Targeting Helicase-Dependent Amplification Products with an Electrochemical Genosensor for Reliable and Sensitive Screening of Genetically Modified Organisms

Abstract

Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops

“…In short, the genosensor was prepared on a gold thin-film previously washed with ethanol and water, and dried under a stream of nitrogen gas. After conditioning, a binary self-assembled monolayer composed of a thiolated capture probe (CP) and p -aminothiophenol ( p -ATP) was built following a backfilling strategy described elsewhere [14]. This thioaromatic agent leads to lower background signals in comparison to the typically used 6-mercaptohexanol, and improvement in sensing surface stability [16].…”

“…A pair of primers, previously designed for isothermal amplification of a P35S specific sequence, was here re-examined for exponential heterothermic DNA amplification. Despite leading to a 21-nucleotide protruding tail adjacent to the transducer surface, no detrimental effect on surface hybridization efficiency was observed [14]. …”

“…Genomic DNA was extracted and purified from 100 mg of homogenized flour using the Nucleospin ® food kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions with minor modifications as described in Reference [14]. All extractions included a blank for controlling potential contamination during the procedure.…”

“…The DNA-modified surfaces thus obtained were further incubated with 6 µL of 1 mM p -aminothiophenol in 2× SSPE, pH 7.4 for 50 min [14]. The genosensors were used in a sandwich-type hybridization assay, schematically depicted in Figure 1.…”

<strong>Electrochemical Detection of <em>Salmonella</em> via On-surface Isothermal Amplification of its Genetic Material onto Highly Stable and Reproducible Indium Tin Oxide Platforms</strong>

“…13 Most HDA applications involve the enzymatic extension of primers in solution. [14][15][16] Although it is known that the immobilization of the primers on a solid surface contributes to the minimization of the typical problems associated with primer-dimer formation, only on one occasion on-chip HDA amplification in combination with fluorescence detection has been described, 17 showing poor sensitivity. Here we present data demonstrating for the first time on-surface HDA amplification with electrochemical detection for Salmonella genome quantification with a detection limit that matches the real-time PCR.…”