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Development and Evaluation of a Combinatorial RT-qPCR Multiplex for Forensic Body Fluid Identification

NCJ Number
Date Published
May 2022

This project synergized on the benefits of microRNAs and microbial DNA to identify body fluids using DNA extracts.


Current presumptive tests lack the sensitivity and specificity molecular techniques can achieve; therefore, molecular methods, such as microRNA and microbial signatures, have been extensively researched in the forensic community. The limitations of each method suggest combining molecular markers to increase discrimination efficiency of multiple body fluids from a single assay. Although microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have been promising in fluids with low microbial abundance. A disadvantage of RNA analysis in forensic casework is RNA extraction; however, several reports have shown that microRNAs co-extract with DNA, increasing implementation potential. The current project first confirmed microRNA detection in DNA extracts, demonstrating that RNA extraction and DNase-treatment are not necessary. A reverse transcription (RT)-qPCR duplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius was designed to identify vaginal/menstrual secretion, feces, and saliva, respectively. The developed classification regression tree model classified five body fluids with 94.6 percent overall accuracy, thus providing proof of concept that microRNAs and microbial DNA can identify multiple body fluids at the quantification step of the current forensic DMA workflow.


Date Published: May 13, 2022