Developmental validation of an mRNA kit: A 5-dye multiplex assay designed for body-fluid identification

• Published in: Forensic science international : genetics 2024-07, Vol.71, p.103045-103045, Article 103045
• Main Authors: Xiao, Yuanyuan, Tan, Mengyu, Song, Jinlong, Huang, Yihang, Lv, Meili, Liao, Miao, Yu, Zailiang, Gao, Zhixiao, Qu, Shengqiu, Liang, Weibo
• Format: Article
• Language: English
• Subjects: Blood Chemical Analysis; Body-fluid identification; Cervix Mucus - chemistry; Female; Fluorescent Dyes; Forensic Genetics - methods; Forensic science; Genetic Markers; Humans; Machine learning; Male; mRNA kit; mRNA profiling; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Reproducibility of Results; RNA, Messenger - genetics; Saliva - chemistry; Semen - chemistry; Sensitivity and Specificity
• ISSN: 1872-4973; 1878-0326
• DOI: 10.1016/j.fsigen.2024.103045

Identifying the sources of biosamples found at crime scenes is crucial for forensic investigations. Among the markers used for body fluid identification (BFI), mRNA has emerged as a well-studied marker because of its high specificity and remarkable stability. Despite this potential, commercially available mRNA kits specifically designed for BFI are lacking. Therefore, we developed an mRNA kit that includes 21 specific mRNA markers of body fluids, along with three housekeeping genes for BFI, to identify four forensic-relevant fluids (blood, semen, saliva, and vaginal fluids). In this study, we tested 451 single-body-fluid samples, validated the universality of the mRNA kit, and obtained a gene expression profile. We performed the validation studies in triplicates and determined the sensitivity, specificity, stability, precision, and repeatability of the mRNA kit. The sensitivity of the kit was found to be 0.1 ng. Our validation process involved the examination of 59 RNA mixtures, 60 body fluids mixtures, and 20 casework samples, which further established the reliability of the kit. Furthermore, we constructed five classifiers that can handle single-body fluids and mixtures using this kit. The classifiers output possibility values and identify the specific body fluids of interest. Our results showed the reliability and suitability of the BFI kit, and the Random Forest classifier performed the best, with 94% precision. In conclusion, we developed an mRNA kit for BFI which can be a promising tool for forensic practice. •A novel mRNA kit of 21 specific mRNAs was developed to classify four body fluids.•Random Forest analysis of over 400 samples resulted in an overall precision of 94%.•Kit has broad applicability and accurate identification of mixtures.