Multiplex PCR assay for identification and quantification of bovine and equine in minced meats using novel specific nuclear DNA sequences

The 2013 horsemeat scandal demonstrated the importance and significance of developing analytical methods to accurately identify and quantify bovine and equine components in meats, In this study, we successfully screened the bovine- and equine-specific nuclear DNA sequences for detection of bovine and equine fractions in minced meats, respectively. Primers and probes designed from bovine- and equine-specific nuclear DNA sequences were critically analysed against 53 species, and then their specificity was evaluated against 23 species through PCR. The conservation of primers and probes binding regions was tested in 44 different individuals of bovine or equine. The limit of detection (LOD) is 0.05 ng in both conventional and real-time PCR, and the limit of quantification (LOQ) is down to 5%. Real-time PCR confirmed that bovine- and equine-specific nuclear DNA sequences are present as a fixed copy number in the genome. A multiplex conventional PCR method was established to effectively discriminate bovine (223 bp), equine (197 bp) and reference (129 bp) in one reaction. A multiplex real-time PCR was developed and confirmed in five binary mixtures of cattle and horse DNA, exhibiting adequate parameters of trueness (R.E. <= 9.35%) and precision (R.S.D. <= 8.88%). Furthermore, we confirmed that our method held good potential in the detection of meat products in real life. The method and strategy developed in this study will play an important role in combating adulteration and maintaining a fair market.