基于微生物标记物鉴别唾液、阴道分泌物的多重荧光标记复合扩增检测体系构建

Study on an eight-plex microbial markers fluorescence multiplex identification system for the discrimination of saliva and vaginal fluid

目的构建基于微生物标记物的荧光复合检测体系,实现对唾液斑迹、阴道分泌物斑迹的区分鉴别。方法经文献检索结合本实验室前期工作,根据特异性和丰度筛选唾液和阴道分泌物微生物标记物;制备唾液斑迹、阴道分泌物斑迹样本,提取总DNA,利用毛细管电泳技术构建基于微生物标记物鉴别唾液、阴道分泌物的多重荧光标记复合扩增检测体系,并使用体液斑迹样本对体系特异性、灵敏度进行检验。结果本研究筛选出5个唾液微生物标记物(微黄奈瑟氏菌Neisseria subflava、非典型韦荣氏菌Veillonella atypica、唾液链球菌Streptococcus salivarius、口腔链球菌Streptococcus oralis、凯托卟啉单胞菌Porphyromonas catoniae)和3个阴道分泌物微生物标记物(卷曲乳杆菌Lactobacillus crispatus、加氏乳杆菌Lactobacillus gasseri、短双歧杆菌Bifidobacterium breve),利用毛细管电泳技术构建了8重微生物荧光标记复合扩增检测体系,并使用140份样本对体系特异性进行验证;测试结果显示,唾液和阴道分泌物的微生物标记物特异性良好。体系对唾液斑迹检测灵敏度为0.065 ng总DNA,对阴道分泌物斑迹检测灵敏度为0.125 ng总DNA。本体系检测过程中提取的总DNA能够同时满足法医DNA检验中通过常染色体短串联重复序列分型进行个体识别的需求。结论本研究构建的8重微生物荧光标记复合扩增检测体系可以实现唾液和阴道分泌物斑迹样本的有效区分,为犯罪现场体液斑迹组织属性推断鉴定技术方法开发提供了新思路。

Objective A fluorescent complex system based on microbial markers was setup for the discrimination of saliva and vaginal fluid.Methods The specific microbial markers in saliva and vaginal fluid were screened based on specificity and abundance according to the literatures and our former research.Stain samples of saliva and vaginal fluid were prepared,and total DNA was extracted.The multiple microbial markers fluorescence complex system was constructed by capillary electrophoresis,and the body fluid stain samples were used to evaluate the specificity and sensitivity of the system.Results In this study,five salivary microbial markers(Neisseria subflava,Veillonella atypica,Streptococcus salivarius,Streptococcus oralis and Porphyromonas catoniae),and three vaginal fluid microbial markers(Lactobacillus crispatus,Lactobacillus gasseri,Bifidobacterium breve)were selected.An eightplex microbial markers fluorescence multiplex identification system was constructed by capillary electrophoresis technology,and the specificity of the system were verified using 140 samples and proved to be excellent.The detection sensitivity of saliva was 0.065 ng of total DNA,and that of vaginal fluid was 0.125 ng of total DNA.The total DNA extracted during the process of this system can meet the needs of forensic short tandem repeat sequence typing as well.Conclusion The eight-plex microbial markers fluorescence multiplex identification system can discriminate saliva and vaginal fluid effectively and provide a new method for body fluid stain identification at crime scene.