| Saliva and vaginal fluid often appear in sexual crime cases,and their identification is of great judicial significance.However,because both of them are derived from epithelial cells in terms of tissue occurrence,they have a high similarity,so accurate identification is still a difficult problem in forensic science.Studies have shown that the human oral and vaginal microenvironments are significantly different and have relatively stable flora structures.Therefore,the distinction between saliva and vaginal fluid based on the specific microorganisms has a good theoretical basis and application potential.Based on the application requirements of microbial forensic testing,this study collected and produced140 samples of saliva,vaginal fluid,semen stains and skin swabs from adult healthy individuals,and used 16 S r RNA gene sequencing technology to detect these four body fluids or tissue samples,in order to understand the microbial community structure characteristics of saliva stains,vaginal fluid stains,semen stains,and skin swab samples,and to screen microbial with significant differences in saliva stains and vaginal fluid stains.Real-time fluorescence quantitative PCR technology was used to verify the specificity and effectiveness of the microbial markers,and an eight-plex microbial markers fluorescence multiplex identification system based on capillary electrophoresis technology was constructed to distinguish saliva stains and vaginal fluid stains.The main results are as follows:(1)The microbial community structures of the four different types of samples were significantly different.Alpha diversity analysis showed that the microbial diversity of skin was the highest,followed by semen stains and saliva stains,and the microbial community diversity of vaginal fluid stains was the lowest.Beta diversity analysis showed that the inter-group differences of the four body fluid tissue types were significantly greater than the intra-groups differences.The samples of the same tissue types were gathered together.The analysis of microbial community composition showed that the dominant bacteria in saliva stain samples were Streptococcus,Neisseria and Haemophilus.The dominant bacteria in vaginal fluid stain samples were Lactobacillus,Bifidobacterium and Prevotella,and the dominant bacteria in semen stain samples were Lactobacillus and Ralstonia.The dominant bacteria in skin swab samples were Propionibacterium,Corynebacterium and Enhydrobacter.In addition,gender had no significant effect on the composition and relative abundance of salivary microbial communities.For skin microbial communities,the microbial diversity of female skin was higher than that of male skin,and the gender difference in community structure showed that Corynebacterium and Enhydrobacter had higher relative abundance in male skin samples.And Gp I and Sphingobium were statistical differences between the two groups.(2)Saliva and vaginal fluid can be distinguished by differential microorganisms.Haemophilus parainfluenzae,Neisseria subflava,Veillonella dispar,Porphyromonas catoniae,Streptococcus oralis,Fusobacterium nucleatum may be used as effective salivary microbial markers.Lactobacillus iners,Bifidobacterium breve may serve as microbial markers of vaginal fluid.(3)An eight-plex microbial markers fluorescence multiplex identification system has a good effect in the identification of saliva and vaginal fluid.The results showed that five salivary microbial markers(Neisseria subflava,Streptococcus salivarius,Veillonella dispar,Porphyromonas catoniae,Streptococcus oralis)and three vaginal fluid microbial markers(Lactobacillus crispatus,Lactobacillus gasseri,Bifidobacterium breve)could be detected in the corresponding target body fluids.There was no cross reaction between saliva and vaginal fluids,and the accuracy of identification of saliva and vaginal fluids was 100%.The sensitivity of this system was 0.125 ng for vaginal fluid samples,and 0.0625 ng for saliva samples.In addition,the DNA extracted in the detection process of this system can also obtain the STR site information of the donor,which can meet the needs of individual identification through STR typing in forensic DNA testing.This study revealed the microbial community structure characteristics of various body fluid tissue samples in the human body,and the eight-plex microbial markers fluorescence multiplex identification system for the identification of saliva and vaginal fluid has a good application prospect. |
