Identification of menstrual blood and vaginal fluid using a 4-plex MSRE-PCR system applicable to alleged sexual assault cases

Menstrual blood and vaginal fluid are frequently encountered in conjunction with semen in alleged sexual assault cases. There are few tests with high specificity for these two body fluids and here we report on a 4-plex MSRE-PCR system for the detection of menstrual blood and vaginal fluid. The assay uses four markers in a multiplex PCR: a positive control (PC), digestive control (DC), menstrual blood-specific (MB) marker and vaginal fluid-specific (VF) marker. Menstrual blood and vaginal fluid samples were identified by the MB and VF markers as expected. Additionally, the results using samples from saliva, semen and blood were also correctly identified, with an absence of both the MB and VF markers as expected, even though MB and VF signals can occur within a few non-target body fluids (urine, nasal fluid and skin). It was found that the methylation levels at the markers for both menstrual blood and vaginal fluid have differences between individuals. The methylation level of the marker for menstrual blood was affected by the day during the menstruation cycle, which also affected the sensitivity of the test. DNA required for correct identification of menstrual blood increased from 1 ng at day 1 to 1.5 ng for day 5. The sensitivity of the assay using samples from vaginal fluid was at least 0.5 ng of DNA for confident identification. Menstrual blood samples deposited on seven types of materials frequently encountered in forensic investigations were all correctly assigned. Also, samples of simulated mixed menstrual blood and vaginal fluid were tested and the results were all concordant as expected. These data help to verify the applicability of the 4-plex MSRE-PCR system in forensic casework.