ABSTRACT
Numerous methods for human body fluid identification using microbiological markers specific to different human body parts are well-established in forensic science. However, method for vaginal fluid screening have not been standardized yet. Therefore, in this study, a real-time polymerase chain reaction based assay for vaginal fluid identification was devised using bacteria residing in human vagina. This method employed three markers, namely Lactobacillus iners, Lactobacillus crispatus, and Bacteroides fragilis. L. iners and L. crispatus were chosen due to their high abundance in the vagina, whereas B. fragilis resides in the rectum. To examine the suitability of the new method for forensic microbial applications, a study of the distribution of vaginal flora in 143 Korean women was performed, along with characterization of the specificity, and performance of the new assay. Additionally, a casework study based on 130, 21, 20 and 17 DNA samples collected from the vagina, anus, saliva, blood, respectively, was carried out. L. iners (80.4%) and L. crispatus (55.2%) were detected with high abundance in the vagina of Korean women. The specificity of these markers was verified using microbial DNA from 23 species. This method could detect at least 1,000 copies/µL of microorganisms for all markers, thereby highlighting its robust sensitivity for vaginal fluid identification. The casework study confirmed these findings, with 89.2% (116/130) detection of vaginal fluid-derived DNA samples, and no false positives identified from the other sources studied. In conclusion, the developed method is expected to be efficient for preliminary microbiological analysis of vaginal samples in forensics.