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Objectives: To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification., Methods: The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained., Results: There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%., Conclusions: In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.

Abstract: Objective To construct a discriminant analysis model based on the differential expression of multiple microRNAs （miRNAs） in two kinds of blood samples （peripheral blood and menstrual blood） and three non-blood samples （saliva, semen and vaginal secretion）, to form an identification solution for peripheral blood and menstrual blood. Methods Six kinds of miRNA （miR-451a, miR-144-3p, miR-144-5p, miR-214-3p, miR-203-3p and miR-205-5p） were selected from literature, the samples of five kinds of body fluids commonly seen in forensic practice （peripheral blood, menstrual blood, saliva, semen, vaginal secretion） were collected, then the samples were divided into training set and testing set and detected by SYBR Green real-time qPCR. A discriminant analysis model was set up based on the expression data of training set and the expression data of testing set was used to examine the accuracy of the model. Results A discriminant analysis statistical model that could distinguish blood samples from non-blood samples and distinguish peripheral blood samples from menstrual blood samples at the same time was successfully constructed. The identification accuracy of the model was over 99%. Conclusion This study provides a scientific and accurate identification strategy for forensic fluid identification of peripheral blood and menstrual blood samples and could be used in forensic practice., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Objective: For the purpose of solving a problems of DNA testing of burned bones., Methods: We present a novel strategy to obtain DNA from burned bones based on the use of cetyltrimethylammonium bromide (CTAB) lysis buffer and isoamyl alcohol-chlorophorm extraction with subsequent DNA purification using the DNA IQ System., Results: The methods were found to be effective in removing the PCR inhibitors from the burned bone. Then the extracted DNA was successfully genotyped by using the florescence labeling STR multiplex method., Conclusion: The results of this research will assist forensic scientists in the identification of DNA from victims whose bodies underwent significant trauma or burning, precluding the utilization of traditional forensic DNA identification techniques.