CN107630072A - The detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis - Google Patents
The detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis Download PDFInfo
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Abstract
The present invention relates to the detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis, belong to technical field of biological.This method prepares the artificial saliva containing porphyromonas gingivalis first, and for the handling characteristics of mouth containing type product, using the use process of artificial saliva simulation buccal tobacco product, and quantitative fluorescent PCR analysis is carried out to the porphyromonas gingivalis content of saliva, so as to investigate the change of the porphyromonas gingivalis content before and after using buccal tobacco product in saliva, reference is provided for the oral health of buccal tobacco product.The present invention can add the porphyromonas gingivalis of different content, the different actual conditions of different human mouth Porphyromonas gingivalis contents be simulated, so as to give comprehensive guidance.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of buccal tobacco product is to porphyromonas gingivalis
The detection method of influence.
Background technology
Shown according to national third time oral health epidemiological investigation result, chronic periodontitis (chronic period
Ontitis, CP) illness rate reach more than 80%, and the main reason for be more than 35 years old crowd's absence of tooth.Modern medicine
Research shows that chronic periodontitis is due to that patient's oral hygiene environment is poor more, and periodontium caused by the factor such as Occlusal Trauma is gradually
The property entered is destroyed, and shows as halitosis, gum redness, bleeding initial stage, long is not treated, and tooth mobility, alveolar bone can be caused to break more
It is bad, even have tooth pulled out, it is also relate to the function of the important organs such as the heart, lung, kidney in addition.Periodontium damage with it is micro- under a variety of gums
Biology is relevant, and its Porphyromonas gingivalis (Porphyromonas gingivalis, P.gingivalis) is considered as slow
One of property most important pathogenic bacteria of periodontitis, the bacterium often detects in patients with periodontitis subgingival plaque and saliva, in periodontal health
It can also be detected in crowd.Porphyromonas gingivalis has a series of causes such as lipopolysaccharides, capsular polysaccharide, pili, gingipain
Cause of disease, can succeed attack to host cell, and escape immune system.
Buccal smokeless tobacco product is a kind of tobacco powder product of moistening, originating from 19th century Sweden.Sweden's mouth
Thin graininess is presented containing smoke product, for water content generally more than 40%, water content is also on sale less than 40% semi-dry products.Production
Product are placed between upper lip and gum, do not need saliva to go out saliva during use, there is in bulk and bagged product.In the process that product uses
In, influence of the product to human mouth health is an important inspection target.Current tobacco product is carried out in itself only for product
The detection of some physical and chemical index, and it is less to the microbiological effect research in human mouth, in particular for gum in oral cavity
The research of Detection of Porphyromonas was not related to.Therefore how overcome the deficiencies in the prior art, which is current technical field of biological, is needed badly
Solve the problems, such as.
The content of the invention
The invention aims to solve the deficiencies in the prior art, there is provided a kind of buccal tobacco product is to porphyromonas
The detection method that monad influences, this method prepares the artificial saliva containing porphyromonas gingivalis first, and is directed to mouth containing type
The handling characteristics of product, the use process of buccal tobacco product is simulated using artificial saliva, and to the porphyromonas list of saliva
Born of the same parents' bacterial content carries out quantitative fluorescent PCR analysis, so as to investigate the porphyromonas before and after using buccal tobacco product in saliva
The change of unit cell bacterial content, reference is provided for the oral health of buccal tobacco product.
To achieve the above object, the technical solution adopted by the present invention is as follows：
The detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*104-1*106CFU/mL, obtain the artificial saliva containing porphyromonas gingivalis；
Step (2), mouth containing tobacco products are entered using the artificial saliva containing porphyromonas gingivalis that step (1) obtains
Row extraction, extraction is multiple, collects extract solution respectively；
Step (3), each extract solution that step (2) is collected into and the artificial saliva containing porphyromonas gingivalis of step (1)
Liquid is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract the porphyromonas gingivalis in each nutrient solution respectively afterwards
DNA, while extract the reference culture original DNA of porphyromonas gingivalis；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient are used as template
Quantitative fluorescent PCR analysis is carried out, draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile fluorescent quantitation P CR analyses are carried out using the porphyromonas gingivalis DNA in each nutrient solution as template respectively,
Blank control template calculates containing for the porphyromonas gingivalis DNA in each nutrient solution using sterilizing distilled water according to standard curve
Amount；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagatagg c-TAMRA.
It is further preferred that the specific method of step (2) is：
Into mouth containing tobacco products 0.8-1.2g, the artificial saliva containing porphyromonas gingivalis of implantation step (1) is in 37
Isothermal vibration is extracted at DEG C, and extraction is multiple, every time the extraction artificial saliva 10mL containing porphyromonas gingivalis used, often
Secondary extraction time is 5min, collects extract solution respectively.
It is further preferred that described concussion speed is 150-300rpm；Extraction time is 3 times.
It is further preferred that the specific method of step (3) is：
(3.1) co-culture：Take each extract solution that step (2) is collected into and the people containing porphyromonas gingivalis of step (1)
Work saliva, every kind of liquid are divided into 3 equal portions, are respectively put into anaerobic culture box and are co-cultured at 37 DEG C, every kind of equal portions of liquid 3
Incubation time be respectively tri- periods of 0h, 2h, 6h；
(3.2) extraction of the porphyromonas gingivalis DNA in nutrient solution：Take out the nutrient solution after the culture of step (3.1)
In 4 DEG C of centrifugation, supernatant is removed, adds microorganism PCR lysates, after being well mixed, be placed in thermal denaturation 15min at 75-85 DEG C,
Then centrifuge again, take supernatant, that is, obtain the porphyromonas gingivalis DNA in nutrient solution；Described microorganism PCR lysates
Addition volume be nutrient solution volume 5%；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：Take with step (3.2) used in it is identical
The microorganism PCR lysates of volume, reference culture to the concentration for adding porphyromonas gingivalis is 107CFU/mL, at 75-85 DEG C
Lower thermal denaturation 15min, is then centrifuged for, and takes supernatant, that is, obtains the reference culture original DNA of porphyromonas gingivalis；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
It is further preferred that the rotating speed of the centrifugation of nutrient solution is 11000-12000r/min in (3.2), the time is
10min；The rotating speed centrifuged after thermal denaturation is 800-1000r/min, time 1min.
It is further preferred that the rotating speed centrifuged in (3.3) after thermal denaturation is 800-1000r/min, time 10s.
It is further preferred that the reference culture to porphyromonas gingivalis is needed before quantitative fluorescent PCR analysis is carried out
The purity of porphyromonas gingivalis DNA in original DNA, each nutrient solution judges that decision method is：By the DNA of testing sample points
The OD value not determined under wavelength 260nm, 280nm；When the OD value under wavelength 260nm and the light under wavelength 280nm
When the ratio of density value is 1.7~1.9, shows that purity is qualified, quantitative fluorescent PCR analysis can be carried out, conversely, then showing purity not
It is qualified, it is impossible to carry out quantitative fluorescent PCR analysis.
It is further preferred that the specific method of step (4) is：
The reference culture that volume is porphyromonas gingivalis is added into the reference culture original DNA of porphyromonas gingivalis
The water that 9 times of original DNA volume, and fully mix, then 10 times of concentration gradient dilutions are taken turns doing, dilute 6 times altogether, as standard curve
Template；
Take the porphyromonas gingivalis DNA in each nutrient solution with sterilized water by 1:Test specimens are used as after the dilution of 8 volume ratios
Product template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
It is further preferred that during quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes it average
Value is calculated.
Compared with prior art, its advantage is the present invention：
The invention provides the detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis, for mouth containing type
Influence of the tobacco product to oral health provides reference.Present invention employs fluorescence quantifying PCR method to porphyromonas in saliva
Monad is analyzed, can real-time quantitative tracking saliva in content of microorganisms change, ensure that experiment accuracy and efficiently
Property.3 co-cultivation periods after devising 3 dynamic saliva collection points simultaneously and chewing, dynamic saliva collection point is to simulate people
Dynamic process when using buccal tobacco product, the period is co-cultured to simulate oral cavity static process of the people after chewing,
Make detection more fully accurate.The present invention can add the porphyromonas gingivalis of different content, simulate different human mouth denss in dente
The different actual conditions of gum Porphyromonas bacterial content, so as to give comprehensive guidance.
Brief description of the drawings
Fig. 1 is real-time fluorescence quantitative PCR detection porphyromonas gingivalis canonical plotting；
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.
The reference culture of porphyromonas gingivalis employed in the embodiment of the present invention is protected purchased from Guangdong Province microorganism fungus kind
Tibetan center；
Buccal tobacco product is Snus General White Mini Portion, S in the embodiment of the present invention
WEDISH。
Blank control template of the present invention is for the baseline value of calibration standard curve using sterilizing distilled water.
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.
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.
Embodiment 1
The detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*104CFU/mL, obtain the artificial saliva containing porphyromonas gingivalis；
Step (2), mouth containing tobacco products are entered using the artificial saliva containing porphyromonas gingivalis that step (1) obtains
Row extraction, extraction is multiple, collects extract solution respectively；Specific method is：
Into mouth containing tobacco products 0.8g, the artificial saliva containing porphyromonas gingivalis of implantation step (1) is at 37 DEG C
Isothermal vibration is extracted, and concussion speed is 150rpm, is extracted 2 times, every time the extraction people containing porphyromonas gingivalis used
Work saliva 9mL, each extraction time are 4min, collect extract solution respectively；
Step (3), each extract solution that step (2) is collected into and the artificial saliva containing porphyromonas gingivalis of step (1)
Liquid is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract the porphyromonas gingivalis in each nutrient solution respectively afterwards
DNA, while extract the reference culture original DNA of porphyromonas gingivalis；Specific method is：
(3.1) co-culture：Take each extract solution that step (2) is collected into and the people containing porphyromonas gingivalis of step (1)
Work saliva, every kind of liquid are divided into 3 equal portions, are respectively put into anaerobic culture box and are co-cultured at 37 DEG C, every kind of equal portions of liquid 3
Incubation time be respectively tri- periods of 0h, 2h, 6h；
(3.2) extraction of the porphyromonas gingivalis DNA in nutrient solution：Take out the nutrient solution after the culture of step (3.1)
In 4 DEG C of centrifugations, supernatant is removed, adds microorganism PCR lysates, after being well mixed, be placed in thermal denaturation 15min at 75 DEG C, so
Centrifuge again afterwards, take supernatant, that is, obtain the porphyromonas gingivalis DNA in nutrient solution；Described microorganism PCR lysates
Add 5% that volume is nutrient solution volume；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：Take with step (3.2) used in it is identical
The microorganism PCR lysates of volume, reference culture to the concentration for adding porphyromonas gingivalis is 107CFU/mL, at 75 DEG C
Thermal denaturation 15min, is then centrifuged for, and takes supernatant, that is, obtains the reference culture original DNA of porphyromonas gingivalis；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient are used as template
Quantitative fluorescent PCR analysis is carried out, draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile fluorescent quantitation P CR analyses are carried out using the porphyromonas gingivalis DNA in each nutrient solution as template respectively,
Blank control template calculates containing for the porphyromonas gingivalis DNA in each nutrient solution using sterilizing distilled water according to standard curve
Amount；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagatagg c-TAMRA；Tool
Body method is：
The reference culture that volume is porphyromonas gingivalis is added into the reference culture original DNA of porphyromonas gingivalis
The water that 9 times of original DNA volume, and fully mix, then 10 times of concentration gradient dilutions are taken turns doing, dilute 6 times altogether, as standard curve
Template；
Take the porphyromonas gingivalis DNA in each nutrient solution with sterilized water by 1:Test specimens are used as after the dilution of 8 volume ratios
Product template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
The reference culture original DNA to porphyromonas gingivalis, each nutrient solution are needed before quantitative fluorescent PCR analysis is carried out
In porphyromonas gingivalis DNA purity judge that decision method is：By the D NA 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.
Wherein, the rotating speed of the centrifugation of nutrient solution is 11000r/min, time 10min in (3.2)；Centrifuged after thermal denaturation
Rotating speed is 800r/min, time 1min.(3.3) rotating speed centrifuged in after thermal denaturation is 8 00r/min, time 10s.
Embodiment 2
The detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*106CFU/mL, obtain the artificial saliva containing porphyromonas gingivalis；
Step (2), mouth containing tobacco products are entered using the artificial saliva containing porphyromonas gingivalis that step (1) obtains
Row extraction, extraction is multiple, collects extract solution respectively；Specific method is：
Into mouth containing tobacco products 1.2g, the artificial saliva containing porphyromonas gingivalis of implantation step (1) is at 37 DEG C
Isothermal vibration is extracted, and concussion speed is 300rpm, is extracted 3 times, every time the extraction people containing porphyromonas gingivalis used
Work saliva 10mL, each extraction time are 5min, collect extract solution respectively；
Step (3), each extract solution that step (2) is collected into and the artificial saliva containing porphyromonas gingivalis of step (1)
Liquid is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract the porphyromonas gingivalis in each nutrient solution respectively afterwards
DNA, while extract the reference culture original DNA of porphyromonas gingivalis；Specific method is：
(3.1) co-culture：Take each extract solution that step (2) is collected into and the people containing porphyromonas gingivalis of step (1)
Work saliva, every kind of liquid are divided into 3 equal portions, are respectively put into anaerobic culture box and are co-cultured at 37 DEG C, every kind of equal portions of liquid 3
Incubation time be respectively tri- periods of 0h, 2h, 6h；
(3.2) extraction of the porphyromonas gingivalis DNA in nutrient solution：Take out the nutrient solution after the culture of step (3.1)
In 4 DEG C of centrifugations, supernatant is removed, adds microorganism PCR lysates, after being well mixed, be placed in thermal denaturation 15min at 85 DEG C, so
Centrifuge again afterwards, take supernatant, that is, obtain the porphyromonas gingivalis DNA in nutrient solution；Described microorganism PCR lysates
Add 5% that volume is nutrient solution volume；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：Take with step (3.2) used in it is identical
The microorganism PCR lysates of volume, reference culture to the concentration for adding porphyromonas gingivalis is 107CFU/mL, at 85 DEG C
Thermal denaturation 15min, is then centrifuged for, and takes supernatant, that is, obtains the reference culture original DNA of porphyromonas gingivalis；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient are used as template
Quantitative fluorescent PCR analysis is carried out, draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile fluorescent quantitation P CR analyses are carried out using the porphyromonas gingivalis DNA in each nutrient solution as template respectively,
Blank control template calculates containing for the porphyromonas gingivalis DNA in each nutrient solution using sterilizing distilled water according to standard curve
Amount；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagatagg c-TAMRA；Tool
Body method is：
The reference culture that volume is porphyromonas gingivalis is added into the reference culture original DNA of porphyromonas gingivalis
The water that 9 times of original DNA volume, and fully mix, then 10 times of concentration gradient dilutions are taken turns doing, dilute 6 times altogether, as standard curve
Template；
Take the porphyromonas gingivalis DNA in each nutrient solution with sterilized water by 1:Test specimens are used as after the dilution of 8 volume ratios
Product template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
The reference culture original DNA to porphyromonas gingivalis, each nutrient solution are needed before quantitative fluorescent PCR analysis is carried out
In porphyromonas gingivalis DNA purity judge that decision method is：By the D NA 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.
Wherein, the rotating speed of the centrifugation of nutrient solution is 12000r/min, time 10min in (3.2)；Centrifuged after thermal denaturation
Rotating speed is 1000r/min, time 1min.(3.3) rotating speed centrifuged in after thermal denaturation is 1 000r/min, time 10s.
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 buccal tobacco product influences on porphyromonas gingivalis, comprises the following steps：
Step (1), artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, make porphyromonas unit cell
Amount of the bacterium in artificial saliva is 1*105CFU/mL, obtains the artificial saliva containing porphyromonas gingivalis, and numbering is 1# liquid；
Step (2), mouth containing tobacco products are entered using the artificial saliva containing porphyromonas gingivalis that step (1) obtains
Row extraction, extraction is multiple, collects extract solution respectively；Specific method is：
Into mouth containing tobacco products 1g, the artificial saliva containing porphyromonas gingivalis of implantation step (1) is at 37 DEG C
Isothermal vibration is extracted, and concussion speed is 200rpm, is extracted 3 times, every time the extraction people containing porphyromonas gingivalis used
Work saliva 10mL, each extraction time are 5min, collect extract solution, numbering 2#, 3#, 4# liquid respectively；
Step (3), each extract solution that step (2) is collected into and the artificial saliva containing porphyromonas gingivalis of step (1)
Liquid is put into anaerobic culture box and co-cultured at 37 DEG C together；Extract the porphyromonas gingivalis in each nutrient solution respectively afterwards
DNA, while extract the reference culture original DNA of porphyromonas gingivalis；Specific method is：
(3.1) co-culture：Take 2#, 3#, 4# liquid that step (2) is collected into and step (1) contains porphyromonas gingivalis
Artificial saliva 1# liquid, every kind of liquid is divided into 3 equal portions, is respectively put into anaerobic culture box and co-cultured at 37 DEG C, often
The incubation time of the kind equal portions of liquid 3 is respectively tri- periods of 0h, 2h, 6h；
(3.2) extraction of the porphyromonas gingivalis DNA in nutrient solution：Take out the nutrient solution after the culture of step (3.1)
1mL is placed in sterile 1.5mL EP pipes in 4 DEG C of centrifugations, is removed supernatant, is added 50 μ L microorganism PCR lysates in EP pipes,
After well mixed, thermal denaturation 15min at 80 DEG C is placed in, is then centrifuged again, takes supernatant, that is, obtains the gum porphin in nutrient solution
Quinoline monad DNA；
(3.3) extraction of the reference culture original DNA of porphyromonas gingivalis：50 μ L microorganisms P CR lysates are taken in going out
In the 1.5mL centrifuge tubes of bacterium, reference culture to the concentration for adding porphyromonas gingivalis is 107CFU/mL, the thermal change at 80 DEG C
Property 15min, is then centrifuged for, takes supernatant, that is, obtain the reference culture original DNA of porphyromonas gingivalis；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR；
Step (4), the reference culture original DNA that 10 times of porphyromonas gingivalis is diluted using concentration gradient are used as template
Quantitative fluorescent PCR analysis is carried out, draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile fluorescent quantitation P CR analyses are carried out using the porphyromonas gingivalis DNA in each nutrient solution as template respectively,
Blank control template calculates containing for the porphyromonas gingivalis DNA in each nutrient solution using sterilizing distilled water according to standard curve
Amount；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Downstream
Primer is 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagatagg c-TAMRA；Tool
Body method is：
Take the μ L of reference culture original DNA 5 of porphyromonas gingivalis to add 45 μ L water, and fully mix, then take turns doing 10
The dilution of times concentration gradient, dilutes 6 times, as standard curve template altogether；
Take the porphyromonas gingivalis DNA in each nutrient solution with sterilized water by 1:Test specimens are used as after the dilution of 8 volume ratios
Product template；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s at 95 DEG C of denaturation, at 60 DEG C of annealing/extension
1min, carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point is calculated according to standard curve
Nutrient solution Porphyromonas gingivalis content.
The reference culture original DNA to porphyromonas gingivalis, each nutrient solution are needed before quantitative fluorescent PCR analysis is carried out
In porphyromonas gingivalis DNA purity judge that decision method is：By the D NA of testing sample respectively wavelength 260nm,
The OD value (optical density, OD) determined under 280nm；When the OD value under wavelength 260nm and in wavelength
When the ratio of OD value is 1.7~1.9 under 280nm (O D260 and OD280 ratios are 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.
Wherein, the rotating speed of the centrifugation of nutrient solution is 11500r/min, time 10min in (3.2)；Centrifuged after thermal denaturation
Rotating speed is 900r/min, time 1min.(3.3) rotating speed centrifuged in after thermal denaturation is 9 00r/min, time 10s.
During quantitative fluorescent PCR analysis detection, each pattern detection in triplicate, takes its average value to be calculated.
As a result as shown in figure 1 and table 1, table 1 is the testing result of each extract solution Porphyromonas gingivalis.
Wherein, standard curve is y=-3.960log (x)+36.90, r2=0.9791；Afterwards by each time point nutrient solution
Ct values be updated in standard curve, try to achieve corresponding to porphyromonas gingivalis content.
Table 1
Note：Unit is CFU/mL, M ± SD.
It is little greatly using the saliva Porphyromonas gingivalis changes of contents after buccal tobacco product by table 1 but raw
Long speed decreases, and wherein 2# liquid reduces the most notable, and this is probably because buccal tobacco product extract has suppression
The effect of porphyromonas gingivalis growth processed.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table is as shown in table 2.
Table 2
| Primer and probe | Sequence 5 ' -3 ' |
| Forward primer | 5’-tacccatcgtcgccttggt-3’(SEQ ID NO.1) |
| Reverse primer | 5’-cggactaaaaccgcatacacttg-3’(SEQ ID NO.2) |
| Probe | FAM-atttatagctgtaagataggc-TAMRA(SEQ ID NO.3) |
Sequence table
<110>Cigarette industry Co., Ltd in Yunnan
<120>The detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis
<160> 3
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tacccatcgt cgccttggt 19
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<212> DNA
<213>Artificial sequence ()
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cggactaaaa ccgcatacac ttg 23
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<212> DNA
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atttatagct gtaagatagg c 21
Claims (9)
1. the detection method that a kind of buccal tobacco product influences on porphyromonas gingivalis, it is characterised in that including following step
Suddenly：
Step（1）, artificial saliva is prepared, and porphyromonas gingivalis is added into artificial saliva, porphyromonas gingivalis is existed
Amount in artificial saliva is 1*104-1*106CFU/mL, obtain the artificial saliva containing porphyromonas gingivalis；
Step（2）, using step（1）The obtained artificial saliva containing porphyromonas gingivalis carries to mouth containing tobacco products
Take, extraction is multiple, collects extract solution respectively；
Step（3）, by step（2）Each extract solution and step being collected into（1）The artificial saliva one containing porphyromonas gingivalis
Rise and be put into anaerobic culture box and co-cultured at 37 DEG C；Extract the porphyromonas gingivalis DNA in each nutrient solution respectively afterwards,
The reference culture original DNA of porphyromonas gingivalis is extracted simultaneously；
Step（4）, carried out using the reference culture original DNA of 10 times of porphyromonas gingivalis of concentration gradient dilution as template glimmering
Fluorescent Quantitative PCR is analyzed, and draws porphyromonas gingivalis quantitative fluorescent PCR standard curve afterwards；
Meanwhile quantitative fluorescent PCR analysis, blank are carried out using the porphyromonas gingivalis DNA in each nutrient solution as template respectively
Contrast template is using sterilizing distilled water, the content of the porphyromonas gingivalis DNA in each nutrient solution of standard curve calculating；
Wherein, sense primer used in quantitative fluorescent PCR analysis is 5 '-tacccatcgtcgccttggt-3 '；Anti-sense primer
For 5 '-cggactaaaaccgcatacacttg-3 '；Probe is FAM-atttatagctgtaagataggc-TAMRA.
2. the detection method that buccal tobacco product according to claim 1 influences on porphyromonas gingivalis, its feature
It is, step（2）Specific method be：
The implantation step into mouth containing tobacco products 0.8-1.2g（1）The artificial saliva containing porphyromonas gingivalis at 37 DEG C
Isothermal vibration is extracted, and extraction is multiple, and the extraction artificial saliva 10mL containing porphyromonas gingivalis used, is carried every time every time
It is 5min to take the time, collects extract solution respectively.
3. the detection method that buccal tobacco product according to claim 2 influences on porphyromonas gingivalis, its feature
It is, described concussion speed is 150-300 rpm；Extraction time is 3 times.
4. the detection method that buccal tobacco product according to claim 2 influences on porphyromonas gingivalis, its feature
It is, step（3）Specific method be：
（3.1）Co-culture：Take step（2）Each extract solution and step being collected into（1）The artificial saliva containing porphyromonas gingivalis
Liquid, every kind of liquid are divided into 3 equal portions, are respectively put into anaerobic culture box and are co-cultured at 37 DEG C, the training of every kind of equal portions of liquid 3
The foster time is respectively tri- periods of 0h, 2h, 6h；
（3.2）The extraction of porphyromonas gingivalis DNA in nutrient solution：Take out step（3.1）Culture after nutrient solution at 4 DEG C
Centrifugation, removes supernatant, adds microorganism PCR lysates, after being well mixed, is placed in thermal denaturation 15min at 75-85 DEG C, then
Centrifuge again, take supernatant, that is, obtain the porphyromonas gingivalis DNA in nutrient solution；Described microorganism PCR lysates add
Enter 5% that volume is nutrient solution volume；
（3.3）The extraction of the reference culture original DNA of porphyromonas gingivalis：Take and step（3.2）Used same volume
Microorganism PCR lysates, it is 10 to add the reference culture of porphyromonas gingivalis to concentration7CFU/mL, at 75-85 DEG C
Thermal denaturation 15min, is then centrifuged for, and takes supernatant, that is, obtains the reference culture original DNA of porphyromonas gingivalis；
Described microorganism PCR lysates are Lysis Buffer for Microorganism to Direct PCR.
5. the detection method that buccal tobacco product according to claim 4 influences on porphyromonas gingivalis, its feature
It is,（3.2）The rotating speed of the centrifugation of middle nutrient solution is 11000-12000r/min, time 10min；What is centrifuged after thermal denaturation turns
Speed is 800-1000r/min, time 1min.
6. the detection method that buccal tobacco product according to claim 4 influences on porphyromonas gingivalis, its feature
It is,（3.3）The rotating speed centrifuged after middle thermal denaturation is 800-1000r/min, time 10s.
7. the detection method that the buccal tobacco product according to claim 1 or 4 influences on porphyromonas gingivalis, it is special
Sign is, the reference culture original DNA to porphyromonas gingivalis, each nutrient solution are needed before quantitative fluorescent PCR analysis is carried out
In porphyromonas gingivalis DNA purity judge that 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.
8. the detection method that the buccal tobacco product according to claim 4,5 or 6 influences on porphyromonas gingivalis, its
It is characterised by, step（4）Specific method be：
Into the reference culture original DNA of porphyromonas gingivalis, addition volume is that the reference culture of porphyromonas gingivalis is original
The water that 9 times of DNA volumes, and fully mix, then 10 times of concentration gradient dilutions are taken turns doing, dilute 6 times altogether, as standard curve mould
Plate；
Take the porphyromonas gingivalis DNA in each nutrient solution with sterilized water by 1:Test sample mould is used as after the dilution of 8 volume ratios
Plate；
Using sterilizing distilled water as blank control template；
Quantitative fluorescent PCR reaction system：
μ L of 200 nmol/L of sense primer 0.175,
μ L of 200 nmol/L of anti-sense primer 0.175,
μ L of 250 nmol/L of probe 0.175,
μ L of sterilized water 1.475,
μ L of template 8,
The μ L of Master Mix (2X) Kit 10 in PCR kit for fluorescence quantitative, totally 20 μ L；
Quantitative fluorescent PCR reaction condition is：5min at 95 DEG C of pre-degeneration, 15s, 1min at 60 DEG C of annealing/extension at 95 DEG C of denaturation,
Carry out 45 circulations；
Porphyromonas gingivalis quantitative fluorescent PCR standard curve is drawn afterwards, and each time point culture is calculated according to standard curve
Liquid Porphyromonas gingivalis content.
9. the detection method that gum base type tobacco product according to claim 8 influences on porphyromonas gingivalis, its feature
It is, when quantitative fluorescent PCR analysis detects, each pattern detection in triplicate, takes its average value to be calculated.
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