We report the development of a swine-specific hybrid nanobioprobe through a covalent integration of a fluorophore-labeled 27-nucleotide<jats:italic>Alu</jats:italic>I-fragment of swine cytochrome b gene to a 3 nm gold nanoparticle for the determination of pork adulteration in processed meat products. We tested the probe to estimate adulterated pork in ready-to-eat pork-spiked beef burgers. The probe quantitatively detected 1–100% spiked pork in burger formulations with ≥90% accuracy. A plot of observed fluorescence against the known concentration of<jats:italic>Alu</jats:italic>I-digested pork DNA targets generated a concave curve, demonstrating a power relationship (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mi>y</mml:mi><mml:mo>=</mml:mo><mml:mn>2.956</mml:mn><mml:msup><mml:mi>x</mml:mi><mml:mrow><mml:mn>0.509</mml:mn></mml:mrow></mml:msup></mml:math>) with a regression coefficient (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mi>R</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:math>) of 0.986. No cross-species detection was found in a standard set of pork, beef, chicken, mutton, and chevon burgers. The method is suitable for the determination of very short-length nucleic acid targets which cannot be estimated by conventional and real-time PCR but are essential for the determination of microRNA in biodiagnostics and degraded DNA in forensic testing and food analysis.
