Exploration and application of microorganisms related to the inference of the time since deposition (TsD) in semen and blood stains

Determining the time since deposition (TsD) of body fluid stains can provide crucial criminal information to forensic researchers. Although there are studies on inferring residual time through DNA and RNA markers, this requires high sample quality, and microorganisms, as a new type of marker with individual and tissue identification capabilities, have the potential for body fluid recognition and TsD inference. Blood and semen are the most common types of bodily fluid stains at crime scenes, but research on the inference of the TsD of these two types of stains through microorganisms still needs to be explored. Thus, this study collected samples of body fluid stains exposed indoors for up to 56 days and selected several microorganisms that were both liquid specific and related to residual time inference in blood (Methylobacterium and Sphingomonas) and semen (Gardnerella) stains via 16 S rRNA high-throughput sequencing. Furthermore, the microorganisms’ ability to infer TsD was verified using qPCR in validation group samples stored under the same conditions, and two multiple logistic regression models were constructed. The average absolute deviation of differences between the predicted and actual retention times of the three types of body fluids in the test set using two estimation methods was 2.15 and 2.06 days, respectively. In conclusion, this study has discovered four novel microorganisms related to the retention time of blood and semen and has preliminarily constructed the TsD prediction models, providing a new direction for future forensic research on the inference of TsD in blood and semen stains.