Microbial Diversity Of Oral Saliva From Healthy Youths

BackgroundOral environment is an important habitat for microorganisms in the human body, and the oral microbial community is one of the most complex microbial community in the human body, consisting of more than 700 bacteria.Once without the balance of oral microorganisms, it affects not only oral health, but also general health.It has recently proposed that microbial communities might lead to some chronic diseases. Staley et al pointed out that less than 1% of microorganisms can be culutured, suggesting that studies based on culture is not fitable studies on the oral microbial diversity and gene.In 1998, Handelsman first proposed metagenome, which is the genomes of the total microbita found in nature.In the metagenome,the samples include culturable and unculturable microorganisms.Association of the U.S. National Academy of Sciences in 2007 called for a global metagenomics research project.At present, it is limited about the knowledge of the composition of the oral microbial community, especially for the healthy youths.Denaturing gradient gel electrophoresis (DGGE) is a technique based on electrophoretic analysis of PCR product, which was put forward by Fischer in 1779. Originally DGGE was only used in the medical field for the analysis of gene mutations, and in 1993, Muyzer was used in microbial community analysis for the first time. Then, there are increasing reports on different micro-ecological environment fields applicated by the technologyObjectivesThis study is to analysis of two methods for the extraction of microbial genomic DNA from different-period human saliva, in order to choose the better method for the different study objective, and provide the study basis of the relationship between oral microorganisms and disease.In this study, PCR-DGGE (denaturing gradient gel eletrophoresis) is applied to analyze the oral microbial diversity of different-period different-individual's saliva from healthy youths with aim to provide informations on DGGE applied to the oral microbial phylogenetic analysis of healthy youths。Part I An attempt to prepare oral microbial genomic DNA from healthy youthsMethods1,Sampling:We collect 90 saliva samples over a two-day period from 30 students (oral health,21-25Y, college of stomatology, Southern Medical University) with informed consent. Saliva samples are collected by expectoration into sterile plastic 15-ml tube and frozen at-20℃until processing.2,The oral microbial genomic DNA extraction:Two methods (the phenol and chloroform extraction method and QIAamp DNA Micro Kit extraction method), which have modified by preliminary experiments, are used on 90 saliva samples of 30 healthy youths from three periods, to prepare the oral microbial genomic DNA. 3,The detection of genomic DNA:The genomic DNA prepared by the two methods is evaluated and compared comprehensively for the quantity, purity, yield and length of the DNA and the PCR amplification effect by visual, Agarose gel electrophoresis, spectrophotometer et al.Results1,Part of the oral microbial genomic DNA samples, prepared by the modified phenol chloroform extraction method, have little visual floc; the agarose gel electrophoresis shows the length of DNA fragments is about 23kb, but with the condition of smearing; the average yield of genomic DNA is 10.03±7.31 ng.μl-1; the majority of the A260nm/A280nm ratios of prepared genomic DNA are less than 1.7; the specific bands of PCR amplification is not clear enough.2,The oral microbial genomic DNA samples, prepared by the modified QIAamp DNA Micro Kit extraction method, are visually close to colorless; the agarose gel electrophoresis shows the length of DNA fragments is about 23kb, and present in a relatively clear lane but still with the condition of smearing; the average yield of genomic DNA is 53.49±34.78 ng.μl-1; the A260nm/A280nm ratio of prepared genomic DNA are all larger than 1.7, mostly in between 1.7～1.8; the specific bands of PCR amplification is also not clear enough, but more clear than those of the oral microbial genomic DNA samples prepared by the modified phenol chloroform extraction method.3,The two groups of oral microbial genomic DNA samples, prepared by the two methods(the modified phenol and chloroform extraction method and QIAamp DNA Micro Kit extraction method) are significant differences (P<0.001) on the aspects of the visual observation, the agarose gel electrophoresis of the crude DNA and the PCR amplification products, the ratio of A260nm/A280nm and the genomic DNA yield.4,The three periods of oral microbial genomic DNA samples are no significant differences (P> 0.05) on the aspects of the visual observation, the agarose gel electrophoresis of the crude DNA and the PCR amplification products, the ratio of A260nm/A280nm and the genomic DNA yield. But the agarose gel electrophoresis shows that the length of DNA fragments are about 23kb。Conclusions1,The two groups of oral microbial genomic DNA samples, prepared by the two methods(the modified phenol and chloroform extraction method and QIAamp DNA Micro Kit extraction method) can be successfully applied to the PCR amplification and maybe used for the further study on the microbial diversity and metagenome.2,The oral microbial genomic DNA samples, prepared by the modified phenol chloroform extraction method, are small fragments, high impurity, but high concentration. And the method is time-consuming, complex, but economic and can be extracted simultaneously a large number of samples. So the modified phenol chloroform extraction method is more suitable for a large number of samples.3,The oral microbial genomic DNA samples, prepared by the modified QIAamp DNA Micro Kit extraction method are low concentration, but high purity, large segment. And the method is money-consuming and more suitable for the small samples and molecular technology, such as metagenomics, DGGE.4,It showed that DNA extracted from human saliva was no significant difference along the three different-period groups. So the saliva, collected in a short period can be mixed, in order to increase the microbiological concentration, and improve the DNA yield, which is benefit for the molecular techniques study, such as metagenomic library construction。Part II An attempt to test extraction methods impact on the oral microbial diversity analysisMethods1,Sampling:We collect 8 saliva samples from 8 students (oral health,21-25Y, college of stomatology, Southern Medical University) with informed consent. Saliva samples are collected by expectoration into sterile plastic 15-ml tube and frozen at-20℃until processing.2,The oral microbial genomic DNA extraction:Two methods (the phenol and chloroform extraction method and QIAamp DNA Micro Kit extraction method), which have modified by preliminary experiments, are used on16 saliva samples of 8 healthy youths, to prepare the oral microbial genomic DNA.3,PCR-DGGE of the oral microbial 16S rDNA gene:PCR amplification is carried out in a 50 u 1 PrimeStar HS Premix (Takara) containing 5μl of lysate and 0.5 u M of each forward (784DEG) and reverse(880RDEG) primers. The samples were run in two separate PCRs for 15cycles using following parameters:98℃for 10s,46℃for 15s and 72℃for 1 min. Then conduct DGGE.4,DGGE fingerprint analysis:DGGE fingerprints have obtained and analysised.Results1,The PCR amplification products for all the 16 extracted DNA samples(without dilution) by 8 students can obtain the DGGE electrophoresis, which shows, between the two methods, there are significant differences on the straps'number and lightness.2,For the phenol and chloroform extraction method, the mean numbers of detected PCR amplicons are 6.63±2.07. For QIAamp DNA Micro Kit extraction method, the mean numbers of detected PCR amplicons are 10.50±1.41. The mean species richnessof the microbial population is greater by QIAamp DNA Micro Kit extraction method than by the phenol and chloroform extraction method, the difference was statistically significant (P=0.001).Conslusions1,The microbial genomic DNA extraction method significantly impacts on the oral microbial DGGE results from the aspects of the microbial diversity and complexity of the oral microbial biota.2,PCR-based DGGE has been shown to be an more excellent means of rapidly and accurately assessing oral microbial diversity and complexity by QIAamp DNA Micro Kit extraction method than by the phenol and chloroform extraction method.PartⅢAn attempt on the oral microbial diversity in healthy youthsMethods1,Sampling:We collect 16 saliva samples over a three-month period from 14 students (oral health,21-25Y, college of stomatology, Southern Medical University) with informed consent. Saliva samples are collected by expectoration into sterile plastic 15-ml tube and frozen at-20℃until processing. 2,The oral microbial genomic DNA extraction:QIAamp DNA Micro Kit extraction method, which has modified by preliminary experiments, is used on 16 saliva samples of 14 healthy youths from three periods, to prepare the oral microbial genomic DNA.3,PCR-DGGE of the oral microbial 16S rDNA gene:PCR amplification is carried out in a 50μl PrimeStar HS Premix (Takara) containing 5 u 1 of lysate and 0.5μM of each forward (784DEG) and reverse(880RDEG) primers. The samples were run in two separate PCRs for 15cycles using following parameters:98℃for 10s,46℃for 15s and 72℃for 1 min. Then conduct DGGE.4,DGGE fingerprint analysis:DGGE fingerprints have obtained, then make DGGE ideography. The Phyltools software is used to calculate similarity between samples with DGGE fingerprints。The data is made the UPMGA by Multivariate statistical package (MVSP).Results1,All 16 extracted DNA samples(without dilution) by 14 students could be effectively amplified with 784DEG/880RDEG primers. The agarose gel electrophoresis for the PCR amplification products shows a single band without obvious side-band, and the lightness and the length in line with expectations.2,The PCR amplification products for all the 16 extracted DNA samples(without dilution) by 14 students can obtain the DGGE electrophoresis, which shows, among the 14 individuals, there are significant differences on the straps'number, lightness and site. But there are some brilliant straps in every sample; even the DGGE electrophoresis shows the presense of several bright bands in different samples.3,The DGGE electrophoresis shows, between the different periods of the same individual, there are still slightly differents, but no significant differences on the straps' number, lightness and site.4,The UPMGA cluster analysis of microbial community structure from the 11 individuals shows that the similarity indexs of the different period from the same individual reach to more than 0.95, while those from the different individuals are less than 0.90.Conslusions1,The oral microbial genomic DNA samples, prepared by the modified QIAamp DNA Micro Kit extraction method can be applied to PCR amplification and DGGE. In the DGGE electrophoresis could be observed the oral microbial diversity.2,In the same individual, the oral microbial community composition is relatively stable over a long period.3,The different individuals have large differences in oral microbial diversity, which means that every individual has a unique oral microbial community. However, the different individuals have some common microbial predominant communities.