Integrating LAMP-coupled Modification SPCE with SiNWs/PtNPs in an Electrochemical DNA Biosensor for Real-time Monitoring of Porcine DNA Amplification

The present study describes the development and optimization of a loop-mediated isothermal amplification (LAMP) technique for the rapid and sensitive detection of porcine DNA, addressing critical needs in food safety and compliance with dietary laws such as halal and kosher. The primer sets were carefully designed to bind selectively to specific region of Sus scrofa mitochondrial DNA, thereby ensuring high specificity and amplification efficiency. The selection of LAMP was driven by its advantages, including rapid amplification time and isothermal conditions, which simplify the equipment requirements and reduce overall costs. To achieve optimal performance, the primers (F3, B3, FIP, BIP, and LF) were carefully designed to initiate strand displacement and DNA synthesis under isothermal conditions. The amplification parameters, such as temperature and incubation time, were systematically optimized, resulting in successful DNA amplification at 63°C for 60 minutes. The reaction's progress was monitored by measuring the turbidity associated with the accumulation of magnesium pyrophosphate, a reaction byproduct. Validation of the amplified products was performed using gel electrophoresis, confirming the presence of the expected DNA fragments. The amplified DNA products were subsequently detected using an advanced electrochemical DNA biosensor. This biosensor employed silicon nanowires and platinum nanoparticles (SiNWs/PtNPs) modified screen-printed carbon electrode (SPCE), with ferrocenylnaphthalene diimide (FND) serving as an intercalator for the detection of double-stranded DNA (dsDNA). The integration of LAMP with this biosensor enabled precise quantification and real-time monitoring of the DNA amplification process. The Limit of Detection (LOD) of the optimized LAMP method was determined to be 175.2 ng/µL, demonstrating its ability to detect low quantities of porcine DNA with high sensitivity. Cross-reactivity studies involving a range of meat sources and processed food matrices demonstrated the system's high reliability and specificity for detecting porcine DNA, with no false positives observed. Additionally, the biosensor effectively detected porcine DNA in binary meat mixtures, simulating real-world scenarios, and underscoring its practical applicability.