DNA mixtures containing semen and vaginal fluid are common biological samples in forensic analysis. However, the analysis of semen–vaginal fluid mixtures remains challenging. In this study, to solve these problems, it is proposed to combine semen-specific CpG sites and closely related microhaplotype sites to form a new composite genetic marker (semen-specific methylation-microhaplotype). Six methylation-microhaplotype loci were selected. To further improve discrimination power, five methylation-SNP loci were also included. The methylation levels and genotypes of these selected loci were obtained using massively parallel sequencing technology. Except for loci MMH04ZHA019 and MMH17ZHA059, the remaining nine loci were successfully sequenced. For the successfully sequenced loci, they performed well in identifying individuals and body fluids. An allele categorization model was developed using K-nearest neighbour algorithm, which was then used to predict allele types in semen–vaginal fluid mixtures. These loci were able to confirm the presence of semen and link semen to a true donor in semen–vaginal fluid mixtures with mixing ratios of 10:1, 9:1, 5:1, 4:1, 1:1, 1:3, 1:4, 1:8 and 1:9 (semen:vaginal fluid). This preliminary study suggests that this new composite genetic marker has great potential as a supplementary tool to commonly used genetic markers (STR, etc.) for analysing semen–vaginal fluid mixtures.

Estimating the postmortem interval (PMI) is critical in the field of forensic science, and necrophagous insects play a significant role in this process. Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) is a common necrophagous insect species, making its rapid and accurate identification essential. However, commonly used molecular biology methods, such as DNA barcode, still have some limitations in identifying necrophagous insects as they are often complex, time-consuming, and reliant on laboratory instruments. Therefore, in this study, we have developed an innovative detection system for the rapid and accurate identification of C. megacephala based on the Cytochrome b gene using recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) in combination. The developed RPA-LFD detection system achieved complete amplification in just 15 min at 37 °C with good sensitivity and specificity. Only 7.8 × 10⁻⁴ ng or more of target DNA fragments were required, and a positive detection rate of 100% was achieved in 18 C. megacephala samples from actual cases. In addition, the ability of the developed RPA-LFD detection system in combination with rapid DNA extraction methods to enable on-site detection was preliminarily explored. The results suggested that when the RPA-LFD detection system was combined with the grinding ddH₂O extraction method (a rapid DNA extraction method), the process from species acquisition to visualization of detection results could be completed in less than 20 min. In conclusion, this innovative RPA-LFD detection system outperforms commonly used molecular biology methods for C. megacephala identification in terms of speed, sensitivity and convenience, making it suitable for direct application at crime scenes, promising to provide important assistance in estimating PMI and expanding the impact of forensic entomological evidence.

In forensic practice, the unequivocal identification of questioned biological stains is indispensable for evidence triage, investigative guidance, and the attribution of criminal responsibility. Among these traces, saliva is one of the most prevalent yet challenging biological matrices to detect. Classical analytical strategies—chemical colorimetry, enzymatic assays, immunochromatography, vibrational spectroscopy, and microscopical techniques—frequently suffer from elevated false-positive and false-negative rates, insufficient analytical sensitivity, and stringent demands regarding sample integrity and environmental background. These limitations are further exacerbated by the intrinsic scarcity and unpredictable degradation of forensic specimens, which often preclude acquisition of material in requisite quantity and quality. To overcome these constraints, we have developed an integrated assay that couples recombinase polymerase amplification with lateral-flow dipstick technology, selectively targeting Neisseria subflava —a microorganism highly enriched in human saliva—for definitive identification of saliva stains. The optimised system exhibits exceptional analytical parameters: 100% specificity, an overall accuracy of 87.1%, and a limit of detection at 0.625 copies/μL, while retaining functionality on samples aged under ambient conditions for extended periods. Importantly, the isothermal reaction proceeds efficiently at human body temperature, enabling on-scene deployment. This capability allows immediate probative determination at the crime scene and provides critical guidance for subsequent targeted DNA extraction and individual identification workflows.