Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.info:eu-repo/semantics/publishedVersio

Detecting harmful algal blooms with isothermal molecular strategies

The use of isothermal nucleic acid amplification strategies to detect harmful algal blooms (HABs) is in its infancy. We describe recent advances in these systems and highlight the challenges for the achievement of simple, low-cost, compact, and portable devices for field applications.info:eu-repo/semantics/acceptedVersio

Detection of isothermally amplified ostreid herpesvirus 1 DNA in Pacific oyster (Crassostrea gigas) using a miniaturised electrochemical biosensor

Given the threat that ostreid herpesvirus 1 (OsHV-1) poses to shellfish aquaculture, the need for rapid, user-friendly and cost-effective methods to detect this marine pathogen and minimise its impact is evident. In this work, an electrochemical biosensor for the detection of OsHV-1 based on isothermal recombinase polymerase amplification (RPA) was developed. The system was first tested and optimised on maleimide microtitre plates as a proof-of-concept, before being implemented on miniaturised gold electrodes. Amperometric detection of the isothermally amplified product was achieved through a sandwich hybridisation assay with an immobilised thiolated capture probe and a horseradish peroxidase (HRP)-labelled reporter probe. Calibration curves were constructed using PCR-amplified OsHV-1 DNA, achieving a limit of detection of 207 OsHV-1 target copies. The biosensor was applied to the analysis of 16 oyster samples from an infectivity experiment, and results were compared with those obtained by qPCR analysis, showing a strong degree of correlation (r = 0.988). The simplicity, rapidity, cost-effectiveness and potential for in-situ testing with the developed biosensor provide a valuable tool for the detection of OsHV-1 in aquaculture facilities, improving their management.info:eu-repo/semantics/acceptedVersio

Detection of Ostreopsis cf. ovata in environmental samples using an electrochemical DNA-based biosensor

Ostreopsis cf. ovata is a benthic microalga distributed in tropical and temperate regions worldwide which produces palytoxins (PlTXs). Herein, an electrochemical biosensor for the detection of this toxic microalga is described. The detection strategy involves isothermal recombinase polymerase amplification (RPA) of the target using tailed primers and a sandwich hybridisation assay on maleimide-coated magnetic beads immobilised on electrode arrays. The biosensor attained a limit of detection of 9 pg/μL of O. cf. ovata DNA (which corresponds to ~640 cells/L), with no interferences from two non-target Ostreopsis species (O. cf. siamensis and O. fattorussoi). The biosensor was applied to the analysis of planktonic and benthic environmental samples. Electrochemical O. cf. ovata DNA quantifications demonstrated an excellent correlation with other molecular methods (qPCR and colorimetric assays) and allowed the construction of a predictive regression model to estimate O. cf. ovata cell abundances. This new technology offer great potential to improve research, monitoring and management of O. cf. ovata and harmful algal blooms.info:eu-repo/semantics/acceptedVersio

Amplified plasmonic and microfluidic setup for DNA monitoring

Plasmonic nanosensors for label-free detection of DNA require excellent sensing resolution, which is crucial when monitoring short DNA sequences, as these induce tiny peak shifts, compared to large biomolecules. We report a versatile and simple strategy for plasmonic sensor signal enhancement by assembling multiple (four) plasmonic sensors in series. This approach provided a fourfold signal enhancement, increased signal-to-noise ratio, and improved sensitivity for DNA detection. The response of multiple sensors based on AuNSpheres was also compared with AuNRods, the latter showing better sensing resolution. The amplification system based on AuNR was integrated into a microfluidic sequential injection platform and applied to the monitoring of DNA, specifically from environmental invasive species—zebra mussels. DNA from zebra mussels was log concentration-dependent from 1 to 1 × 106 pM, reaching a detection limit of 2.0 pM. In situ tests were also successfully applied to real samples, within less than 45 min, using DNA extracted from zebra mussel meat. The plasmonic nanosensors’ signal can be used as a binary output (yes/no) to assess the presence of those invasive species. Even though these genosensors were applied to the monitoring of DNA in environmental samples, they potentially offer advantage in a wide range of fields, such as disease diagnostics.info:eu-repo/semantics/acceptedVersio

Dual quantitative PCR assay for identification and enumeration of Karlodinium veneficum and Karlodinium armiger combined with a simple and rapid DNA extraction method

Karlodinium is a dinoflagellate genus responsible for massive fish mortality events worldwide. It is commonly found in Alfacs Bay (NW Mediterranean Sea), where the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. Microscopy analysis is not able to differentiate between these two species. Therefore, new and rapid methods that accurately and specifically detect and differentiate these two species are crucial to facilitate routine monitoring, to provide early warnings and to study population dynamics. In this work, a quantitative real-time PCR (qPCR) method to detect and enumerate K. veneficum and K. armiger is presented. The ITS1 region of the ribosomal DNA was used to design species-specific primers. The specificity of the primers together with the melting curve profile provided a reliable qualitative identification and discrimination between the two Karlodinium species. Additionally, a simple and rapid DNA extraction method was used. Standard curves were constructed from 10-fold dilutions of cultured microalgae cells. Finally, the applicability of the assay was tested with field samples collected from Alfacs Bay. Results showed a significant correlation between qPCR determinations and light microscopy counts (y = 2.838 x + 564; R2 = 0.936). Overall, the qPCR method developed herein is specific, rapid, accurate, and promising for the detection of these two Karlodinium species in environmental samples.info:eu-repo/semantics/acceptedVersio

Electrochemical genosensor for the direct detection of tailed PCR amplicons incorporating ferrocene labelled dATP

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2′-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade−1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.info:eu-repo/semantics/acceptedVersio

First report of Gambierdiscus in the Western Mediterranean Sea (Balearic Islands)

Gambierdiscus (Dinophyceae) species are benthic dinoflagellates living in marine littoral zones of circumtropical areas and have recently been described in temperate waters [1]. Some species are producers of potent neurotoxins: ciguatoxins (CTXs) and maitotoxins (MTXs). Ciguatoxins are linked to Ciguatera Fish Poisoning (CFP). Ciguatera used to be restricted to tropical and subtropical areas, but since the last decade, it appears to be expanding to more temperate latitudes. For example, outbreaks of ciguatera have been reported in the Canary Islands and Madeira (eastern Atlantic Ocean), where several species of the genus Gambierdiscus have been identified.info:eu-repo/semantics/publishedVersio

Assessment of cytotoxicity in ten strains of Gambierdiscus australes from Macaronesian Islands by neuro-2a cell-based assays

Within the dinoflagellate genus Gambierdiscus several species are well-known producers of ciguatoxins (CTXs) and maitotoxins (MTXs). These compounds are potent marine toxins that accumulate through the food chain, leading to a foodborne disease known as ciguatera when contaminated fish is consumed. Given the threat that the presence of these toxins in seafood may pose to human health and fisheries, there is an evident necessity to assess the potential toxicity of Gambierdiscus sp. in a particular area. Thus, the purpose of this work was to evaluate the production of CTX and MTX of 10 strains of Gambierdiscus australes isolated from the Selvagem Grande Island (Madeira, Portugal) and El Hierro Island (Canary Islands, Spain). The strains were first characterized by light microscopy and species were confirmed by molecular biology, being identified as G. australes. Following the species identification, CTX and MTX production of G. australes extracts was evaluated at the exponential growth phase using neuro-2a cell-based assays. Additionally, the production of MTX was also investigated in two of the G. australes strains collected at the stationary growth phase. Interestingly, 9 out of 10 strains were found to produce CTX-like compounds, ranging from 200 to 697 fg equiv. CTX1B cell−1. None of the G. australes strains showed MTX-like activity at the exponential phase, but MTX production was observed in two strains at the stationary growth phase (227 and 275 pg equiv. MTX cell−1). Therefore, the presence of G. australes strains potentially producing CTX and MTX in these Macaronesian Islands was confirmed.info:eu-repo/semantics/acceptedVersio

Colorimetric DNA-based assay for the specific detection and quantification of Ostreopsis cf. ovata and Ostreopsis cf. siamensis in the marine environment

Ostreopsis is a toxic benthic dinoflagellate largely distributed worldwide in tropical and temperate areas. In the Mediterranean Sea, periodic summer blooms have been reported and have become a serious concern due to their direct impact on human health and the environment. Current microalgae identification is performed via light microscopy, which is time-consuming and is not able to differentiate among Ostreopsis species. Therefore, there is mature need for rapid, specific and easy-to-use detection tools. In this work, a colorimetric assay exploiting a combination of recombinase polymerase amplification (RPA) and a sandwich hybridisation assay was developed for O. cf. ovata and O. cf. siamensis detection and quantification. The specificity of the system was demonstrated by cross-reactivity experiments and calibration curves were successfully constructed using genomic DNA, achieving limits of detection of 10 and 14 pg/μL for O. cf. ovata and O. cf. siamensis, respectively. The assay was applied to the analysis of planktonic and benthic environmental samples from different sites of the Catalan coast. Species-specific DNA quantifications were in agreement with qPCR analysis, demonstrating the reliability of the colorimetric approach. Significant correlations were also obtained between DNA quantifications and light microscopy counts. The approach may be a valuable tool to provide timely warnings, facilitate monitoring activities or study population dynamics, and paves the way towards the development of in situ tools for the monitoring of harmful algal blooms.info:eu-repo/semantics/acceptedVersio