In criminal investigations there are some cases in which identifying the presence of vaginal secretions
provides crucial evidence in proving sexual assault. However, there are no methods for definitively
identifying vaginal secretions. In the present study, we focused on Lactobacillus levels in vaginal
secretions and developed a novel identification method for vaginal secretions by relative quantification
based on real time PCR. We designed a Lactobacillus conserved region primer pair (LCP) by aligning 16S
rRNA gene sequences from major vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus
gasseri, Lactobacillus iners and Lactobacillus jensenii), and selected the human specific primer pair (HSP) as
an endogenous control for relative quantification. As a result, the DCt (DCt = Ct[LCP] Ct[HSP]) values of
vaginal secretions (11 out of 12 samples) were significantly lower than those of saliva, semen and skin
surface samples, and it was possible to discriminate between vaginal secretions and other body fluids.
For the one remaining sample, it was confirmed that the predominant species in the microflora was not
of the Lactobacillus genus. The DCt values in this study were calculated when the total DNA input used
from the vaginal secretions was 10 pg or more. Additionally, the DCt values of samples up to 6-months-
old, which were kept at room temperature, remained unchanged. Thus, we concluded in this study that
the simple DCt method by real time PCR is a useful tool for detecting the presence of vaginal secretions.