CN111187808A

CN111187808A - Research method for human oral cavity microorganism group structure and composition

Info

Publication number: CN111187808A
Application number: CN201911423139.6A
Authority: CN
Inventors: 舒毅
Original assignee: Stomatological Hospital of Chongqing Medical University
Current assignee: Stomatological Hospital of Chongqing Medical University
Priority date: 2019-08-08
Filing date: 2019-12-31
Publication date: 2020-05-22

Abstract

The invention discloses a research method of human oral cavity microorganism group structure and composition, comprising the following steps: step 1, collecting a saliva sample; step 2, extracting DNA of the salivary bacteria assembly; step 3, PCR amplification of target gene fragments; step 4, performing DGGE electrophoresis; step 5, performing back cutting sequencing on the difference bands; and 6, analyzing a DGGE map and data. The method adopts DGGE technology to carry out diversity of salivary microorganism groups on saliva samples of sample population, and learns the structure and composition of the salivary microorganism groups of the population. The present invention reaches the following conclusions: the DGGE can comprehensively reflect the oral microbial community structure, is simple and convenient to operate, and is an economic and accurate technology for evaluating the oral building condition and predicting the occurrence of oral diseases.

Description

Research method for human oral cavity microorganism group structure and composition

Technical Field

The invention belongs to the technical field of medicine, relates to a research method of a human oral cavity microorganism group structure and composition, and particularly relates to a research method of a human oral cavity microorganism group structure and composition in different growth and development stages and individual oral cavity health condition evaluation, which can be used for disease screening or oral cavity sub-health condition evaluation and disease prediction.

Background

A large number of microorganisms inhabit on the body surface and in the body, participate in the maintenance of human health and the occurrence process of diseases in the form of microbial communities, and play an important role, so the gene is called as a 'human second gene'. The microorganisms planted in the oral cavity form a specific complex micro-ecological environment to participate in the regulation and control process of the health and diseases of the oral cavity of a host. The oral microecological system with stable balance plays an important role in maintaining oral health, the imbalance of balance is an initiation factor for the occurrence and development of various oral infectious diseases, and the excessive growth of oral pathogenic bacteria or the growth of probiotics can cause the occurrence of the oral infectious diseases and even cause systemic diseases.

The transition of the micro-ecological environment from a "healthy" state to a "diseased" state is not a single or individual action of a certain number of bacteria, but rather involves a symbiotic flora, even due to the action of certain potentially pathogenic bacteria that cannot be isolated by traditional culture techniques. Denaturing Gradient Gel Electrophoresis (DGGE) is a molecular biology technology widely applied to diversity analysis of oral microbial groups, and can simultaneously analyze multiple strains in the same bacterial plaque biomembrane aiming at a microbial 16SrRNA/16S rDNA fragment so as to conveniently and quickly know the composition and the structure of the bacterial plaque biomembrane, and can also quickly compare the structural diversity of a healthy sample and a diseased sample so as to monitor the ecological change of the oral microbes under a certain pathogenic condition.

Disclosure of Invention

The invention aims to provide a research method for the structure and composition of a human oral microbial group. From the microecology perspective, the influence of physiological factors of dentition deletion on the diversity of oral microecological structures is researched by adopting different research modes, the oral dominant flora and core microorganisms of the old people are searched, and a certain theoretical basis is provided for the research of the pathogenesis of the oral diseases and the systemic diseases of the old people and a new method for clinical prevention and diagnosis and treatment.

The technical scheme is as follows:

a research method for human oral cavity microorganism group structure and composition comprises the following steps:

step 1, collecting saliva samples: collecting non-irritant saliva samples 3ml 12 hours after brushing teeth and 2 hours after meal drinking of the target population, sending back to the laboratory within 2 hours of refrigerated transportation, and freezing and storing at-80 ℃ for extracting collective DNA.

Step 2, extracting DNA of the salivary bacteria assembly: by MasterPure^TMDNA Purification Kit (Epicentre Biotechnologies) extracted genomic DNA of salivary bacteria.

Step 3, PCR amplification of target gene fragments: with dreamcaq^TMThe PCR Master Mix kit amplifies 16SrDNA V3 region of DNA collected from salivary bacteria, and the primers are designed as universal primers of different types of bacteria: the nucleotide sequence of BAC1 with the upstream primer of which the 5' end is specifically added with a GC clip is shown as SEQ: ID: 1, the nucleotide sequence of the downstream primer BAC2 is shown as SEQ: ID: 2, the size of the amplified bacterial V3 hypervariable region fragment is about 340 bp. PCR reaction (50. mu.l): the whole reaction system was filled with 100ng of DNA template, 1. mu.l of forward primer (25. mu.M), 1. mu.l of reverse primer (25. mu.M), and 25. mu.l of premixed PCR reaction solution. The PCR reaction parameters were as follows:

the first step is as follows: pre-denaturation at 94 ℃ for 4 min;

the second step is that: denaturation at 94 ℃ for 1 min;

the third step: annealing at 56 ℃ for 1 minute;

the fourth step: extension at 72 ℃ for 2 min;

circulating the second step to the fourth step for 25 times;

the fifth step: extension at 72 ℃ for 5 min;

after the reaction is completed, the PCR product is subjected to electrophoresis for 30 minutes at 100 volts by using a 1% common agarose gel added with gold view, a 1 XTAE buffer solution, and a gel imaging system to detect the product and the concentration thereof. The PCR product was purified using the QIAGEN QIAquick PCRPoint Kit for use.

Step 4, DGGE electrophoresis: the spotting was performed by using a Bio-Rad DCode gene mutation detection system (Bio-Rad Co.). 16ml of DGGE denatured solution with concentration of 60% and 40% respectively, adding 10% APS 18. mu.l as inducer and TEMED 150. mu.l as catalyst, mixing, fixing on gel infusion device, pouring gel at constant speed, carefully inserting comb, and allowing gel polymerization for about one hour. And the electrophoresis control device is turned on, and the electrophoresis buffer is preheated to 58 ℃. After the gel was completely solidified, 20. mu.l of PCR product was mixed with 2X sample solution and spotted into the wells. The constant voltage was 60V and the temperature was 60 ℃ for electrophoresis overnight. After about 16h, the glass plates were separated, the gel was stained in 0.5% μ g/ml hexadiene bromide staining solution (EB) for 10min, rinsed in 1 XTAE buffer for 10min, excess hexadiene bromide was rinsed off, the gel was removed, and imaged in a gel imaging system. To verify the reproducibility of the experiment, each set of samples was run in duplicate 2-3 times.

Step 5, back cutting sequencing of the difference bands: cutting different bands on DGGE glue under an ultraviolet lamp, placing the bands in a sterile EP tube, adding 20 mu l of distilled water for soaking, mashing to fully dissolve DNA in the bands, taking 5 mu l of leaching liquid (about 100ng) for sequencing PCR reaction after overnight at 4 ℃, and carrying out the same system and reaction conditions as the above (50 mu l): the whole reaction system was filled with 100ng of DNA template, 1. mu.l of forward primer (25. mu.M), 1. mu.l of reverse primer (25. mu.M), and 25. mu.l of premixed PCR reaction solution. The PCR reaction was purified using QIAGEN QIAquick PCR Purification Kit and the product was sequenced by Beijing Zhongkoschin technologies, Inc. Sequencing results are subjected to Sequence alignment analysis in the identity 16S rRNAplast Sequence of a Human Oral Microbiome Database, and the most similar microbial species are found.

Step 6, DGGE map and data analysis: the collected DGGE electrophoretic images were analyzed using Quantity One 1-D analysis software 4.6.2(Bio-Rad, USA). Experimental reproducibility evaluation sample population richness and balance were reflected using Sorenson's similarity coefficient, using sample average band number (No.), and Shannon index (H').

Further, step 2 is specifically that ① samples from which DNA is to be extracted after discarding food residues are centrifuged at high speed (14000rpm) for 10 minutes, supernatant is discarded, precipitate is retained, ② 300. mu.l of cell lysate containing 50. mu.g proteinase K is added, the mixture is mixed by a shaker, water bath at 65 ℃ is carried out for 40 minutes, 5 seconds are shaken and mixed evenly every 5-10 minutes, bacterial cell walls are fully lysed, ③ samples are cooled to 37 ℃, 1.5. mu.l of RNase A (5. mu.g/. mu.l) is added and mixed evenly by a shaker, water bath at 37 ℃ is carried out for 30 minutes, RNA is degraded, sample RNA contamination is removed, ④ minutes later, samples are placed on ice to be cooled for 3-5 minutes, 175. mu.l of protein precipitation solution is added and mixed evenly, low temperature high speed centrifugation (4 ℃; 14,000rpm) is carried out for 10 minutes, protein precipitation is discarded, supernatant is added with 500. mu.l of isopropanol and mixed evenly, low temperature high speed centrifugation (4 ℃; 14,000rpm) is carried out for 10 minutes, DNA pellet is washed in dry air twice, DNA is dissolved in 25. mu.20. mu.l of NOOD alcohol, DNA is added into a vacuum tube, and the residual volume of TE is determined, and then dissolved in 5920. mu.20. mu.l of DNA is added into a sample.

As described in the step 2, the research method (1) has simple and convenient material taking, can obtain saliva after eating, has noninvasive sampling and saves wound injuries such as traditional blood samples; (2) the technology is mature, the result stability is good, and the method is widely applied to the structural diversity of microbial communities and has high popularization.

Further, the oral health of the sample is judged according to the results of the map analysis and sequencing comparison of the strains, and the possibility of future oral diseases is estimated. The research method can be used in the process of preparing products for diagnosing oral diseases.

The invention has the beneficial effects that:

the microbial community structure in the saliva is relatively stable and can not be obviously changed along with the difference of time and space, so that the specific micro-ecological environment structure of a certain crowd in a specific period can be relatively accurate and real, and the saliva is convenient to obtain, so that the saliva is widely used for researching the diversity of the oral microbial structure. The Denaturing Gradient Gel Electrophoresis (DGGE) technology is a molecular biology technology widely applied to diversity analysis of oral microbial groups, can simultaneously analyze multiple strains in the same bacterial plaque biomembrane aiming at a microbial 16S rRNA/16S rDNA fragment so as to conveniently and quickly know the composition and the structure of the bacterial plaque biomembrane, can also quickly compare the structural diversity of a healthy sample and a diseased sample, monitors the ecological change of the oral microbial under a certain pathogenic condition, and is suitable for being applied to one of effective means for clinically screening and monitoring the oral health of people.

Drawings

FIG. 1 is a flow chart of a method of studying the structure and composition of the human oral microbiome of the present invention;

FIG. 2 is a DGGE electrophoretogram:

FIG. 3 is a UPGAM cluster analysis of DGGE electropherograms.

Detailed Description

The technical solutions of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.

Referring to fig. 1, a method for studying the structure and composition of a human oral microbial group comprises the following steps:

Step 2, extracting DNA of the salivary bacteria assembly: by MasterPure^TMDNA Purification Kit (Epicentre Biotechnologies) extraction of salivary bacterial genomic DNA the main steps are ① high speed centrifugation (14000rpm) of the sample from which DNA is to be extracted after discarding food residues for 10 minutes, discarding the supernatant, retaining the precipitate ② adding 300. mu.l of cell lysate containing 50. mu.g proteinase K, shaking and mixing, water bath at 65 ℃ for 40 minutes, shaking and mixing for 5 seconds every 5-10 minutes, fully lysing bacterial cell walls, ③ cooling the sample to 37 ℃, adding 1.5. mu.l RNase A (5. mu.g/. mu.l) shaking and mixing, water bath at 37 ℃ for 30 minutes, degrading RNA, removing RNA contamination of the sample, ④ minutes later, placing the sample on ice to cool and mix for 3-5 minutes later, adding 175. mu.l of protein precipitation solution, high speed centrifugation at low temperature (4 ℃; 14,000rpm) for 10 minutes, discarding protein precipitate at 56, adding 500. mu.l of isopropanol to the supernatant and mixing, high speed centrifugation at low temperature (4,000 rpm) for 10 minutes, sucking out DNA from the sample, adding 260. mu.l of dry DNA in a dry tube, washing the bottom with alcohol to remove DNA, adding 25. mu.l of DNA, washing the residual DNA in a dry tube, removing the DNA, and removing the DNA, removing the residual DNA in 5920. mu.g/75. mu.g of DNA,and dispensed into 4 EP tubes in 5. mu.l volumes for storage at-20 ℃ until use.

Step 3, PCR amplification of target gene fragments: with dreamcaq^TMThe PCR Master Mix kit amplifies 16SrDNA V3 region of DNA collected from salivary bacteria, and the primers are designed as universal primers of different types of bacteria: the upstream primer 5 ' end is specifically added with a GC clip BAC1 (5'-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCCGAC TAC GTG CCA GCA GCC-3') and a downstream primer BAC2 (5'-GGA CTA CCA GGG TAT CTA ATC C-3'), and the size of the amplified bacterial V3 hypervariable region fragment is about 340 bp. PCR reaction (50. mu.1): the whole reaction system was filled with 100ng of DNA template, 1. mu.l of forward primer (25. mu.M), 1. mu.l of reverse primer (25. mu.M), and 25. mu.l of premixed PCR reaction solution. The PCR reaction parameters were as follows:

the first step is as follows: pre-denaturation at 94 ℃ for 4 min;

the second step is that: denaturation at 94 ℃ for 1 min;

the third step: annealing at 56 ℃ for 1 minute;

the fourth step: extension at 72 ℃ for 2 min;

circulating the second step to the fourth step for 25 times;

the fifth step: extension at 72 ℃ for 5 min;

Step 4, DGGE electrophoresis: the spotting was performed by using a Bio-Rad DCode gene mutation detection system (Bio-Rad Co.). 16ml of DGGE denatured solution with concentration of 60% and 40% respectively, adding 10% APS 18. mu.l as inducer and TEMED 150. mu.l as catalyst, mixing, fixing on gel infusion device, pouring gel at constant speed, carefully inserting comb, and allowing gel polymerization for about one hour. And the electrophoresis control device is turned on, and the electrophoresis buffer is preheated to 58 ℃. After the gel was completely solidified, 20. mu.l of PCR product was mixed with 2X sample solution and spotted into the wells. The constant voltage was 60V and the temperature was 60 ℃ for electrophoresis overnight. After about 16h, the glass plates were separated, the gel was stained in 0.5% μ g/ml hexadiene bromide staining solution (EB) for 10min, rinsed in 1 XTAE buffer for 10min, excess hexadiene bromide was rinsed off, the gel was removed, and imaged in a gel imaging system (FIG. 2). To verify the reproducibility of the experiment, each set of samples was run in duplicate 2-3 times.

And judging the oral health state of the sample according to the results of map analysis and sequencing comparison of the strains, and predicting the possibility of oral diseases in the future.

The result is that ① DGGE fingerprint analysis shows that the average band number and Shannon index of saliva microorganisms of the non-dentition elderly are less than the average band number of saliva microorganisms of the whole dentition and dentition defect elderly, and the reduction trend has statistical difference, the cluster analysis result shows that the saliva microorganisms of the whole dentition and dentition defect elderly have more common microorganism populations, while the saliva microorganism communities of the non-dentition elderly are single clusters, ③ difference band sequencing shows that detected bacteria are all oral resident multi-visible bacteria, wherein bacteria related to cardiovascular diseases and respiratory diseases are detected in saliva of the non-dentition elderly.

And (4) conclusion: the DGGE technology finds that the diversity of oral flora is inversely proportional to the defect degree of dentition, the dentition can influence the complexity of oral micro-ecological structures, except for the conventional normal oral resident bacteria, bacteria related to the cardiovascular and respiratory diseases are separated from the oral cavity of the elderly people without dental occlusion (the screened bacteria are not new bacteria), but the specific mechanism needs to be further explored. In conclusion, the DGGE technology can be used as one of the means for preventing and evaluating oral diseases and systemic diseases.

The invention has the following advantages:

(1) and non-invasive: 3ml saliva samples are convenient and quick to take, and the oral health condition of an individual can be comprehensively reflected;

(2) and accuracy: the 16S rDNA fragment is amplified by adopting a PCR technology, the technology is mature and stable, and the amplified fragment has species genetic stability, can reflect individual difference and has higher accuracy;

(3) and economy: the PCR technology belongs to a mature technology, each laboratory can operate, the cost for analyzing species genetic information by DGGE electrophoresis is low, and the cost of the whole operation process is low;

(4) and social benefits are as follows: the oral cavity microbial community structure and composition are described, the oral cavity health condition of an individual can be reflected, even the occurrence of oral cavity diseases of certain specific groups is estimated, and preventive measures are taken in a targeted manner to reduce the cost of disease diagnosis and treatment, so that the oral cavity microbial community structure and composition have certain social value.

For example, it is found out by analyzing saliva samples of an age group without a tooth and a tooth, that the specific sample group shows relatively simple flora diversity compared with a normal adult group (as shown in fig. 1 and fig. 2), sequencing is performed on a specific strip, and a sequencing result (table 1) shows that the DGGE can accurately separate different species of bacteria in the oral saliva sample, so that the rapid detection method is suitable for rapid detection of oral clinical samples; unnamed bacteria associated with endocarditis have also been found in the edentulous group, which may be associated with a loss of masticatory efficiency due to loss of dentition, limited food intake and relatively low body immunity, in the elderly, mostly in the edentulous group.

TABLE 1 differential band sequencing results

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

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Claims

1. A research method for human oral cavity microorganism group structure and composition is characterized in that: the method comprises the following steps:

step 1, collecting saliva samples: collecting non-irritant saliva samples 3ml of 12 hours after brushing teeth and 2 hours after meal drinking of a target group, sending the samples back to a laboratory within 2 hours of cold storage and transportation, and performing freezing storage at-80 ℃ to extract aggregate DNA;

step 2, extracting DNA of the salivary bacteria assembly: by MasterPure^TMExtracting salivary bacteria genome DNA by using the DNA Purification Kit;

step 3, PCR amplification of target gene fragments: with dreamcaq^TMThe PCR Master Mix kit amplifies 16SrDNA V3 region of DNA collected from salivary bacteria, and the primers are designed as universal primers of different types of bacteria: the nucleotide sequence of BAC1 with the upstream primer of which the 5' end is specifically added with a GC clip is shown as SEQ: ID: 1, the nucleotide sequence of the downstream primer BAC2 is shown as SEQ: ID: 2, the size of the amplified V3 hypervariable region fragment is 340 bp; and (3) PCR reaction system: 100ng of DNA template, 1 mul of upstream primer and 1 mul of downstream primer, and 25 mul of premixed PCR reaction solution, and the whole reaction system is filled up with water; the PCR reaction parameters were as follows:

the first step is as follows: pre-denaturation at 94 ℃ for 4 min;

the second step is that: denaturation at 94 ℃ for 1 min;

the third step: annealing at 56 ℃ for 1 minute;

the fourth step: extension at 72 ℃ for 2 min;

circulating the second step to the fourth step for 25 times;

the fifth step: extension at 72 ℃ for 5 min;

after the reaction is finished, the PCR product uses 1% common agarose gel added with gold view, 1 XTAE buffer solution, 100V electrophoresis for 30 minutes, and a gel imaging system detects the product and the concentration thereof; purifying the PCR product by using a QIAGENQIAquick PCR purification kit for later use;

step 4, DGGE electrophoresis: the spotting is carried out by using a Bio-Rad DCode gene mutation detection system; 16ml of DGGE denatured solution with the concentration of 60 percent and 40 percent respectively, adding 18 mul of 10 percent APS as an inducer and 150 mul of TEMED as a catalyst, uniformly mixing, fixing the mixture in a glue infusion device, pouring glue at a constant speed as much as possible, carefully inserting a comb, and polymerizing the gel for one hour; opening an electrophoresis control device, and preheating an electrophoresis buffer solution to 58 ℃; after the glue is completely solidified, uniformly mixing 20 mu l of PCR product with 2X sample loading liquid, and adding into a sample adding hole; performing electrophoresis at a constant voltage of 60V and a temperature of 60 ℃ overnight; after 16h, separating the glass plate, placing the glue in 0.5% mu g/ml ethidium bromide staining solution EB for staining for 10min, rinsing with 1 × TAE buffer solution for 10min, rinsing off redundant ethidium bromide, taking out the glue, and imaging in a glue imaging system; in order to verify the repeatability of the experiment, each group of samples is run for 2-3 times;

step 5, back cutting sequencing of the difference bands: cutting different bands on the DGGE glue under an ultraviolet lamp, placing the bands in a sterile EP tube, adding 20 mu l of distilled water for soaking, mashing to fully dissolve DNA in the bands, taking 5 mu l of leaching liquid after overnight at 4 ℃ for sequencing PCR reaction, wherein the system and the reaction conditions are as the same as the above: 100ng of DNA template, 1 mul of upstream primer and 1 mul of downstream primer, and 25 mul of premixed PCR reaction solution, and the whole reaction system is filled up with water; the PCR reaction was purified using QIAGEN QIAquick PCR Purification Kit and the product was sequenced by Beijing Zhongke Schilin science and technology Co., Ltd; performing Sequence comparison analysis on the sequencing result in the identity 16S rRNA Blast Sequence of a Human Oral Microbiol Database to find out the most similar microbial species;

step 6, DGGE map and data analysis: analyzing the collected DGGE electrophoresis image by adopting Quantity One 1-D analysis software 4.6.2; experimental repeatability evaluation sample population richness and balance were reacted using Sorenson's similarity coefficient, sample average number of bands No. and Shannon index H'.

2. The method of claim 1, wherein the population of human oral microorganisms comprises:

the step 2 is specifically that ① samples which are to be extracted with food residues are centrifuged at high speed and centrifuged at 14000rpm for 10 minutes, supernatant is discarded and precipitates are reserved, ② 300 mul of cell lysate containing 50 mul of proteinase K is added, the cell lysate is evenly mixed by an oscillator, water bath is carried out at 65 ℃ for 40 minutes, 5-10 minutes is carried out every 5-10 minutes, bacterial cell walls are fully cracked, ③ samples are cooled to 37 ℃, 1.5 mul of RNase A oscillator is added and evenly mixed, water bath is carried out at 37 ℃ for 30 minutes, RNA is degraded, sample RNA pollution is removed, ④ minutes is carried out, the samples are placed on ice to be cooled for 3-5 minutes, 175 mul of protein precipitation solution is added, the samples are evenly mixed and centrifuged at high speed and low temperature, the samples are centrifuged at 39000 rpm for 10 minutes, the supernatant is added with 500 mul of isopropanol and evenly mixed, the samples are centrifuged at high speed and low temperature, the samples are centrifuged at 4 ℃, 10 minutes at ⑤, the supernatant is added with 75% ethanol, DNA blocks attached to the bottom of an EP tube, dry DNA blocks are washed in dry air twice, the dry DNA is added into a vacuum tube, the EP tube, the dry DNA mass concentration of 260-20 mul TE is determined, the residual DNA is completely dissolved in a 5920 mul of TE tube, and the DNA is added, the 25-20 mu 2 mol of TE, the.

3. The method of claim 1, wherein the population of human oral microorganisms comprises:

judging the oral health state of the sample according to the results of the map analysis and sequencing comparison of the strains, and the application of the research method in the process of preparing products for diagnosing oral diseases.