Development of two step reverse transcription droplet digital PCR (RT-ddPCR) for simultaneous identification of saliva and semen

Determination of the type of body fluids is essential for crime scene reconstruction and for improving the reliability of expert testimonies. Messenger RNA (mRNA) analysis by reverse transcription quantitative PCR (RT-qPCR) has been used in forensic genetics, particularly for body fluid identification. It is a relative quantification method that compares the Ct values of target and reference gene. Thus, the method is unsuitable for determining exact copy numbers of the target gene. To address this limitation, this study performed body fluid-specific mRNA analysis using two-step reverse transcription droplet digital PCR (RT-ddPCR), which is capable of absolute quantification. We found that RT-ddPCR was accurate and sensitive enough to detect as little as 1.5 copies/μl of complementary DNA (cDNA), making it suitable for application using casework samples. It was also highly specific for body fluids, as non-specific amplification did not occur. In addition, saliva-semen mixtures with ratios ranging from 1:50 to 50:1 were successfully identified. When comparing the results of RT-qPCR and RT-ddPCR, some samples were difficult to interpret because of the high Ct values of RT-qPCR. However, when the same samples were analyzed using RT-ddPCR, saliva and semen were distinctly identified. Thus, RT-ddPCR is useful for mixed samples (e.g., in sexual assault cases) with low amounts of DNA, which often leads to ambiguous results when using RT-qPCR. Other body fluids (e.g., vaginal fluid and menstrual blood) can also be identified by including additional markers. This study demonstrates the potential of RT-ddPCR for applications in forensic science.