Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes

• Published in: Legal medicine (Tokyo, Japan) Japan), 2022-09, Vol.58, p.102087-102087, Article 102087
• Main Authors: Akutsu, Tomoko, Yokota, Isao, Watanabe, Ken, Toyomane, Kochi, Yamagishi, Takayuki, Sakurada, Koichi
• Format: Article
• Language: English
• Subjects: Body fluid identification; Capillary electrophoresis; Cutoff value; Forensic biology; Multiplex RT-PCR
• ISSN: 1344-6223; 1873-4162
• DOI: 10.1016/j.legalmed.2022.102087

•Multiplex RT-PCR for multiple forensically relevant body fluids was re-designed.•Determination of the multiple markers was performed by SeqStudio Genetic Analyzer.•Cutoff value was set to eliminate the slight amplification in non-targeted samples.•Targeted body fluid specificities were drastically improved in this procedure.•It could be a powerful and convenient tool for forensic body fluid identification. Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.