is another to there is research to gather
26 kinds of samples such as the supragingival plaque of specific tooth position and subgingival plaque and saliva are simultaneously mixed, and identify 247 kinds of 9 doors
There is obvious individual difference in bacterium, these bacteriums, exist significantly in the level of category with enteric bacteria in kind and strain level
Difference.Have researcher by core microorganism by abundance height be arranged in order for：Streptococcus (25%), Prey irrigate Pseudomonas
(16%), hemophilus (12%), Luo Si Pseudomonas (7%), Veillonella (6%), neisseria (6%), Fusobacterium
And porphyrin Pseudomonas (4%) (6%).
Researcher thinks that when internal environment of oral cavity changes original Oral health behaviours balance is formed newly by destruction
Ecological environment or stagnant area, counterpart intracavitary resident microorganisms can have an impact, the species of oral microorganism, quantity, microorganism with
Correlation between microorganism, between microorganism and host changes, and these changes all may result in abutment generation
Dental caries and cementopathia.Therefore the change of oral cavity related microorganisms is one of important indicator of microenvironment.
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 the total bacterium of saliva 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 always thin to saliva
The detection method that bacterium influences, this method gather the saliva of volunteer first, and for the handling characteristics of gum base type product, using complete
Analogue simulation chews machine to simulate the mastication processes of gum base type tobacco product, and total bacterial content in saliva in mastication processes is carried out
Quantitative fluorescent PCR is analyzed, and is glue so as to investigate the change of total bacterial content before and after using gum base type tobacco product in saliva
The oral health of fundamental mode tobacco product provides reference.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of gum base type tobacco product comprises the following steps to the detection method of the total bacteria effect of saliva：
Step (1), healthy volunteer's saliva is gathered, numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) collects several times
Saliva, manually chewed, in mastication processes, collect the saliva of different chew time points；
Step (3), the saliva for the different chew time points that step (2) is collected into is put into together with the saliva collected detests
Oxygen incubator is co-cultured at 37 DEG C；Extract the total DNA of bacteria of saliva in the nutrient solution at each time point respectively afterwards, carry simultaneously
Take the original DNA of a variety of oral bacteria reference culture mixtures；
Step (4), using concentration gradient dilute 10 times a variety of oral bacteria reference culture mixtures original D NA as
Template carries out quantitative fluorescent PCR analysis, draws quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the total DNA of bacteria of saliva in the nutrient solution at each time point as masterplate respectively
Analysis, blank control template calculate the total bacterial content of saliva 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 '-cgctagtaatcgtggatcaga atg-
3’；Anti-sense primer is 5 '-tgtgacgggcggtgtgta-3 '；Probe is FAM-cacggtgaatacgttccc gggc-
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) is adopted
The saliva 5mL collected, is manually chewed；The mechanical tooth of chewing is set to be ground with occlusion above and below frequency progress once in 5 seconds and left and right
The 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 left and right
The mill changing of the relative positions, which is done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect solution ware in
Liquid, numbering is 2# liquid；
The saliva 5mL that step (1) collects is reinjected for the second time, is manually chewed；Set and chew mechanical tooth with 10 seconds
Frequency once carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10 mm；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 the rotation of rolling clamping plate every time
Gyration is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the saliva 5mL that step (1) collects, and is manually chewed；Set and chew mechanical tooth with 5 seconds one
Secondary frequency carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8m m；Meanwhile start opening and closing left and right rotation
Turn component, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, gather the rotation of rolling clamping plate every time
Angle 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 the total DNA of bacteria of saliva extraction：The μ L of nutrient solution 50 after the culture of step (3.1) are taken out to put
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 microbial lytic buffer solutions, 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 the total DNA of bacteria of saliva in nutrient solution；
(3.3) extraction of the original DNA of a variety of oral bacteria reference culture mixtures：Take and be no less than 5 kinds of oral bacteria marks
The mixture 1mL of quasi- bacterial strain, every kind of oral bacteria reference culture concentration is 10 in mixture8CFU/mL, be placed in 1.5mL from
In heart pipe, after 12000r/m centrifugations 5min, supernatant is abandoned, adds 50 μ L microorganism PCR lysates, the thermal denaturation at 80 DEG C
15min, 10s then is centrifuged under 1000r/m, take supernatant liquor, that is, obtain the original of a variety of oral bacteria reference culture mixtures
Beginning DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
It is further preferred that the mixture of oral bacteria reference culture includes streptococcus mutans, porphyromonas unit cell
At least one of bacterium, staphylococcus aureus, actinomyces.
It is further preferred that need to mix a variety of oral bacteria reference cultures before quantitative fluorescent PCR analysis is carried out
The purity of the total DNA of bacteria of saliva judges in the original DNA of thing, each time point nutrient solution, and 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 original DNA 5 of a variety of oral bacteria reference culture mixtures are taken, adds the μ L of water 45 and fully mixes, then take turns doing
10 times of concentration gradient dilutions, dilute 6 times, as standard curve template altogether；
The total DNA of bacteria of saliva 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；
Quantitative fluorescent PCR standard curve is drawn afterwards, and it is total according to saliva in each time point nutrient solution of standard curve calculating
Bacterial content.
It is further preferred that during quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes it average
Value is calculated.
All bacteriums in the saliva having been found at present that the total bacterium of saliva refers in the present invention.
Detection method is to be directed to the changes of contents of total bacterium in saliva, and primer is universal primer.It can examine
All bacterial contents surveyed in saliva.
Compared with prior art, its advantage is the present invention：
The present invention chews gum base type tobacco product using chewing simulating machine, not only allows for thin in product leachable and saliva
The interaction of bacterium, adhesive attraction of the mastication processes gum base type product to bacterium is also fully simulated, so that entirely changing
Chewing of the journey closer to people.Fluorescence quantifying PCR method is employed to analyze total bacterium in saliva, can real-time tracking saliva
The change of middle content of microorganisms, it ensure that the accuracy and high efficiency of experiment.3 dynamic saliva collections when devising chewing simultaneously
Point and chew after 3 co-cultivation the periods, dynamic saliva collection point with simulate people using tobacco product when chewing dynamic mistake
Journey, the period is co-cultured to simulate oral cavity static process of the people after chewing, makes detection more fully accurate.
The present invention can simulate human mouth and chew the change that the total bacterium of saliva 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, suppression can be provided according to testing result
The product of harmful bacteria, play a part of health protection.
Brief description of the drawings
Fig. 1 is the real-time fluorescence quantitative PCR detection total bacterium canonical plotting of saliva；
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.
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.
Streptococcus mutans, porphyromonas gingivalis, staphylococcus aureus, actinomyces and blood used in the embodiment of the present invention
Streptococcic reference culture is purchased from Guangdong Province's Culture Collection.
Embodiment 1
A kind of gum base type tobacco product comprises the following steps to the detection method of the total bacteria effect of saliva：
Step (1), healthy volunteer's saliva is gathered, numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) collects several times
Saliva, manually chewed, in mastication processes, collect 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, implantation step (1) collects
Saliva 5mL, manually chewed；Set and chew mechanical tooth with occlusion and the left and right mill changing of the relative positions up and down of frequency progress once in 5 seconds
Make, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every about 4 times occlusions and left and right mill mistake
Action, which is done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect solution ware in liquid
Body, numbering are 2# liquid；
The saliva 5mL that step (1) collects is reinjected for the second time, is manually chewed；Set and chew mechanical tooth with 10 seconds
Frequency once carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10 mm；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 the rotation of rolling clamping plate every time
Gyration is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the saliva 5mL that step (1) collects, and is manually chewed；Set and chew mechanical tooth with 5 seconds one
Secondary frequency carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8m m；Meanwhile start opening and closing left and right rotation
Turn component, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, gather the rotation of rolling clamping plate every time
Angle 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 is put into together with the saliva collected detests
Oxygen incubator is co-cultured at 37 DEG C；Extract the total DNA of bacteria of saliva in the nutrient solution at each time point respectively afterwards, carry simultaneously
Take the original DNA of a variety of oral bacteria reference culture mixtures；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 the total DNA of bacteria of saliva extraction：The μ L of nutrient solution 50 after the culture of step (3.1) are taken out to put
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 microbial lytic buffer solutions, 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 the total DNA of bacteria of saliva in nutrient solution；
(3.3) extraction of the original DNA of a variety of oral bacteria reference culture mixtures：Take and be no less than 5 kinds of oral bacteria marks
The mixture 1mL of quasi- bacterial strain, every kind of oral bacteria reference culture concentration is 10 in mixture8CFU/mL, be placed in 1.5mL from
In heart pipe, after 12000r/m centrifugations 5min, supernatant is abandoned, adds 50 μ L microorganism PCR lysates, the thermal denaturation at 80 DEG C
15min, 10s then is centrifuged under 1000r/m, take supernatant liquor, that is, obtain the original of a variety of oral bacteria reference culture mixtures
Beginning DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
Step (4), using concentration gradient dilute 10 times a variety of oral bacteria reference culture mixtures original D NA as
Template carries out quantitative fluorescent PCR analysis, draws quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the total DNA of bacteria of saliva in the nutrient solution at each time point as masterplate respectively
Analysis, blank control template calculate the total bacterial content of saliva 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 '-cgctagtaatcgtggatcaga atg-
3’；Anti-sense primer is 5 '-tgtgacgggcggtgtgta-3 '；Probe is FAM-cacggtgaatacgttccc gggc-
TAMRA。
Original D NA to a variety of oral bacteria reference culture mixtures, each is needed before quantitative fluorescent PCR analysis is carried out
The purity of the total DNA of bacteria of saliva judges in time point nutrient solution, and 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 original DNA 5 of a variety of oral bacteria reference culture mixtures are taken, adds the μ L of water 45 and fully mixes, then take turns doing
10 times of concentration gradient dilutions, dilute 6 times, as standard curve template altogether；
The total DNA of bacteria of saliva 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；
Quantitative fluorescent PCR standard curve is drawn afterwards, and it is total according to saliva in each time point nutrient solution of standard curve calculating
Bacterial content.
The mixture of oral bacteria reference culture includes streptococcus mutans and porphyromonas gingivalis, and its excess-three kind is unlimited
System.
Embodiment 2
A kind of gum base type tobacco product comprises the following steps to the detection method of the total bacteria effect of saliva：
Step (1), healthy volunteer's saliva is gathered, numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) collects several times
Saliva, manually chewed, in mastication processes, collect 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, implantation step (1) collects
Saliva 5mL, manually chewed；Set and chew mechanical tooth with occlusion and the left and right mill changing of the relative positions up and down of frequency progress once in 5 seconds
Make, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every about 4 times occlusions and left and right mill mistake
Action, which is done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect solution ware in liquid
Body, numbering are 2# liquid；
The saliva 5mL that step (1) collects is reinjected for the second time, is manually chewed；Set and chew mechanical tooth with 10 seconds
Frequency once carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10 mm；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 the rotation of rolling clamping plate every time
Gyration is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the saliva 5mL that step (1) collects, and is manually chewed；Set and chew mechanical tooth with 5 seconds one
Secondary frequency carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8m m；Meanwhile start opening and closing left and right rotation
Turn component, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, gather the rotation of rolling clamping plate every time
Angle 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 is put into together with the saliva collected detests
Oxygen incubator is co-cultured at 37 DEG C；Extract the total DNA of bacteria of saliva in the nutrient solution at each time point respectively afterwards, carry simultaneously
Take the original DNA of a variety of oral bacteria reference culture mixtures；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 the total DNA of bacteria of saliva extraction：The μ L of nutrient solution 50 after the culture of step (3.1) are taken out to put
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 microbial lytic buffer solutions, 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 the total DNA of bacteria of saliva in nutrient solution；
(3.3) extraction of the original DNA of a variety of oral bacteria reference culture mixtures：Take and be no less than 5 kinds of oral bacteria marks
The mixture 1mL of quasi- bacterial strain, every kind of oral bacteria reference culture concentration is 10 in mixture8CFU/mL, be placed in 1.5mL from
In heart pipe, after 12000r/m centrifugations 5min, supernatant is abandoned, adds 50 μ L microorganism PCR lysates, the thermal denaturation at 80 DEG C
15min, 10s then is centrifuged under 1000r/m, take supernatant liquor, that is, obtain the original of a variety of oral bacteria reference culture mixtures
Beginning DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
Step (4), using concentration gradient dilute 10 times a variety of oral bacteria reference culture mixtures original D NA as
Template carries out quantitative fluorescent PCR analysis, draws quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the total DNA of bacteria of saliva in the nutrient solution at each time point as masterplate respectively
Analysis, blank control template calculate the total bacterial content of saliva 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 '-cgctagtaatcgtggatcaga atg-
3’；Anti-sense primer is 5 '-tgtgacgggcggtgtgta-3 '；Probe is FAM-cacggtgaatacgttccc gggc-
TAMRA。
Original D NA to a variety of oral bacteria reference culture mixtures, each is needed before quantitative fluorescent PCR analysis is carried out
The purity of the total DNA of bacteria of saliva judges in time point nutrient solution, and 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 original DNA 5 of a variety of oral bacteria reference culture mixtures are taken, adds the μ L of water 45 and fully mixes, then take turns doing
10 times of concentration gradient dilutions, dilute 6 times, as standard curve template altogether；
The total DNA of bacteria of saliva 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；
Quantitative fluorescent PCR standard curve is drawn afterwards, and it is total according to saliva in each time point nutrient solution of standard curve calculating
Bacterial content.
During quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes its average value to be calculated.Oral cavity
The mixture of bacterium reference culture includes staphylococcus aureus and actinomyces, and its excess-three kind does not limit.
Embodiment 3
A kind of gum base type tobacco product comprises the following steps to the detection method of the total bacteria effect of saliva：
Step (1), healthy volunteer's saliva is gathered, numbering is 1# liquid；
Step (2), gum base type tobacco product is put into chewing simulating machine, and implantation step (1) collects several times
Saliva, manually chewed, in mastication processes, collect 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) collects
Saliva 5mL, is manually chewed；The mechanical tooth of chewing is set to carry out occlusion up and down and left and right mill changing of the relative positions work with 5 seconds frequencies once,
Totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, make every about 4 times occlusions and the left and right mill changing of the relative positions
Do and once gather tumbling action, the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect solution ware in liquid,
Numbering is 2# liquid；
The saliva 5mL that step (1) collects is reinjected for the second time, is manually chewed；Set and chew mechanical tooth with 10 seconds
Frequency once carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 10 times, occlusion width is 10 mm；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 the rotation of rolling clamping plate every time
Gyration is 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects the saliva 5mL that step (1) collects, and is manually chewed；Set and chew mechanical tooth with 5 seconds one
Secondary frequency carries out occlusion up and down and the left and right mill changing of the relative positions is made, and totally 30 times, occlusion width is 8m m；Meanwhile start opening and closing left and right rotation
Turn component, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, gather the rotation of rolling clamping plate every time
Angle 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 is put into together with the saliva collected detests
Oxygen incubator is co-cultured at 37 DEG C；Extract the total DNA of bacteria of saliva in the nutrient solution at each time point respectively afterwards, carry simultaneously
Take the original DNA of a variety of oral bacteria reference culture mixtures；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 the total DNA of bacteria of saliva extraction：The μ L of nutrient solution 50 after the culture of step (3.1) are taken out to put
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 microbial lytic buffer solutions, 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 the total DNA of bacteria of saliva in nutrient solution；
(3.3) extraction of the original DNA of a variety of oral bacteria reference culture mixtures：Take and be no less than 5 kinds of oral bacteria marks
The mixture 1mL of quasi- bacterial strain, every kind of oral bacteria reference culture concentration is 10 in mixture8CFU/mL, be placed in 1.5mL from
In heart pipe, after 12000r/m centrifugations 5min, supernatant is abandoned, adds 50 μ L microorganism PCR lysates, the thermal denaturation at 80 DEG C
15min, 10s then is centrifuged under 1000r/m, take supernatant liquor, that is, obtain the original of a variety of oral bacteria reference culture mixtures
Beginning DNA；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
Step (4), using concentration gradient dilute 10 times a variety of oral bacteria reference culture mixtures original D NA as
Template carries out quantitative fluorescent PCR analysis, draws quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR point is carried out using the total DNA of bacteria of saliva in the nutrient solution at each time point as masterplate respectively
Analysis, blank control template calculate the total bacterial content of saliva 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 '-cgctagtaatcgtggatcaga atg-
3’；Anti-sense primer is 5 '-tgtgacgggcggtgtgta-3 '；Probe is FAM-cacggtgaatacgttccc gggc-
TAMRA。
Original D NA to a variety of oral bacteria reference culture mixtures, each is needed before quantitative fluorescent PCR analysis is carried out
The purity of the total DNA of bacteria of saliva judges in time point nutrient solution, and 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 original DNA 5 of a variety of oral bacteria reference culture mixtures are taken, adds the μ L of water 45 and fully mixes, then take turns doing
10 times of concentration gradient dilutions, dilute 6 times, as standard curve template altogether；
The total DNA of bacteria of saliva 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；
Quantitative fluorescent PCR standard curve is drawn afterwards, and it is total according to saliva in each time point nutrient solution of standard curve calculating
Bacterial content.
During quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes its average value to be calculated.
In the mixture of oral bacteria reference culture it is streptococcus mutans in the present embodiment, porphyromonas gingivalis, golden yellow
Color staphylococcus, actinomyces and Streptococcus sanguis.
As a result as shown in Fig. 1, table 1, table 1 is the testing result of the total bacterial content of saliva in nutrient solution.
Wherein, standard curve is y=-4.030log (x)+42.74, r2=0.9981；Afterwards by each time point nutrient solution
Ct values be updated in standard curve, try to achieve corresponding to saliva total bacterial content.
Table 1
Note：Unit is CFU/mL, M ± SD.
By table 1, total bacterial content decreases in the saliva after chewing, and wherein 2# liquid reduces the most notable, and this can
Can be due to that gum base type tobacco product has the function that a variety of oral bacterias of absorption.
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’-cgctagtaatcgtggatcagaatg-3’(SEQ ID NO.1) |
| Reverse primer | 5’-tgtgacgggcggtgtgta-3’(SEQ ID NO.2) |
| Probe | FAM-cacggtgaatacgttcccgggc-TAMRA(SEQ ID NO.3) |
Sequence table
<110>Cigarette industry Co., Ltd in Yunnan
<120>A kind of detection method of gum base type tobacco product to the total bacteria effect of saliva
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence ()
<400> 1
cgctagtaat cgtggatcag aatg 24
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 2
tgtgacgggc ggtgtgta 18
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 3
cacggtgaat acgttcccgg gc 22
Claims (7)
1. a kind of gum base type tobacco product is to the detection method of the total bacteria effect of saliva, it is characterised in that comprises the following steps：
Step（1）, healthy volunteer's saliva is gathered, numbering is 1# liquid；
Step（2）, gum base type tobacco product is put into chewing simulating machine, and implantation step several times（1）The saliva collected,
Manually chewed, in mastication processes, collect the saliva of different chew time points；
Step（3）, by step（2）The saliva for the different chew time points being collected into is put into anaerobism training together with the saliva collected
Case is supported to be co-cultured at 37 DEG C；Extract the total DNA of bacteria of saliva in the nutrient solution at each time point respectively afterwards, while extract more
The original DNA of kind oral bacteria reference culture mixture；
Step（4）, entered using the original DNA of 10 times of a variety of oral bacteria reference culture mixtures of concentration gradient dilution as template
Row quantitative fluorescent PCR is analyzed, and draws quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR analysis is carried out using the total DNA of bacteria of saliva in the nutrient solution at each time point as masterplate respectively, it is empty
White contrast template calculates the total bacterial content of saliva 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 '-cgctagtaatcgtggatcagaatg-3 '；Under
Trip primer is 5 '-tgtgacgggcggtgtgta-3 '；Probe is FAM-cacggtgaatacgttcccgggc-TAMRA.
2. gum base type tobacco product according to claim 1 is to the detection method of the total bacteria effect of saliva, 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）Collect
Saliva 5mL, manually chewed；Set and chew mechanical tooth with occlusion and the left and right mill changing of the relative positions up and down of frequency progress once in 5 seconds
Make, totally 20 times, occlusion width is 15mm；Meanwhile start opening and closing left and right rotary components, every about 4 times occlusions and left and right mill mistake
Action, which is done, once gathers tumbling action, and the anglec of rotation for gathering rolling clamping plate every time is 45 °；After the completion of collect solution ware in liquid
Body, numbering are 2# liquid；
Step is reinjected for the second time（1）The saliva 5mL collected, is manually chewed；Set and chew mechanical tooth with 10 seconds once
Frequency carry out up and down occlusion and left and right mill the changing of the relative positions make, totally 10 times, occlusion width is 10mm；Meanwhile start opening and closing left and right rotation
Component, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, gather the anglec of rotation of rolling clamping plate every time
Spend for 30 °；After the completion of collect solution ware in liquid, numbering is 3# liquid；
Third time reinjects step（1）The saliva 5mL collected, is manually chewed；Set and chew mechanical tooth with 5 seconds once
Frequency carries out occlusion up and down 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 rotation group
Part, do every about 5 times occlusions and the left and right mill changing of the relative positions and once gather tumbling action, gather the anglec of rotation of rolling clamping plate every time
For 90 °；After the completion of collect solution ware in liquid, numbering is 4# liquid.
3. gum base type tobacco product according to claim 2 is to the detection method of the total bacteria effect of saliva, 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）The extraction of the total DNA of bacteria of saliva in nutrient solution：Take out step（3.1）Culture after the μ L of nutrient solution 50 be placed in nothing
In bacterium 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
Microbial lytic buffer solution, 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 the total DNA of bacteria of saliva in nutrient solution；
（3.3）The extraction of the original DNA of a variety of oral bacteria reference culture mixtures：Take and be no less than 5 kinds of oral bacteria standard bacterias
The mixture 1mL of strain, every kind of oral bacteria reference culture concentration is 10 in mixture8CFU/mL, it is placed in 1.5mL centrifuge tubes
In, after 12000 r/m centrifugations 5min, abandon supernatant, add 50 μ L microorganism PCR lysates, the thermal denaturation 15min at 80 DEG C, so
10s is centrifuged under 1000r/m afterwards, takes supernatant liquor, that is, obtains the original DNA of a variety of oral bacteria reference culture mixtures；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
4. gum base type tobacco product according to claim 3 is to the detection method of the total bacteria effect of saliva, it is characterised in that
The mixture of oral bacteria reference culture includes streptococcus mutans, porphyromonas gingivalis, staphylococcus aureus, actinomyces
At least one of.
5. gum base type tobacco product according to claim 1 or 3 exists to the detection method of the total bacteria effect of saliva, its feature
In needing the original DNA to a variety of oral bacteria reference culture mixtures, each time point before quantitative fluorescent PCR analysis is carried out
The purity of the total DNA of bacteria of saliva judges in nutrient solution, and decision method is：By the DNA 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 glimmering
Fluorescent Quantitative PCR is analyzed.
6. gum base type tobacco product according to claim 3 is to the detection method of the total bacteria effect of saliva, it is characterised in that
Step（4）Specific method be：
The μ L of original DNA 5 of a variety of oral bacteria reference culture mixtures 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；
The total DNA of bacteria of saliva 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；
Quantitative fluorescent PCR standard curve is drawn afterwards, and the total bacterium of saliva in each time point nutrient solution is calculated according to standard curve
Content.
7. gum base type tobacco product according to claim 1 or 6 exists to the detection method of the total bacteria effect of saliva, its feature
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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| 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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| Title |
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| 高茜: "滇橄榄对人口腔唾液流率、pH及细菌的影响研究", 《食品工业科技》 * |
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