Explore variation of salivary bacteria across time and geolocations.

Saliva is an informative body fluid that can be found at various crime scenes, and the salivary bacterial community has been revealed it is a potential auxiliary target for forensic identification. However, the variation of salivary bacterial community composition across time and geolocation needs to be explored. The study was designed to be carried out during the winter vacation that was across about 50 days and eight geographic locations. The high throughput sequencing was performed with the V3-V4 region of the16S rRNA gene to explore salivary bacterial community composition. An overall slight fluctuation of the salivary bacteria was observed, which primarily occurred in the relative abundance of the salivary bacterial taxa. The results of principal coordinate analysis and hierarchical clustering showed samples were clustered by the individuals. All individuals could be correctly identified with the random forest model. In summation, although the relative abundance of salivary bacteria varied across the changes of time and geolocation, the individualized characteristic of salivary bacteria remained steady, which is beneficial for the salivary bacterial application in personal identification.

Validation of the AGCU Expressmarker 16 + 22Y Kit: a new multiplex for forensic application.

Genotyping by targeting short tandem repeats (STRs) has been widely used in forensic applications. However, most commercial kits detect autosomal STRs or Y-STRs alone, which waste both time and opportunity. The AGCU Expressmarker 16 + 22Y Kit includes 16 autosomal and 22 Y-chromosomal STR loci and is designed for the forensic science field and obtaining quicker results. Here, we conducted the validation study according to Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. Validation of PCR-based studies, species specificity, sensitivity, DNA mixture studies, inhibitors, precision, and sizing accuracy were performed. Furthermore, this system was also tested in 346 random male samples from Han, Hui, Tibetan, and Zhuang populations in China, showing its high power for forensic discrimination in the Chinese population. In addition, this system was able to deal with AMELY deletion cases, which can correctly identify sex in forensic criminal investigations. Our results suggested that the AGCU Expressmarker 16 + 22Y Kit is a useful tool for rapid criminal investigation.

A multiplex microbial profiling system for the identification of the source of body fluid and skin samples.

Determining the source of body fluids is crucial in forensic investigations, as it provides valuable information about suspects and the nature of the crime. Microbial markers that trace the source of tissues and body fluids based on site specificity and temporal stability are often used effectively for this purpose. In this study, a multiplex system comprising seven microbial markers (Finegoldia magna, Corynebacterium tuberculostearicum, Cutibacterium acnes, Haemophilus parainfluenzae, Streptococcus oralis, Prevotella melaninogenica and Faecalibacterium prausnitzii) was developed to distinguish between skin, saliva, and feces samples. Based on these markers, the system produces electropherograms that are specific for each sample type. We collected 492 samples from six different skin sites (palm, antecubital crease, inguinal crease, cheek, upper back, and toe web space), the buccal mucosa, and stool were collected to further test the system. Beta diversity analysis revealed distinct clustering among the three sample groups. Additionally, skin microenvironment cluster analysis was used to identify skin sites accurately. This analysis classified skin samples into four distinct microenvironments: dry, moist, oily, and foot. Finally, we established a machine learning prediction model based on random forest regression to identify the skin microenvironment, achieving an overall prediction accuracy of 79 %. The multiplex system developed in this study accurately identifies the sources of body fluids, and the skin microenvironment. These findings offer new insights into the application of microbial markers in forensic science.

Exploring a multiplex DNA methylation-based SNP typing method for body fluids identification: As a preliminary report.

In forensic investigation, identification of the cellular origin from body fluid can be essential in the crime scene reconstruction. Recently, DNA methylation could potentially be used as a novel marker for body fluid identification. The simultaneous analysis of CpGs and neighboring single nucleotide polymorphisms (SNPs) has been proposed as an efficient assay for body fluids identification. In this study, a multiplex DNA methylation-based SNP typing system was developed. The specificity, sensitivity and detectability in mixtures and degraded samples were explored in our study. As results, four DNA methylation-based semen-specific SNP (SE1-4) showed good specificity, but two markers associative with saliva (SA1) and vaginal fluid (VA3) was observed cross-reactivity sporadically. Interesting, VA3 were found only presented in the female which may be useful for sexual identification. Moreover, this multiplex system successfully amplification in mixtures and aged samples which proves it be used as a valuable protocol in the identification of actual forensic samples. The strategy indicated that the approach was suitable and reliable for the body fluids analysis in mix stains in Han Chinese for forensic purposes.

Developed and evaluated a multiplex mRNA profiling system for body fluid identification in Chinese Han population.

In forensic casework, identification the cellular origin from a biological sample is crucial to the case investigation and reconstruction in crime scene. DNA/RNA co-extraction for STR typing and human body fluids identification has been proposed as an efficient and comprehensive assay for forensic analysis. Several cell-specific messenger RNA (mRNA) markers for identification of the body fluids have been proposed by previous studies. In this study, a novel multiplex mRNA profiling system included 19 markers was developed and performed by reverse transcription endpoint polymerase chain reaction (RT-PCR). The multiplex combined 3 housekeeping gene markers and 16 cell-specific markers that have been used to identify five types of human body fluids: peripheral blood, semen, saliva, vaginal secretions and menstrual blood. The specificity, sensitivity, stability and detectability of the mixture were explored in our study. Majority of the cell-specific mRNA markers showed high specificity, although cross-reactivity was observed sporadically. Specific profiling for per body fluid was obtained. Moreover, the interpretation guidelines for inference of body fluid types were performed according to the A. Lindenbergh et al. The scoring guidelines can be applied to any RNA multiplex, which was based on six different scoring categories (observed, observed and fits, sporadically observed and fits, not observed, sporadically observed, not reliable, and non-specific due to high input). The simultaneous extraction of DNA showed positive full or partial profiling results of all samples. It demonstrated that the approach of combined STR-profiling and RNA profiling was suitable and reliable to detect the donor and origin of human body fluids in Chinese Han population.

Identification of Saliva Using MicroRNA Biomarkers for Forensic Purpose.

In the forensic science community, microRNA (miRNA) profiling has started to be explored as an alternative tool for body fluid identification. Several origins of body fluid can be distinguished by measuring differential expression patterns of particular miRNAs. However, most of reported saliva miRNAs are nonoverlapping and debatable. The aim of this study was to develop a strategy of identifying saliva using miRNA biomarkers for forensic purpose. Eight miRNA candidates were selected to examine expression abundance in forensically relevant body fluids using hydrolysis probes quantitative real-time PCR (TaqMan qPCR). Results revealed that none of them was truly saliva specific, and only miR-200c-3p, miR-203a, and miR-205-5p were higher or more moderate expression in saliva. A stepwise strategy that combines each of three miRNAs with different body fluid-specific miRNAs was developed, and three miRNA combinations could effectively differentiate saliva from other body fluids.

A model for data analysis of microRNA expression in forensic body fluid identification.

MicroRNAs (miRNAs, 18-25 bases in length) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. MiRNA expression patterns, including presence and relative abundance of particular miRNA species, provide cell- and tissue-specific information that can be used for body fluid identification. Recently, two published studies reported that a number of body fluid-specific miRNAs had been identified. However, the results were inconsistent when different technology platforms and statistical methods were applied. To further study the role of miRNAs in identification of body fluids, this study sets out to develop an accurate and reliable model for data analysis of miRNA expression. To that end, the relative expression levels of three miRNAs were studied using the mirVana™ miRNA Isolation Kit, high-specificity stem-loop reverse transcription (RT) and high-sensitivity hydrolysis probes (TaqMan) quantitative real-time polymerase chain reaction (qPCR) in forensically relevant biological fluids, including venous blood, vaginal secretions, menstrual blood, semen and saliva. Accurate quantification of miRNAs requires not only a highly sensitive and specific detection platform for experiment operation, but also a reproducible methodology with an adequate model for data analysis. In our study, the efficiency-calibrated model that incorporated the impact of the quantification cycle (Cq) values and PCR efficiencies of target and reference genes was developed to calculate the relative expression ratio of miRNAs in forensically relevant body fluids. Our results showed that venous blood was distinguished from other body fluids according to the relative expression ratio of miR16 using as little as 50pg of total RNA, while the expression level of miR658 was unstable and that of miR205 was nonspecific among different body fluids. Collectively, the findings may constitute a basis for future miRNA-based research on body fluid identification and show miRNAs as a promising biomarker in forensic identification of body fluids.