Development of HyBeacon^® probes for specific mRNA detection using body fluids as a model system

HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA profiling. This work explores their use for the identification of expressed gene sequences through mRNA profiling. The use of mRNA is becoming increasingly common in forensic casework to identify body fluids on evidence items, as it offers higher specificity and fewer false positives than current chemical presumptive testing methods. The work presented here details the development of a single-step one-tube RT-PCR assay to detect the presence of body fluids of forensic interest (saliva, blood, seminal fluid, vaginal fluid and menstrual blood) using HyBeacon^® probes and melt curve analysis. Each assay shows a high degree of specificity to the target body fluid mRNA suggesting there is no requirement to remove genomic DNA prior to analysis. Of the five assays developed, four were able to detect between 10 and 100 copies of target cDNA, the fifth 1000 copies of target. The results presented here demonstrate that such an approach can be optimised for non-expert users and further areas of work are discussed.