Improvement and evaluation of the identification method for vaginal secretions by relative quantification targeting <i>Lactobacillus</i> DNA

Identification of vaginal secretions from a suspect’s penis, fingers, sexual toys, or other possessions is crucial evidence in proving sexual crimes. A decade earlier, we focused on the Lactobacillus species present in the vagina and developed a method for identifying vaginal secretions by the relative quantification of Lactobacillus DNA, which was introduced into forensic practice. In this study, after confirming the feasibility of this method on QuantStudio 5, a different real-time PCR device from that used in the development of the method, we examined the annealing temperature to improve the specificity for vaginal secretions. Increasing the annealing temperature to 63 °C (compared to 55 °C in the original development study) reduced the non-specific amplification caused by DNA from microorganisms closely related to the Lactobacillus genus present in saliva, providing a clearer distinction between vaginal secretions and saliva than that obtained during the development phase. Furthermore, based on the comparison of DNA extraction methods, it was recommended that the DNA solution used in this method be purified from lysozyme-treated solution and then PK- or DTT-treated solution. The rectal mucosa samples from women contained a significant number of Lactobacillus and were difficult to distinguish from vaginal secretions. In conclusion, although caution is required when anal intercourse is suspected, this improved method with higher annealing temperature is anticipated to be increasingly used in forensic practice, because it is not only simple and highly sensitive but also has improved specificity.