Development of a multiplex RT-PCR assay and statistical evaluation of its use in forensic identification of vaginal fluid.

The identification of vaginal fluid from casework samples of sexual assaults provides important probative evidence of vaginal intercourse. The aim of this study was to establish a more specific procedure for identifying vaginal fluids for forensic purposes. Vaginal fluid marker candidates have been evaluated quantitatively and five of these markers (ESR1, SERPINB13, KLK13, CYP2B7P1, MUC4) have been amplified simultaneously by a multiplex reverse transcription-polymerase chain reaction (RT-PCR) procedure. Each amplicon has been separated and quantified automatically using chip electrophoresis. Subsequently, in the present study, detectability and cross-reactivity of the developed multiplex procedure were assessed in detail using various forensically relevant body fluids. Then, a cutoff value for the positive detection of vaginal fluids was set for each marker by Youden index. The ability of the multiplex RT-PCR assay to distinguish between vaginal and other body fluids was evaluated statistically using a likelihood ratio (LR) that was estimated using a Bayesian estimation approach to consider the infrequency of detection. A high LR was obtained when all five markers showed positive results (LR = 4.33 × 109; 95% credible interval, 3.95 × 107 -2.87 × 1012). The developed procedure was validated using vaginal fluid samples under various conditions. High LRs were found for aged vaginal fluid stains, although each amplicon peak was low. It was also able to identify vaginal stains mixed with other body fluids. In conclusion, the multiplex RT-PCR-based procedure followed by the statistical evaluation using LR could be a powerful tool for the objective identification of vaginal fluids.