Quantitative evaluation of candidate genes and development of a multiplex RT-PCR assay for the forensic identification of vaginal fluid

The identification of vaginal fluid from a mixed sample containing semen provides important probative evidence of vaginal intercourse in sexual assaults. In studies on the mRNA-based identification of body fluids, vaginal characteristic genes, such as MUC4 and HBD1, have been used to identify vaginal fluid; however, these are insufficient to discriminate vaginal fluid from other body fluids because of incomplete specificity and detectability. The aim of this study was to develop a more specific procedure for identifying vaginal fluid. First, previously reported and newly selected candidate genes were evaluated quantitatively using real-time reverse transcription-polymerase chain reaction (RT-PCR). Then, we developed a multiplex RT-PCR assay to detect probable candidates simultaneously. Each amplicon was detected and quantified by chip electrophoresis. Furthermore, we examined the specificity and robustness of the developed multiplex RT-PCR assay using various body fluids and aged vaginal fluid stains. As a result of this real-time RT-PCR assay, we selected five candidate genes—MUC4, CYP2B7P1, KLK13, ESR1, and SERPINB13—on the basis of their specificity and sensitivity. Then, the simultaneous amplification of these genes was performed successfully, and each fragment could be separated and quantified automatically by chip electrophoresis. The specificity and detectability of the multiplex detection of vaginal characteristic genes were almost comparable to those of real-time RT-PCR and it could be applied to vaginal fluid samples stored at room temperature for 1.5 years. Although discrimination criteria should be set, the multiplex RT-PCR assay developed in this study could be an effective tool for the identification of vaginal fluid.