Development and validation of simultaneous identification of 26 mammalian and poultry species by a multiplex assay

A multiplex PCR assay was developed to simultaneously identify 22 mammalian species (alpaca, Asiatic black bear, Bactrian camel, brown rat, cat, cattle, common raccoon, dog, European rabbit, goat, horse, house mouse, human, Japanese badger, Japanese wild boar, masked palm civet, pig, raccoon dog, red fox, sheep, Siberian weasel, and sika deer) and four poultry species (chicken, domestic turkey, Japanese quail, and mallard), even from a biological sample containing a DNA mixture of multiple species. The assay was designed to identify species through multiplex PCR and capillary electrophoresis, with a combination of amplification of a partial region of the mitochondrial D-loop by universal primer sets and a partial region of the cytochrome b (cyt b) gene by species-specific primer sets. The assay was highly sensitive, with a detection limit of 100 copies of mitochondrial DNA. The assay's ability to identify species from complex DNA mixtures was demonstrated using an experimental sample consisting of 10 species. Efficacy, accuracy, and reliability of the assay were validated for use in forensic analysis with the guidelines of Scientific Working Group on DNA Analysis Methods (SWGDAM). The multiplex PCR assay developed in this study enables cost-effective, highly sensitive, and simultaneous species identification without massively parallel sequencing (MPS) platforms. Thus, the technique described is straightforward and suitable for routine forensic investigations.