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Real-time PCR is the gold standard technique for the unambiguous detection of sequence-specific fragments of pathogen DNA in many fields but it does not always fulfill the extremely low detectability legislation demands. Herein we demonstrate that rationally designed genosensors can rival or even supersede it when combined with previous amplification strategies even isothermal ones. Among the latter, helicase-dependent amplification (HDA) is a simple and powerful DNA amplification using helicase instead of temperature cycles to unwind DNA; it follows an amplification scheme very similar to PCR, which could facilitate its widespread adoption for point-of-need applications. Unfortunately, the primer design for HDA is more restrictive than for PCR. We show that it is possible to adapt PCR primers to isothermal amplification of a DNA sequence specific for Salmonella . We design an electrochemical DNA sensor, based on a sandwich hybridization assay on the surface of indium-tin oxide electrodes, which allows monitoring both PCR and HDA amplicons. The resulting sequence-specific detection strategy not only increased the selectivity of the detection but also its sensitivity, improving real-time PCR in two-fold and a hundred-fold when combined with HDA and PCR, respectively, achieving the single molecule detection. [ABSTRACT FROM AUTHOR]

Simple, cost-effective and reliable tools for the quantification of genetically modified organisms (GMO) in food and feed are highly demanded to enforce labelling legislation in the EU. Herein, we report a novel method for quantitative analysis of genetically modified soybean with the event GTS-40-3-2, also known as Roundup Ready (RR) soybean, using magnetoassays with electrochemical detection, coupled to DNA amplification by end-point polymerase chain reaction (PCR). For the proposed work, two DNA sequences were targeted via hybridisation onto magnetic beads, one specific for the transgenic event and the other for the taxon or species-specific lectin gene. Enzymatic labelling was performed to obtain an electrochemically active product measured by chronoamperometry. By optimising the number of PCR cycles, among other parameters, two magnetoassays coupled to PCR were successfully accomplished and linearity was obtained in the ranges of 53–4425 and 1093–88,496 DNA copies for the event-specific and lectin sequences, respectively. The proposed method provides accurate and precise RR soybean quantitative results, being effectively compared to those obtained by real-time PCR, as the reference method. These findings confirm the suitability of the method as an alternative tool for GMO quantification. [ABSTRACT FROM AUTHOR]