A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA

Area of Science:

• Forensic science
• Microbial forensics
• Molecular biology

Background:

• Current microbial forensics studies primarily rely on DNA sequence analysis.
• Quantification of human and bacterial DNA in forensic samples is often overlooked.
• Determining DNA quantities is crucial for selecting appropriate analysis methods, especially when human DNA is limited for short tandem repeat (STR) typing.

Purpose of the Study:

• To develop a simple, cost-effective quantitative PCR (qPCR) assay for simultaneous quantification of human and bacterial DNA.
• To establish a reliable tool for forensic laboratories to assess DNA profiles.

Main Methods:

• Development of a custom multiplex quantitative PCR (qPCR) assay.
• Validation through a reproducibility study comparing results with established qPCR assays.
• Application of the assay in small-scale experiments on common surfaces.

Main Results:

• The multiplex qPCR assay demonstrated comparable reproducibility to existing assays.
• Common surfaces yielded insufficient human DNA (below 250 pg) for STR analysis.
• All tested surfaces provided sufficient bacterial DNA quantities for sequence analysis, even at concentrations as low as 10^-8 ng/uL.

Conclusions:

• The developed qPCR multiplex assay accurately quantifies both human and bacterial DNA.
• This tool aids forensic scientists in deciding between human or bacterial DNA analysis strategies.
• It is particularly valuable in cases with limited human DNA for identification.