Beyond the mix: Advancements in simultaneous detection and quantification of human, dog, and cat DNA

Animal-related crimes have increased with an increase in the number of pets worldwide, underscoring the importance of animal-related biological evidence at crime scenes. Evidence obtained in cases involving dogs and cats often includes a mixture of human and animal DNA. In this study, we developed a method using droplet digital polymerase chain reaction (ddPCR) to simultaneously identify and quantitatively detect human, dog, and cat DNA in mixed samples. HLA-DRA was chosen as a human-specific marker, OR6D7 as a dog-specific marker, and FLAI-K as a cat-specific marker. The species specificity of each target was confirmed using 14 control DNA samples from 11 mammals and 3 birds. Sensitivity tests determined the limit of detection to be 0.0008 ng/μL for human DNA and 0.00061 ng/μL for dog and cat DNA. In the mixture test, each DNA sample was independently and accurately detected in samples containing trace amounts of all three types of DNA. This study demonstrated the effectiveness of applying ddPCR to forensic case samples from dog- and cat-related incidents. We have presented a reliable method for the accurate identification and quantification of human, dog, and cat DNA simultaneously, offering possibilities for advancements in forensic DNA analysis and related fields.