| ObjectPersonal identification is one of the main elements of forensic evidence work,providing investigative clues and trial basis for judicial practice.At present,the common detection method used in forensic personal identification is STR typing technology,which is based on human DNA analysis and detection,high sensitivity,high specificity,fully validated,and the typing results are easy to standardise,with the continuous expansion of domestic and international STR database,STR typing technology has become one of the most widely used detection means in forensic practical examination cases.However,forensic scientists have not stopped their research and have been searching for new biomarkers for personal identification.The human oral cavity serves as an important gateway connecting the human internal environment to the external environment,and is an important gateway for air and food to enter the respiratory and digestive tracts.The human oral cavity provides a suitable environment for the survival of microorganisms and contains a large number of microbial cells,which are far more numerous than the human oral mucosal epithelial cells and can be left on site in the form of human cell shedding and transfer.The greatest factor influencing the composition of human microorganisms is the individual itself,individual microbial composition is individual-specific and site-specific,and human oral microorganisms also consist of specific microbial communities with the potential to distinguish between different individuals.Under normal circumstances,human oral microorganisms are time-stable and do not change significantly over time.Therefore,human oral microorganisms have the potential for homogeneous identification and are potential markers that can be used for forensic personal identification.Based on the two characteristics of individual specificity and temporal stability of human oral microbial communities,the forensic value of human oral microbial communities in personal identification was studied to provide a basis for the subsequent investigation of the use of human oral microbial communities in forensic personal identification.1.The analysis of bacterial 16 S r RNA genes was based on conventional microbiological assays,which include T-RFLP and HRM,and an attempt was made to apply SBE to microbiological assays to investigate the ability of these three methods to detect microorganisms in the human oral cavity.2.Since T-RFLP and HRM have the disadvantages of being time-consuming and requiring large amounts of template,and since the microbiological analysis is considered to be co-assayed with the existing PCR-STR technology platform in forensic laboratories,we started with the bacterial 16 S r RNA gene sequences,from which we conducted a locus screening to screen for loci with differences in base information in different strains of bacteria.3.To analyse the screened loci using the SBE assay,typing the loci results and assessing the ability of these loci to discriminate between individuals4.To investigate the variability in the microbial composition of human saliva and oral mucosa and to provide a basis for subsequent matching studies of mock examination samples.5.In order to accurately and efficiently typing the results of the sample experiments,it is proposed to develop software for automatic typing based on Excel.Based on the SBE test results,the distribution characteristics of the oral microbial community will be obtained in order to facilitate the extension of the method for the application of human oral microorganisms for forensic personal identification.Methods1.Saliva samples and oral swab samples from 22 individuals were collected for the analysis of human oral microbial diversity.2.In this study,the oral microorganisms of different individuals(22 individuals)were studied using the T-RFLP assay for the variable region of the bacterial 16 S r RNA gene,and their results were analysed by PCA and MDS,and the Bray-Curtis phase difference coefficients were calculated,and the samples were clustered and differentiated from each other according to UPGMA.3.In this study,the oral microorganisms of different individuals were analysed using HRM assay for the variable region of bacterial 16 S r RNA gene,and different individuals were differentiated according to the obtained melting curves.4.For the two genera of bacteria commonly found in the human oral cavity,Streptococcus and Veillonella,sequence information of all strains of the two genera inhabiting the oral cavity was found from the 16 S r RNA gene sequence information file in the e HOMD database,and multiple sequence alignment was performed using DNAMAN software to screene loci with polymorphisms in each bacterial genus.5.Amplification primers and single base extension primers were designed for the screened loci following primer design principles.Primers were designed using Oligo7 software and primer blast was performed on the NCBI website to verify the specificity of the amplification primers.PCR amplification of individual DNA samples was performed using amplification primers for each locus,and PAGE was performed on the PCR amplification products to verify the amplification effect and specificity of the amplification primers based on the PAGE results.6.The SBE assay was applied to the study of human oral microorganisms,and the polymorphic loci were screened using the SBE assay,and the loci with variability between individuals were further screened based on the SBE assay results.7.Based on the final screened loci,saliva samples and oral swab samples from 22 individuals were analyzed using the SBE method,and the results were typed and named according to the base composition of each locus by themselves,and an attempt was made to type the results of different individuals,and the typing results of each locus for different individuals were counted.The ability of each locus to differentiate between individuals was calculated based on the typing results expressed as resolution and calculated as: resolution = number of SBE assay results typed/number of individuals tested.8.Saliva samples from another 77 individuals were analysed using the SBE method based on the final screened loci for a population study to investigate the ability of this method to discriminate between individuals in a population.9.Software with functions such as automated typing of test results and frequency calculation of each typing result,using relevant functional formulae in Excel.Results1.Analysis of 16 S r RNA gene variable regions of different individual oral microorganisms by T-RFLP,all samples were successfully detected by capillary electrophoresis.1)The results of PCA and MDS analyses showed that individuals were scattered and not clustered,showing individual differences.2)Cluster analysis of the samples according to Bray-Curtis coefficient of dissimilarity showed that saliva samples from 22 individuals clustered into 15 subclasses and oral swab samples from22 individuals clustered into 12 subclasses.2.The T-RFLP results showed that 46 microbial groups were detected in the saliva samples and 43 groups in the oral swab samples,indicating that the microbial groups in the saliva samples were richer than those in the oral swab samples and had a better ability to differentiate between individuals.The combined analysis of the saliva and oral swab data showed that saliva and oral swab shared some of the same microorganisms,but their respective microbial compositions were not identical,indicating that the microbial composition of saliva was different from that of the oral mucosa.3.The results of the PCR-HRM analysis of the 16 S r RNA variable region of the oral microorganisms showed that the melting curves of different individuals were not consistent.4 melting curves were observed in the saliva samples of 5 individuals,which were divided into 4 groups.In the comparison of the HRM results for oral swab and saliva samples,some individuals had similar melting curves for saliva and oral swab samples and were classified in the same group.4.By comparing the 16 S r RNA gene sequences of all strains,six polymorphic loci were screened in Streptococcus spp.and seven polymorphic loci were screened in Veronococcus spp.for S-SVN1,S-SVN2,S-SVN3,S-SVN4,S-SVN5,S-SVN6,VSVN1,V-SVN2,V-SVN3,V-SVN4,V-SVN5,V-SVN6 and V-SVN7,respectively.The primers were designed according to the flanking sequences of each locus and the PAGE results showed good specificity of amplification primers for each locus and good amplification results.5.The loci with individual differences were further screened among the 13 loci based on the SBE assay.Among the 13 loci,10 loci showed consistent detection results in saliva samples from different individuals,and 3 loci(S-SNV6,V-SNV4,VSNV5)showed that although the base composition of the detection results was the same,the ratio of each base composition was not consistent in different individuals,as reflected by the major and minor component of bases were inconsistent.This indicates that the results of these three loci are inconsistent in saliva samples from different individuals and that these three loci are individual specific.6.After preliminary sequence comparison and experimental detection,three loci were finally retained in this study: S-SNV6 of Streptococcus spp.,V-SNV4 and VSNV5 of Veronococcus spp..Based on the detection results of SBE,the typing results of three loci were named by naming the first four letters of the English name of the bacterial genus(the first letter is capitalized)-the approximate position of the locus on the 16 S r RNA gene(positioning to the hundredth digit is sufficient)-base information/number,and the naming results of each locus were and the naming results of each locus were as follows: locus V-SNV5: Veil-600-C,Veil-600-CT,Veil-600-TC,Veil-600-T;locus V-SNV4: Veil-500-1～28;locus S-SNV6: Stre-1100-1～287.The typing results for locus V-SNV5 in 22 individuals were: in saliva samples,7 individuals were assigned to Veil-600-C,6 to Veil-600-CT,5 to Veil-600-TC and 4to Veil-600-T.In oral swab samples,7 individuals were assigned to Veil-600-C,5 to Veil-600-CT,7 to Veil-600-TC and 3 to Veil-600-T.Apparently,the detection of locus V-SNV5 was not entirely consistent between individuals.The typing results for the different individuals at locus V-SNV4 were as follows: in the saliva samples,22 individuals were assigned to a total of 13 typings: 5 individuals to Veil-500-5,2individuals each to Veil-500-1、Veil-500-6、Veil-500-10、Veil-500-14 and Veil-500-20,and the remaining typings were assigned to 1 individual each.In the oral swab sample,22 individuals were also assigned to 13 subtypes,with 4 individuals assigned to Veil-500-16,3 individuals assigned to Veil-500-7,2 individuals each assigned to Veil-500-2、Veil-500-3、Veil-500-9 and Veil-500-10,and 1 individual assigned to each of the remaining subtypes.Clearly,the typing results for locus V-SNV4 were variable among individuals.The typing results of locus S-SNV6 in individual samples were: in saliva samples,10 individuals were assigned to Stre-1100-8,while 3individuals each were assigned to Stre-1100-3,Stre-1100-4,and Stre-1100-16,and 1individual each was assigned to the remaining typing.In the oral swab samples,9individuals were assigned to Stre-1100-4,7 individuals were assigned to Stre-1100-11,and 1 individual was assigned to each of the remaining subtypes.The results showed that the detection results of locus S-SNV6 were also variable among individuals.Among the above three loci,there were four typing results for locus VSNV5,16 typing results for locus V-SNV4,and 10 typing results for S-SNV6.The richness of typing results for locus V-SNV4 was better than the other two loci.8.Combined analysis of the typing results for loci V-SNV5,V-SNV4 and S-SNV6 showed a total of 19 typings in 22 individuals in the saliva samples and a total of 22 typings in the oral swab samples,with each individual having an individual-specific typing.9.An Excel-based automated typing software "Micro-SNa Pshot Stat" has been developed which allows automated typing of SBE results from multiple samples and the associated statistical calculations.10.In the population study,a total of 4 typing results were detected at locus Veil-5,18 typing results at locus Veil-4 and 11 typing results at locus Stre-6.Combined analysis of the three loci detected 66 unique typing results in 99 individuals with a resolution of 66/99 at 0.67.Conclusion1.In this study,the T-RFLP assay was used to analyze the 16 S r RNA gene variable region of human oral microorganisms to differentiate between individuals.Based on the experimental individuals,the results of T-RFLP clustering analysis showed that the method could distinguish 68% of the individuals in saliva samples and 55% of the individuals in oral swab samples.2.The microbial composition of saliva in the same individual differs from the microbial composition of the oral mucosa,with different microbial habitats in the oral cavity sharing some common microorganisms,but each having a unique microbial composition.3.This study finally screened three loci with individual specificity in Streptococcus spp.and Veronococcus spp.: V-SNV5,V-SNV4,and S-SNV6.Based on the experimental individuals in this project,the resolution of locus V-SNV5 was 0.18,the resolution of locus V-SNV4 was 0.55 and 0.59 in saliva samples and oral swab samples,and the resolution of locus S-SNV6 was 0.32.The resolution of locus V SNV4 had better resolution than the other two loci,and locus V-SNV5 had the lowest resolution.The combined analysis of the typing results of the 3 loci calculated a resolution of 0.86 in the saliva samples and 1 in the oral swab samples of this project.4.This study is based on the SBE method to analyse microbial 16 S r RNA genes to successfully typify different individuals.The assay was able to better analyse the bacterial 16 S r RNA gene-specific regions between different individuals and was able to differentiate between the experimental individuals in this study.The combined analysis of the three loci was superior to the T-RFLP and HRM assays in distinguishing different individuals.5.The automated typing software "Micro-SNa Pshot Stat" developed in this project allows for rapid and automated typing of SBE results for the loci screened in this experiment,and facilitates the promotion of forensic personal identification methods for human oral microbiology.6.The analysis of microbial 16 S r RNA gene-specific loci based on the SBE method can distinguish 67% of individuals,which initially achieves the application of human oral microorganisms for forensic personal identification. |
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