Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis

We developed a simple and rapid method for animal species identification in the forensic science field based on mitochondrial DNA using two multiplex real-time PCRs and analysis of the resultant SYBR Green I melting curves. This method was designed to identify nine domestic animals simultaneously (dog, cat, rabbit, cattle, pig, chicken, goat, sheep and horse) and four wild animals (deer, raccoon-dog, monkey and bear) by comparing the different melting temperatures of the amplicons produced from samples originating from each species. For this analysis, we targeted various mitochondrial genes, including those encoding cytochrome b (cytb), NADH dehydrogenase 5 (ND5), cytochrome c oxidase 3 (COX3), tRNA-ND5, and tRNA-ATP synthase 8 (ATP8). For practical applications, this study presents a validation of this assay including its specificity, sensitivity and robustness. The limits of detection in the multiplex reactions were 10 pg for eight of the nine animals, excluding horse (1 pg for horse). The method was able to correctly identify the animal species from artificial forensic samples including blood stains, saliva, hair and bone, and samples digested in artificial gastric fluid, and for 17 forensic casework samples. The data from the multiplex real-time PCR assays are obtainable only 30 min after DNA extraction of the samples, making the assays useful for screening samples containing DNA from unknown animal origin in the forensic field.