title: A simple identification method for vaginal secretions using relative quantification of Lactobacillus DNA.
abstract: 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 ΔCt (ΔCt=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 ΔCt values in this study were calculated when the total DNA input used from the vaginal secretions was 10pg or more. Additionally, the ΔCt 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 ΔCt method by real time PCR is a useful tool for detecting the presence of vaginal secretions.
authors.affiliation: Forensic Science Laboratory, Ehime Prefectural Police Headquarters, 2-2 Minamihoribatacho, Matsuyama, Ehime 790-8573, Japan. Electronic address: doi.bio.rese@gmail.com.
authors.forename: Masanori
authors.initials: M
authors.lastname: Doi
authors.affiliation: Forensic Science Laboratory, Ehime Prefectural Police Headquarters, 2-2 Minamihoribatacho, Matsuyama, Ehime 790-8573, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Gamo
authors.affiliation: Forensic Science Laboratory, Ehime Prefectural Police Headquarters, 2-2 Minamihoribatacho, Matsuyama, Ehime 790-8573, Japan; Department of Legal Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Okiura
authors.affiliation: Department of Legal Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nishimukai
authors.affiliation: Department of Legal Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Migiwa
authors.initials: M
authors.lastname: Asano
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D005260:Female; D006801:Humans; D007778:Lactobacillus / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014621:Vagina / Q000378:metabolism* / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Body fluid identification; Human DNA; Lactobacillus; Microflora; Real time PCR; Vaginal secretions