Rapid Forensic DNA Profiling via Real-Time Recombinase Polymerase Amplification of InDel Markers

Forensic DNA profiling commonly relies on polymerase chain reaction (PCR) amplification followed by capillary electrophoresis (CE) or massively parallel sequencing (MPS), which requires expensive, laboratory-based equipment that depends on a stable power supply and is unsuitable for field applications. Here, we present a proof-of-concept assay that uses recombinase polymerase amplification (RPA) combined with exo probe detection for rapid, isothermal genotyping of insertion-deletion (InDel) markers. To the best of our knowledge, this study represents the first demonstration of forensic DNA typing using RPA coupled with exo probes. The reaction proceeds at 39 °C and combines amplification and detection in a single 20 min step. Thirteen DNA samples were genotyped in triplicate across eight InDel loci using allele-specific fluorescent probes. Genotypes were derived from differential endpoint fluorescence between matched and mismatched probes. Compared with benchmark genotyping, 97.07% of genotypes (n = 307) were correct at 1 ng DNA input. Accurate profiles were reliably obtained for DNA inputs as low as 250 pg, and partial profiles were still detectable at 31 pg. The results demonstrate that RPA-based InDel genotyping is fast, sensitive, and reproducible. With further optimization, such as refined probe design and selection of robust loci, the assay has clear potential to achieve complete accuracy and to be integrated into portable lab-on-a-chip platforms for rapid, field-deployable forensic identification.