PCR-free and minute-scale electrochemical analysis of porcine DNA adulteration via molecularly amplified DNA sandwich assay

The increasing incidence of meat adulteration and mislabeling poses significant challenges in terms of food safety and consumer trust. This study proposes an electrochemical DNA biosensor for detecting porcine mitochondrial DNA in tainted meat products, offering a novel approach to address the above challenges. Unlike conventional nucleic acid amplification tests that rely on polymerase chain reactions (PCRs), the proposed biosensor employs a molecularly amplified DNA strategy with DNA tracers that bind to two regions of the target DNA, creating an elongated hybridization structure with multiple redox-tagging molecules. This design catalyzes detection signals autonomously, eliminating the need for PCR amplification. One-step DNA probe immobilization using poly-adenine (poly-A) oligonucleotides significantly improves hybridization efficiency and reduces the necessity for extensive sample purification, thereby simplifying the detection process. The proposed biosensor exhibits a linear detection range of 10¹–10⁶ pM and a limit of detection (LOD) of 2.2 pM in controlled settings. Furthermore, the proposed biosensor distinguishes pork from beef in adulterated samples with a LOD of 1 % w/w. With its stability exceeding 9 weeks and a cost of less than 0.5 USD per test, the proposed biosensor offers a highly sensitive, economically viable solution with significant potential for widespread use in the meat industry and by end-users, effectively combating porcine adulteration.