Methods For Detection Of Toxins Research Articles

A Glycoprotein-Based Surface-Enhanced Raman Spectroscopy-Lateral Flow Assay Method for Abrin and Ricin Detection.

Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

Nanoscale Materials Applying for the Detection of Mycotoxins in Foods.

Trace amounts of mycotoxins in food matrices have caused a very serious problem of food safety and have attracted widespread attention. Developing accurate, sensitive, rapid mycotoxin detection and control strategies adapted to the complex matrices of food is crucial for in safeguarding public health. With the continuous development of nanotechnology and materials science, various nanoscale materials have been developed for the purification of complex food matrices or for providing response signals to achieve the accurate and rapid detection of various mycotoxins in food products. This article reviews and summarizes recent research (from 2018 to 2023) on new strategies and methods for the accurate or rapid detection of mold toxins in food samples using nanoscale materials. It places particular emphasis on outlining the characteristics of various nanoscale or nanostructural materials and their roles in the process of detecting mycotoxins. The aim of this paper is to promote the in-depth research and application of various nanoscale or structured materials and to provide guidance and reference for the development of strategies for the detection and control of mycotoxin contamination in complex matrices of food.

Cloning the gene fragment encoding botulinum neurotoxin serotype A and establishment of a detection method for Clostridium botulinum using LAMP technique

The neurotoxins produced by Clostridium botulinum are lethal in minute amounts. Botulinum neurotoxin serotype A accounts for approximately 60% of botulism cases worldwide. Bacterial and toxin detection methods including the gold standard mouse bioassay and routine culture both require tedious procedures and need days to obtain results. Importantly, the development of a rapid and accurate technique to identify the toxin-encoding gene is crucial for timely diagnosis and treatment. This study aimed to clone a gene fragment encoding botulinum neurotoxin serotype A to serve as a positive control and establish a method based on the loop-mediated isothermal amplification (LAMP) technique to rapidly detect the encoding bont/A gene. The LAMP reaction was performed at the optimized temperature of 65˚C for 30 minutes with the in-house recombinant plasmid carrying a 250-bp fragment as the positive control. The developed method showed positive results for all strains provided by the National Institute of Hygiene and Epidemiology, consistent with the conventional polymerase chain reaction (PCR) results using the Vietnam standard with a total time roughly four times faster than the PCR amplification.

Incidence of Aflatoxin in Ready to Eat Nuts from Local Food Markets in Mangaluru, India

Abstract Background and Objective Aflatoxins are a group of naturally occurring mycotoxin which are toxic secondary metabolites produced by certain filamentous fungi like Aspergillus flavus and Aspergillus parasiticus. The main objective of this study was to screen the occurrence of aflatoxin in ready to eat nuts available locally and analyzing for its nutritive value and to evaluate the efficiency of conventional (thin-layer chromatography [TLC]) and sensitive kit-based (enzyme-linked immunosorbent assay [ELISA]) method by detection of the aflatoxin in the sample. Methods A total of 50 samples including peanuts (10), cashew nuts (10), almonds (10), pistachio (10), and walnuts (10) were collected from different stores in Mangalore city. Each sample was divided into three fractions, as for microbiological analysis, proximate analysis, and detection of aflatoxin by following standard method (AOAC2000). Results The present study evidenced the contamination of aflatoxin in all of the five types of ready-to-eat nuts examined and the concentration was within the acceptable limits. But, among the samples analyzed, G10 (groundnut) showed a maximum concentration of 16 µg/L aflatoxin detected by ELISA method. It was also observed that the proximate analysis mainly moisture content did not affect aflatoxin accumulation. Conclusion Our study shows that aflatoxin contamination of food products has become a serious threat. Although several methods for detection and quantification of toxins have been developed, due to their low concentration of toxicity in food commodities, an analytical method for detection and quantification of aflatoxin have to be specific, sensitive, and simple to carry out and among TLC and ELISA, ELISA came out as a suitable for rapid and sensitive detection.

Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins

Insect-resistant genetically modified organisms have been globally commercialized for the last 2 decades. Among them, transgenic crops based on Bacillus thuringiensis crystalline (Cry) toxins are extensively used for commercial agricultural applications. However, less emphasis is laid on quantifying Cry toxins because there might be unforeseen health and environmental concerns. Immunoassays, being the preferred method for detection of Cry toxins, are reviewed in this study. Owing to limitations of traditional colorimetric enzyme-linked immunosorbent assay, the trend of detection strategies shifts to modified immunoassays based on nanomaterials, which provide ultrasensitive detection capacity. This review assessed and compared the properties of the recent advances in immunoassays, including colorimetric, fluorescence, chemiluminescence, surface-enhanced Raman scattering, surface plasmon resonance, and electrochemical approaches. Thus, the ultimate aim of this study is to identify research gaps and infer future prospects of current approaches for the development of novel immunosensors to monitor Cry toxins in food and the environment.

Performance Characteristics of Refined LC-FLD and HILIC-MS/MS Methods for the Determination of Paralytic Shellfish Toxins in Shrimp, Whelk, and Crab.

Paralytic Shellfish Poison (PSP) toxins have been reported in non-bivalve shellfish species, including crustaceans and gastropods. Routine surveillance of these species is currently conducted in parts of England. To date, detection methods have not been validated for these matrices. Validation is required to ensure the test is fit for purpose, to give greater confidence in any results generated and ultimately facilitates accreditation. The aim was to test and validate two independent PSP toxin detection methods previously validated for bivalve shellfish matrices, for applicability to commercial non-bivalve species of interest. Matrices were shrimp (Crangon crangon), common whelk (Buccinum undatum), and edible crab (Cancer pagurus). The two methods assessed were the pre-column oxidation high-performance liquid chromatography-fluorescence detection AOAC 2005.06 Official Method of analysis and an internationally validated hydrophilic interaction chromatography-tandem mass spectrometry method. Brown and white crab meat were assessed separately. A refined extraction protocol was implemented with an increased solvent to sample ratio. The same extraction protocol was utilized for both methods, allowing both methods to be run simultaneously. Method sensitivity, recovery, repeatability, and method uncertainty were characterized in all matrix/toxin combinations. Overall, both methods performed similarly to that previously reported in bivalve mollusks. Acceptability of the majority of toxin/matrix combinations was evidenced through comparison of method performance characteristics against specific performance criteria, including Horwitz ratio values. Both PSP toxin detection methods were found to provide acceptable performance for the monitoring of shrimp, whelk, and crab species. Two PSP toxin detection methods have been single-laboratory validated successfully for three non-bivalve shellfish species.

Using Nanomaterials in Colorimetric Toxin Detection

Exposure to toxins through contaminated food is a serious concern. For the detection of toxins in complex matrices, there are many analytical instrumentation-based methods; however, these approaches are generally expensive, laborious to perform, and require skilled technicians. Thus, they can only be utilized in centralized laboratories. To efficiently prevent the contamination by toxins and improve food safety, the use of on-site toxin detection methods enabling simple, rapid, sensitive, specific, reliable, and affordable identification of toxins is required. A colorimetric toxin detection strategy providing a naked-eye readout platform suits these requirements. Notably, the implementation of nanomaterials in the colorimetric strategy has proven to rapidly generate a higher capacity for detectable color responses owing to their unique physicochemical and catalytic properties. In this review, recent research on colorimetric toxin detection utilizing diverse nanostructures including noble metal nanoparticles and enzyme-like catalytic nanomaterials (nanozymes) is reviewed and discussed. Current challenges and future prospects for the utilization of nanomaterials in colorimetric toxin detection are also discussed.

The Food Poisoning Toxins of Bacillus cereus

Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic type, a food intoxication, manifests in nausea and vomiting, food infections with enteropathogenic strains cause diarrhea and abdominal pain. Causative toxins are the cyclic dodecadepsipeptide cereulide, and the proteinaceous enterotoxins hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe) and cytotoxin K (CytK), respectively. This review covers the current knowledge on distribution and genetic organization of the toxin genes, as well as mechanisms of enterotoxin gene regulation and toxin secretion. In this context, the exceptionally high variability of toxin production between single strains is highlighted. In addition, the mode of action of the pore-forming enterotoxins and their effect on target cells is described in detail. The main focus of this review are the two tripartite enterotoxin complexes Hbl and Nhe, but the latest findings on cereulide and CytK are also presented, as well as methods for toxin detection, and the contribution of further putative virulence factors to the diarrheal disease.

Addressing the Analytical Challenges for the Detection of Ciguatoxins Using an Electrochemical Biosensor.

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 μg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

Acquired knowledge about Ciguatera Fish Poisoning in the Canary Islands population

Acquired knowledge about Ciguatera Fish Poisoning in the Canary Islands population

A single run UPLC-MS/MS method for detection of all EU-regulated marine toxins

A UPLC-MS/MS method has been developed for identification and quantification of hydrophilic and lipophilic marine toxins. The method included the determination of 14 toxins of STX group, 15 lipophilic toxins, 15 toxins of the TTX group and DA. LODs and LOQs for STX group were significantly improved in comparison to the official validated methods and at the same level than other UPLC-MS/MS published methods. The same for lipophilic toxins, with better LODs and LOQs than the EU official method. LOD and LOQ for DA were higher than those obtained with the EU official method. While for TTXs LOD and LOQ were comparable to other validated methods. Validation studies demonstrated acceptable method performance characteristics for linearity, and repeatability between-batch and within-batch. The study demonstrated that the UPLC-MS/MS method provides an excellent tool to determinate hydrophilic and lipophilic toxins and therefore it could be appropriate for routine testing and interlaboratory validation.

From Commensal to Consumer: Staphylococcus aureus Toxins, Diseases, and Detection Methods.

Staphylococcus aureus is a Gram-positive bacterium capable of causing a wide array of infections. Generally a commensal organism, S. aureus encodes several virulence mechanisms that contribute to disease progression. This review highlights toxins as a secreted virulence factor by S. aureus, the diseases that manifest as a result, and the methods used to detect them. In particular, the advantages and limitations of current toxin detection methods are discussed.

Detection of azaspiracids in mussels using electrochemical immunosensors for fast screening in monitoring programs

Given the widespread occurrence of azaspiracids (AZAs), it is clearly necessary to advance in simple and low-cost methods for the rapid detection of these marine toxins in order to protect seafood consumers. To address this need, electrochemical immunosensors for the detection of AZAs based on a competitive direct immunoassay using peroxidase-labelled AZA as a tracer were developed. An anti-AZA polyclonal antibody was immobilised in a controlled and stable manner on protein G or avidin-coated electrodes. Experimental conditions were first optimised using colorimetric immunoassays on microtitre plates, providing intermediate products already applicable to the accurate detection of AZAs. Then, transfer of the protein G and avidin–biotin interaction-based immunoassays to 8-electrode arrays provided compact and miniaturised devices for the high-throughput detection of AZAs. The low amounts of immunoreagents required as well as the potential for reusability of the avidin–biotin interaction-based immunosensors represented significant economic savings as well as a contribution to sustainability. The electrochemical immunosensors enabled the quantification of all regulated AZAs below the regulatory limit, as well as a broad range of other toxic AZA analogues (from 63 ± 3 to 2841 ± 247 μg AZA-1 equiv./kg for the protein G-based immunosensor and from 46 ± 2 to 3079 ± 358 μg AZA-1 equiv./kg for the avidin–biotin interaction-based immunosensor). The good agreement between the results obtained by the immunosensors and LC–MS/MS in the analysis of naturally contaminated mussel samples demonstrated the easy implementation of electrochemical immunosensors for routine analysis of AZAs in food safety monitoring programs.

Journey from food toxins to food safety: Transition over a century in service of nation.

Since the first report of lathyrism in 1926, the ICMR-National Institute of Nutrition (NIN) at Hyderabad, India, has made tremendous contributions in the field of food toxins/food safety for the benefit of the people. The present article highlights the Institute's work on various food toxins/foodborne diseases since its inception and discusses the important contributions made in the context of public health protection that formed the basis for several national policies on their prevention and control. The investigations on food toxins, in the initial decades, were limited to the description of lathyrism and its endemicity. Subsequently, the horizon was broadened to include the problem of mycotoxins and mycotoxicosis, which had received global attention and variety of other disease outbreaks investigations leading to total food safety transition in the forthcoming decades. Important contributions in epidemiological investigations, reproduction of the disease in experimental animals, surveillance and monitoring studies, development of methods for detection of food toxins and contaminants, evolving strategies for prevention and control and developing the concept of risk assessment and risk management for addressing food safety issues in the country are discussed.

RecentTrendsinMarinePhycotoxinsfrom AustralianCoastalWaters.

Phycotoxins, which are produced by harmful microalgae and bioaccumulate in the marine food web, are of growing concern for Australia. These harmful algae pose a threat to ecosystem and human health, as well as constraining the progress of aquaculture, one of the fastest growing food sectors in the world. With better monitoring, advanced analytical skills and an increase in microalgal expertise, many phycotoxins have been identified in Australian coastal waters in recent years. The most concerning of these toxins are ciguatoxin, paralytic shellfish toxins, okadaic acid and domoic acid, with palytoxin and karlotoxin increasing in significance. The potential for tetrodotoxin, maitotoxin and palytoxin to contaminate seafood is also of concern, warranting future investigation. The largest and most significant toxic bloom in Tasmania in 2012 resulted in an estimated total economic loss of ~AUD$23M, indicating that there is an imperative to improve toxin and organism detection methods, clarify the toxin profiles of species of phytoplankton and carry out both intra- and inter-species toxicity comparisons. Future work also includes the application of rapid, real-time molecular assays for the detection of harmful species and toxin genes. This information, in conjunction with a better understanding of the life histories and ecology of harmful bloom species, may lead to more appropriate management of environmental, health and economic resources.

High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been adopted as the EU reference method (No. 15/2011) for detection and quantitation of lipophilic toxins. Traditional LC–MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers.Matrix effects are an important issue to address in LC–MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce matrix effects for all compounds, and was found to be particularly useful for the non-targeted approach.Limits of detection and method accuracy were comparable between the systems tested, demonstrating the applicability of HRMS as an effective tool for screening and quantitative analysis. HRMS offers the advantage of untargeted analysis, meaning that datasets can be retrospectively analyzed. HRMS (full scan) chromatograms of passive samplers yielded significantly less complex data sets than mussels, and were thus more easily screened for unknowns. Consequently, we recommend the use of HRMS in combination with passive sampling for studies investigating emerging or hitherto uncharacterized toxins.

Detection of diarrhetic shellfish poisoning toxins using high-sensitivity human cancer cell-based impedance biosensor

Diarrhetic shellfish poisoning (DSP) toxin is a dangerous contamination in seafood worldwide that can threaten human health and fishing. A large number of animal bioassays and chemical analytical methods are employed for DSP toxin detection. However, these toxin detection methods are low-throughput and high-cost which hamper their wide applications. In this study, HeLa and HepG2 cell lines were selected as the sensitive elements to establish the CIBs for monitoring the cytotoxicity induced by a representative DSP toxin, okadaic acid (OA). The limit of detection (LOD) of HeLa- and HepG2-based biosensors are 10.2μg/L and 3.3μg/L, respectively, which are both lower than the conventional cell-based assay (21.2μg/L of HeLa cells and 9.8μg/L of HepG2 cells). The half maximal inhibitory concentration (IC50) values of OA in HeLa and HepG2 cells which were obtained from CIB (49.9±4.9 and 39.2±4.3μg/L) are both lower than Cell Counting Kit-8 assay (CCK8) (62.7±7.1 and 45.8±6.7μg/L). Besides, CIB measurement presents good correlation with mouse bioassay (MBA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS). In summary, all the results indicate that the CIB technology had great potential to be an effective complement in DSP toxins detection.

A novel Love Wave biosensor for rapid and sensitive detection of marine toxins.

Marine toxins are produced by plankton and do a great harm to human through food chain by accumulating in shellfishes and fishes. It is highly required and favorable to develop novel methods for the rapid and efficient detection of marine toxins to avoid the poisoning cases that have occurred frequently in many countries. This study presents a real-time Love Wave biosensor for the rapid detection of okadaic acid (OA), which used HepG2 cell lines as the sensing elements. The results indicate that this cell-based biosensor can provide real-time information of cellular activities induced by okadaic acid and has a higher sensitivity than the conventional cell-based assay. It is suggested that this cell-based biosensor can be used as a convenient and efficient method for marine toxin detection, which has a great potential to contribute to avoid the harmful effects of marine toxins on the human health.

Development of a novel method for detection ofClostridium difficileusingHS‐SPME‐GC‐MS

A novel method has been developed that allows successful differentiation between Clostridium difficile culture-positive and culture-negative stool samples based on volatile organic compound (VOC) evolution and detection by headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). The method is based on the activation of p-hydroxyphenylacetate decarboxylase produced by Cl. difficile and the detection of a specific VOC, that is 2-fluoro-4-methylphenol from an enzyme substrate. In addition, other VOCs were good indicators for Cl. difficile, that is isocaproic acid and p-cresol, although they could not be used alone for identification purposes. One hundred stool samples were tested, of which 77 were positive by culture. Detection using HS-SPME-GC-MS allowed confirmation of the presence of Cl. difficile within 18 h with a sensitivity and specificity of 83·1 and 100%, respectively. It is recommended that this new approach could be used alongside conventional methods for Cl. difficile detection, including toxin detection methods, which would allow any false-negative results to be eliminated. The ability to identify Cl. difficile-positive stool samples by the analysis of VOCs could allow the development of a VOC detection device which could allow rapid diagnosis of disease and hence prompt treatment with appropriate antibiotics.

Nanoporous membrane based impedance sensors to detect the enzymatic activity of botulinum neurotoxin A.

Botulinum neurotoxins are among the most potent toxic bacterial proteins for humans and there is a great need to develop simple, rapid and sensitive methods for toxin detection and protease activity quantification in field deployment. In this paper, a nanoporous membrane based impedance sensor was developed to monitor the activity of the BoNT serotype A light chain protease (LcA). Synaptosomal-associated protein 25 (SNAP-25) was first immobilized inside nanopore walls via silane linkers. BoNT LcA was then injected over the nanoporous membrane substrate sensor and specifically cleaved SNAP-25. The cleavage activity could be monitored by measuring impedance signals across nanoporous membranes which represented the nanopore blockage degree. This initial device could achieve a 500 pM LcA detection limit within 25 minutes.