Body fluids are crucial pieces of evidence to help evaluate the circumstances of a case; therefore, being able to distinguish between body fluids commonly seen in forensic casework is essential.
Routine forensic serology testing faces drawbacks for body fluid differentiation such as sequential testing leading to sample loss or degradation or lack of confirmatory testing for saliva and vaginal fluid. The goal of this study is to overcome drawbacks of routine serology testing by performing body fluid identification using genomic DNA collected within the typical genotyping workflow. Samples were bisulfite treated and amplified via PCR followed by high-resolution melt (HRM) to produce melting temperature curves based on two tissue specific differentially methylated regions (tDMRs), BCAS4-M and DDX4-U-L. ScreenClust Principal Component Analysis (PCA) was used to determine the efficiency of the tDMR primer sets for differentiating the tested body fluids. By this method, we were able to differentiate blood, semen, saliva, and vaginal fluid. A partial developmental validation will be shown in this presentation. The application of this research demonstrates a method for distinguishing body fluids alone and within mixtures through a low-cost and time-efficient method that could be easily incorporated into a forensic laboratory current workflow.
ISHI Student Ambassador, Beighley Ayers will be presenting on the above during her poster presentation at ISHI 34 this September. We chatted with her to learn a little more, and if you’ll be at the conference, be sure to stop by poster #33 to learn more!
Briefly describe your work/area of interest.
Because body fluids are a source of DNA that is routinely found at crime scenes, it is vital to determine which body fluid (s) are present to fully grasp the circumstances of a case. The current go-to analysis is serology testing which has its advantages but comes with drawbacks as well. Not every biological fluid has a confirmatory test available, false positives are possible with many of the serology tests, and testing could affect the quality and quantity of the sample. Therefore, an additional way to differentiate body fluids would be through polymerase chain reaction (PCR) high resolution melt (HRM). PCR-HRM coupled with the exploitation of DNA methylation patterns is another method to distinguish body fluids from one another in single and multi-source samples. Bisulfite treatment is a way to epigenetically modify the DNA by enforcing the cytosines that are unmethylated to uracil and leaving the methylated cytosines intact. Methylation refers to in this instance, the modification in the promoter region on the 5’ carbon of cytosine, forming 5-methlycytosine. During PCR-HRM, there is a gradual increase in temperature; furthermore, the temperature at which a sample melt is indicative of the body fluid based on its methylation pattern.
This research project was started by Towson University professor, Dr. Cynthia Zeller, along with previous Towson graduate students. For this research project, I have focused on performing a developmental validation to test for specificity, sensitivity, and mixture analysis. I extracted, quantified, and bisulfite treated all four body fluids involved in the study, including blood, saliva, semen, and vaginal fluid. I set-up and ran all PCR-HRM runs as well as helped optimize the conditions for body fluid differentiation. Finally, I analyzed results using principal component analysis and the ScreenClust software.
Through PCR-HRM, we have successfully differentiated single source samples and are continuing our work with samples that include body fluid mixtures. There have been positive results with mixed samples, but we want to continue to try different combinations.
How did you get interested in this work? Why did this particular project appeal?
My research advisor, Dr. Cynthia Zeller, saw the need for this research in for the forensics community. She has been progressing the project every year with different Towson University graduate research assistants. When I got to Towson and started working with her, it was apparent we made a great team. She had told me about the project and was eager to work together to finally wrap up. I was excited to research body fluid differentiation to expand upon the current literature with another method with our particular set of primers.
Can you summarize the impact of your work for the audience (ISHI attendees and some general forensic enthusiasts)? How might this advance the field?
As mentioned in the project background, routine forensic serology testing comes with numerous disadvantages. By using exploiting epigenetics and performing PCR-HRM, body fluids can be differentiated via a lower-cost in-tube methodology that can identify components within mixtures of body fluids in a single run. In addition, the methods I have been studying use a quantitative thermocycler, an instrument that is commonplace in a forensic laboratory. Thus, these factors increase the attainability of incorporating PCR-HRM for body fluid differentiation.
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