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The pig (Sus scrofa) mitochondrial genome was  targeted to design short (15-30 nucleotides) DNA markers that would be suitable  for biosensor-based hybridization detection of target DNA. Short DNA markers  are reported to survive harsh conditions in which longer ones are degraded into  smaller fragments. The whole swine mitochondrial-genome was in silico digested  with AluI restriction enzyme. Among 66 AluI fragments, five were  selected as potential markers because of their convenient lengths, high degree  of interspecies polymorphism and intraspecies conservatism. These were  confirmed by NCBI blast analysis and ClustalW alignment analysis with 11  different meat-providing animal and fish species. Finally, we integrated a  tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter  citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid  nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved  pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in  deliberately contaminated autoclaved pork-beef binary mixtures and no  cross-species detection was recorded, demonstrating the feasibility of this  type of probe for biosensor-based detection of pork adulteration of halal and  kosher foods.

Development of swine-specific DNA markers for biosensor-based halal authentication