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Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. [ABSTRACT FROM AUTHOR]

Affinity characterization is essential to develop reliable aptamers for tumor biomarker detection. For alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC), two DNA aptamers were described with very different affinity. In this work, we estimate the dissociation constant of both of them by means of a direct assay on magnetic beads modified with AFP and electrochemical detection on carbon screen-printed electrodes (SPCE). Unlike previous works, both aptamers showed similar dissociation constant (Kd) values, in the subµM range. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers by both terminal deoxynucleotidyl transferase (TdT) and rolling circle amplification (RCA). Both DNA amplifications improved the sensitivity and also the apparent binding constants from 713 nM to 189 nM for the short aptamer and from 526 nM to 32 nM for the long aptamer. This improvement depends on the true affinity of the binding pair, which ultimately limits the analytical usefulness. [ABSTRACT FROM AUTHOR]

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]

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 106-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. [ABSTRACT FROM AUTHOR]

Genosensing technology has mostly based on mixed self-assembled monolayers (SAMs) of thiol-modified oligonucleotides and alkanethiols on gold surfaces. However, the typical backfilling approach, which incorporates the alkanethiol in a second step, gives rise to a heterogeneous distribution of oligonucleotide probes on the surface, negatively affecting to both hybridization efficiency and surface stability. Despite aromatic thiols present a remarkably different behavior from alkanethiols, with higher rigidity and stronger intermolecular interactions, they have been scarcely explored for the fabrication of DNA sensing platforms. We have investigated different approaches involving SAMs of aromatic thiols, namely p -mercaptobenzoic acid ( p -MBA) and p -aminothiophenol ( p -ATP), to yield DNA sensing layers for sequence-specific detection of target oligonucleotides. The studied monolayers were evaluated by DNA surface coverage and further information was obtained by determining their functionality in a sandwich hybridization assay with enzymatic amplification of the electrochemical read-out. The insertion of thiol-oligonucleotides into p -ATP monolayers previously oxidized, and the covalent binding of amino-oligonucleotides to pure p -MBA monolayers give rise to increased storage stability and better analytical performance. The quantification of RNA from Legionella pneumophila cellular lysates was successfully performed, illustrating the usefulness of these sensing architectures for detecting pathogenic bacteria. [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]

Abstract: Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour. [Copyright &y& Elsevier]