Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification / 法医学杂志

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.

Identification of Peripheral Blood and Menstrual Blood Based on the Expression Level of MicroRNAs and Discriminant Analysis / 法医学杂志

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.

Research Progress on MicroRNA in Forensic Medicine as Molecular Markers / 法医学杂志

MicroRNA （miRNA） belongs to a class of endogenous non-coding small RNA molecules with a length of 18-24 nucleotides. The expression of miRNA is highly conservative, has time sequence and is highly tissue-specific. MiRNA could not be easily degraded by ribonuclease, and is resistant to changes in environmental factors such as temperature and pH value. Moreover, miRNA can even be detected in corrupt tissue. As a result, miRNA has broad application prospects in many fields of forensic medicine such as source identification of body fluid and estimation of cause of death. This article briefly summarizes the application of miRNA in forensic practice, such as body fluid identification, determination of postmortem interval and cause of death analysis.

Efficiency of 27-plex single nucleotide polymorphism multiplex system for ancestry inference in different populations / 南方医科大学学报

<p><b>OBJECTIVE</b>To validate the efficiency of 27-plex single nucleotide polymorphism (SNP) multiplex system for ancestry inference.</p><p><b>METHODS</b>The 27-plex SNP system was validated for its sensitivity and species specificity. A total of 533 samples were collected from African, Southern Chinese Han, China's ethic minorities (Yi, Hui, Miao, Tibet, and Uygur), European, Central Asian, Western Asian, Southern Asian, Southeast Asian and South American populations for clustering analysis of the genotypes by citing 3 representative continental ancestral groups [East Asia (CHB), Europe (CEU), and Africa (YRI)] from HapMap database.</p><p><b>RESULTS</b>The system sensitivity is 0.125 ng. Twenty and six genotypes were detected in chimpanzee and monkeys, respectively. Except in rs10496971, no more products were found in other animals. The system was capable of differentiating intercontinental populations but not of distinguishing between East Asian and Southeast Asian population or between Southern Chinese Han population and Chinese Ethnic populations (Hui, Miao, Yi and Tibet). This system achieved a 100% accuracy for intercontinental population source inference for 46 blind test samples.</p><p><b>CONCLUSION</b>27-plex SNPs multiplex system has a high sensitivity and species specificity and can correctly differentiate the ancestry origins of individuals from African, European and East Asian for criminal case investigation. But this system is not capable of distinguishing subpopulation groups and more specific ancestry-informative markers are needed to improve its recognition of Southeast Asian and Chinese ethnic populations.</p>