Meat species identification by two direct-triplex real-time PCR assays using low resolution melting

- First validated direct multiplex real-time PCR assay for meat species identification.
- Economical and rapid due to no DNA extraction and use of intercalating dye.
- Assay targets six species. No cross-reactivity observed with other meat species.
- Assay showed high accuracy and reproducibility from as low as 0.32 ng of DNA.
- Assay successfully amplified 86 of 93 commercial food samples.

We developed and validated the first direct multiplex real-time PCR assay with melt curve analysis for the identification of different types of meat. The assay detects six commonly eaten meat species and provides the following benefits: ease of use, shortened analysis time, and decreased analysis cost. Species-specific primers were designed from cytochrome b, cytochrome oxidase I, and 16S rRNA genes to generate PCR products with specific melting peaks that differentiate pork, beef, horsemeat, duck meat, ostrich meat, and chicken. Validation of the assay showed that it is robust, reproducible, specific, and sensitive down to 0.32 ng of DNA template. It takes less than one hour from sample to result and costs less than one USD per sample. We also successfully amplified 92.5% of market samples, demonstrating the assay's robustness. The developed assay has the potential to be implemented in food testing laboratories worldwide.