Search

Plasticisers are commonly used to increase the flexibility of a wide variety of food contact materials including the plastic tubing, liners, and gaskets used in the dairy industry. In recent years, some classes of plasticisers have come under scrutiny due to the potential for transfer of these compounds into the milk itself, which can then be further processed into foods such as powdered milks and cheeses, infant formula, and baked goods. One such set of plasticisers that is being evaluated for frequency of use, potential routes of exposure, and risk to consumers is ortho-phthalates, hereafter referred to as phthalates. In order to better understand the actual use of phthalate versus non-phthalate plasticised tubing, a robust, rapid, and portable analytical method is necessary for on-site screening. Laboratory Raman and near-infrared spectrometers have been used extensively for polymer and additive evaluation, and advances in portable/hand-held technology could lead to feasible plasticiser evaluation in the field. This research overviews efforts to evaluate six portable spectroscopy devices for their ability to identify phthalate versus non-phthalate plasticised polyvinyl chloride (PVC) dairy tubing, liners, and gaskets. The most successful method, a hand-held Raman spectrometer along with a plasticiser spectral library or a chemometric model, can rapidly and accurately identify phthalate containing PVC and has the potential to be employed as a future field screening technique for regulators and the dairy industry. [ABSTRACT FROM AUTHOR]

In the fishing industry, there is a need for rapid and accurate screening of seafood freshness that supports the National Seafood Sensory Experts who perform organoleptic measurements. Previous studies employing near-infrared spectroscopy, Raman spectroscopy, and bioelectrical impedance analysis have shown potential success of these analytical techniques to evaluate freshness of seafood. However, the smaller sample sizes and limited sample diversity combined with not having the fish graded by sensory experts could limit the greater validity of these techniques. The present work was undertaken to investigate the suitability of four portable devices (two micro-miniaturized NIR devices, one portable Raman spectrometer, and one bioelectrical impedance analysis device) for analysis of two imported fish species that are commonly suspected of decomposition. Research focused on using large, naturally diverse sample sets to truly evaluate the robustness of each portable device. Results indicated that while techniques initially appeared promising, when more diversity was added into the models (e.g., natural variation (protein, water, fat), fillet thickness (8 to 30 mm), day-to-day variations), the instruments lose ability to correctly identify fresh from spoiled fish. As such, more research and technology innovations are necessary before instrumentation is ready for deployment to industries and regulatory agencies. [ABSTRACT FROM AUTHOR]

This study evaluated an electronic‐nose (e‐nose) sensor in combination with support vector machine (SVM) modeling for predicting the decomposition state of four types of fish fillets: mahi‐mahi, croaker, red snapper, and weakfish. The National Seafood Sensory Expert scored fillets were thawed, 10‐g portions were weighed into glass jars which were then sealed, and the jars were held at approximately 30°C to allow volatile components to be trapped and available for analysis. The measurement of the sample vial headspace was performed with an e‐nose device consisting of nanocomposite, metal oxide semiconductor (MOS), electrochemical, and photoionization sensors. Classification models were then trained based on the sensory grade of each fillet, and the e‐nose companion chemometric software identified that eight MOS were the most informative for determining a sensory pass from sensory fail sample. For SVM, the cross‐validation (CV) correct classification rates for mahi‐mahi, croaker, red snapper, and weakfish were 100%, 100%, 97%, and 97%, respectively. When the SVM prediction performances of the eight MOS were evaluated using a calibration‐independent test set of samples, correct classification rates of 93–100% were observed. Based on these results, the e‐nose measurements coupled with SVM models were found to be potentially promising for predicting the spoilage of these four fish species. Practical Application: This report describes the application of an electronic‐nose sensor as a potential rapid and low‐cost screening method for fish spoilage. It could provide regulators and stakeholders with a practical tool to rapidly and accurately assess fish decomposition. [ABSTRACT FROM AUTHOR]

Simple, fast, and accurate analytical techniques for verifying the accuracy of label declarations for marine oil dietary supplements containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are required because of the increased consumption of these products. We recently developed broad-based partial least squares regression (PLS-R) models to quantify six fatty acids (FAs) and FA classes by using the spectroscopic data from a portable Fourier transform infrared (FTIR) device and a benchtop Fourier transform near infrared (FT-NIR) spectrometer. We developed an improved quantification method for these FAs and FA classes by incorporating a nonlinear calibration approach based on the machine learning technique support vector machines. For the two spectroscopic methods, high accuracy in prediction was indicated by low root mean square error of prediction and by correlation coefficients (R2) close to 1, indicating excellent model performance. The percent accuracy of the support vector regression (SV-R) model predicted values for EPA and DHA in the reference material was 90 to 110%. In comparison to PLS-R, SV-R accuracy for prediction of FA and FA class concentrations was up to 2.4 times higher for both ATR-FTIR and FT-NIR spectroscopic data. The SV-R models also provided closer agreement with the certified and reference values for the prediction of EPA and DHA in the reference standard. Based on our findings, the SV-R methods had superior accuracy and predictive quality for predicting the FA concentrations in marine oil dietary supplements. The combination of SV-R with ATR-FTIR and/or FT-NIR spectroscopic data can potentially be applied for the rapid screening of marine oil products to verify the accuracy of label declarations. A spectroscopic and SV-R method was used to predict concentrations of FAs and FA classes in marine oil products. Excellent model predictions (R2 > 0.97) were obtained. SV-R outperformed PLS-R for the quantification of FAs in marine oil products. This method could be used to verify the accuracy of label declarations in marine oil products. [ABSTRACT FROM AUTHOR]

We recently observed that the weak near-infrared (NIR) band near 5260 cm-1 was relatively more intense for extra virgin olive oil (EVOO) than for refined olive oil (ROO). We also observed that its intensity was diminished upon heating and erroneously presumed that it may be attributed to volatile carbonyl components in EVOO. In the present study we demonstrate for the first time that this band is primarily attributed to a water O-H combination band. To accurately determine the intensity of this weak band, observed on a shifted and sloping baseline, we measured the peak-to-peak (p-p) height of its first derivative. An exponential calibration curve for p-p height versus gravimetrically-determined concentration of spiked water was satisfactorily generated. The calibration curve was first evaluated by using independent sets of gravimetrically prepared test samples. Subsequently, it was used to determine the moisture content, a quality parameter, for a limited set of authenticated reference olive oils whose quality and purity were confirmed by official methods. These concentrations, 0.098-0.12% H2O (w/w) for EVOO, 0.022-0.030% H2O (w/w) for ROO, and 0.028-0.054% H2O (w/w) for pomace olive oil (POO), were consistent with those reported in the literature. For 88 commercial products investigated, the moisture levels fell in the range from 0.026% to 0.13% (w/w). The correlation between moisture content and other olive oil quality parameters has been reported in the literature and has yet to be further investigated. [ABSTRACT FROM AUTHOR]

Two handheld Raman spectroscopic devices in combination with chemometrics were explored as rapid, non-targeted screening tools for the detection of milk powder (MP) adulteration using melamine as an example contaminant. The first device (device 1) employs a unique laser setup and data processing software that decreases the spectral interference from background fluorescence which could allow for improved detection capabilities. The second instrument (device 2) is the first of its kind to combine advanced chemometric data processing directly on-board the portable device which could allow for more rapid analysis of complex samples through “pass/fail” results. In this work, the performance of these two devices were evaluated and compared to each other and to that of a benchtop Raman spectrometer for the detection of melamine-spiked MP samples. The portable devices had similar levels of performance as the benchtop Raman spectrometer in terms of sensitivity and specificity for classifying test samples as genuine MP or MP-melamine blends. High specificity (100%) for melamine adulterated MPs at concentrations ≥1.0% (w/w) for dry-blends and ≥0.30% for wet-blends in the validation set was found for the non-targeted, soft independent modeling of class analogy (SIMCA) model developed for portable device 2. A similar level of specificity was observed when the non-targeted model was tested using a blind set of test samples and on-board chemometrics. Additionally, SIMCA models for devices 1 and 2 correctly classified genuine MPs with 97 and 100% correct classifications, respectively. Device 1 had comparatively lower specificity for MP blends. These results indicate that the developed screening methods using the handheld Raman devices are potentially well-suited for rapid, routine field analysis of MP samples for detection of economically motivated adulteration. [ABSTRACT FROM AUTHOR]

To investigate cases of acute oxalate nephrosis without evidence of ethylene glycol exposure, archived data and tissues from cheetahs (Acinonyx jubatus) from North America (n = 297), southern Africa (n = 257), and France (n = 40) were evaluated. Renal and gastrointestinal tract lesions were characterized in a subset of animals with (n = 100) and without (n = 165) oxalate crystals at death. Crystals were confirmed as calcium oxalate by Raman spectroscopy in 45 of 47 cheetahs tested. Crystals were present in cheetahs from 3.7 months to 15.9 years old. Cheetahs younger than 1.5 years were less likely to have oxalates than older cheetahs (P = .034), but young cheetahs with oxalates had more oxalate crystals than older cheetahs (P < .001). Cheetahs with oxalate crystals were more likely to have renal amyloidosis, interstitial nephritis, or colitis and less likely to have glomerular loop thickening or gastritis than those without oxalates. Crystal number was positively associated with renal tubular necrosis (P ≤ .001), regeneration (P = .015), and casts (P ≤ .001) but inversely associated with glomerulosclerosis, renal amyloidosis, and interstitial nephritis. Crystal number was unrelated to the presence or absence of colitis and was lower in southern African than American and European animals (P = .01). This study found no evidence that coexisting chronic renal disease (amyloidosis, interstitial nephritis, or glomerulosclerosis), veno-occlusive disease, gastritis, or enterocolitis contributed significantly to oxalate nephrosis. Oxalate-related renal disease should be considered as a potential cause of acute renal failure, especially in young captive cheetahs. The role of location, diet, stress, and genetic predisposition in the pathogenesis of oxalate nephrosis in cheetahs warrants further study. [ABSTRACT FROM AUTHOR]

As a result of the increase in the practice of tattooing, the US Food and Drug Administration has identified a need for improved analytical methods to detect the pigments and potential impurities in the inks. Raman spectroscopy allows for nondestructive identification of compounds and is commonly used in art, archaeology, and forensics; however, the technique has only limitedly been applied to the identification of tattoo pigments. In this study, approximately 30 inorganic, organometallic, and organic pigments were evaluated with Raman spectroscopy by using 532, 633, and 780-nm lasers. Individual optimization of the instrumental parameters was performed for each pigment in order to enhance spectral quality. This research highlights the need for multiple laser interrogation, as the spectra of some pigments were difficult to obtain by using a particular wavelength due to interferences from absorption or fluorescence. However, by using these multiple wavelengths, all pigments could be identified by their unique spectral features. A spectral library of the pigments was created for each laser wavelength and then challenged with pigments from multiple manufacturers. All pigments were identified correctly, and the method is poised to be an effective, noninvasive means for qualitatively identifying tattoo pigments. Published 2017. This article is a U.S. Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

Raman spectroscopy in combination with chemometrics was explored as a rapid, non-targeted screening method for the detection of milk powder (MP) adulteration using melamine as an example contaminant. Raman spectroscopy and an unsupervised pattern-recognition method, principal component analysis (PCA), allowed for the differentiation of authentic MPs from adulterated ones at concentrations > 1.0% for dry-blended (DB) samples and > 0.30% for wet-blended (WB) ones. Soft independent modelling of class analogy (SIMCA), a supervised pattern-recognition method, was also used to classify test samples as adulterated or authentic. Combined statistics at a 97% confidence level from the SIMCA models correctly classified adulteration of MP with melamine at concentrations ≥ 0.5% for DB samples and ≥ 0.30% for WB ones, while no false-positives from authentic MPs were found when the spectra in the 600–700 cm–1range were pre-processed using standard normal variate (SNV) followed by a gap-segment derivatisation. The combined technique of Raman spectroscopy and chemometrics proved to be a useful tool for the rapid and cost-efficient non-targeted detection of adulteration in MP at per cent spiking levels. [ABSTRACT FROM PUBLISHER]

There is a need to develop rapid tools to screen milk products for economically motivated adulteration. An understanding of the physiochemical variability within skim milk powder (SMP) and non-fat dry milk (NFDM) is the key to establishing the natural differences of these commodities prior to the development of non-targeted detection methods. This study explored the sources of variance in 71 commercial SMP and NFDM samples using Raman spectroscopy and principal component analysis (PCA) and characterised the largest number of commercial milk powders acquired from a broad number of international manufacturers. Spectral pre-processing using a gap-segment derivative transformation (gap size = 5, segment width = 9, fourth derivative) in combination with sample normalisation was necessary to reduce the fluorescence background of the milk powder samples. PC scores plots revealed no clear trends for various parameters, including day of analysis, powder type, supplier and processing temperatures, while the largest variance was due to irreproducibility in sample positioning. Significant chemical sources of variances were explained by using the spectral features in the PC loadings plots where four samples from the same manufacturer were determined to likely contain an additional component or lactose anomers, and one additional sample was identified as an outlier and likely containing an adulterant or differing quality components. The variance study discussed herein with this large, diverse set of milk powders holds promise for future use as a non-targeted screening method that could be applied to commercial milk powders. [ABSTRACT FROM AUTHOR]

Microcystins (MCs) comprise a group of cyclic heptapeptide toxins that share a common backbone and have two variable l -amino acids that yield at least 21 known analogs of varying potency. These hepatotoxins and potential tumor promoters are produced by certain cyanobacteria, including Microcystis aeruginosa . The cyanobacterium M. aeruginosa blooms in freshwater lakes and can potentially co-occur with other species such as Aphanizomenon flos-aquae , which is targeted and harvested for the production of dietary supplements known as blue-green algae (BGA). BGA supplements are currently marketed in the U.S. and internationally as a product that may elevate mood, increase energy, and alleviate attention deficit hyperactivity disorder. However, the potential for BGA dietary supplements to be contaminated with MCs is of concern, and there are currently no validated methods for detection of MCs in these products. This research focused on establishing screening methods for toxic Microcystis and MCs in BGA supplements. A DNA-based method employing polymerase chain reaction (PCR) was used as a prescreening tool to evaluate the dietary supplements and to detect the presence of toxin genes (i.e., presence of toxic Microcystis ). A rapid, sensitive surface plasmon resonance (SPR) biosensor, directed towards recognition of all MC forms, was also developed and validated. This improved SPR biosensor incorporates a commercial Adda-group antibody (Ab) that has the capacity for broader recognition of MCs than previously developed sensors for BGA supplements that rely solely on an arginine-reactive Ab and can quantitate MC levels down to 0.24 ng/mL (equivalent to 0.24 μg per gram of BGA supplement) in less than 10 min. Such a rapid, quantitative screening method may allow for further surveillance of BGA products to assist risk assessment efforts, establishment of regulatory guidance levels, and response to potential consumer complaints related to BGA products. The PCR technique and SPR biosensor may be used in concert as prescreening and screening tools, respectively or individually, thereby limiting the number of samples that must be evaluated with confirmatory methods. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX), a small molecular weight neurotoxin, is responsible for poisoning events that traditionally occur from consumption of contaminated puffer fish. Recent studies have shown a growing number of foods contaminated with TTX and a larger number of waters and associated countries where the toxin may occur. The apparent expanding prevalence of TTX supports a growing need for screening assays that can be, used to detect potentially harmful food. In the past few years, surface plasmon resonance (SPR) biosensors have been developed for rapid, robust detection of TTX; however, these assays focus on detection of unbound antibody from an inhibition reaction with the toxin. This manuscript introduces the first direct immunoassay for a seafood toxin, specifically TTX. Major advantages of this assay compared to indirect assays include increased speed of analysis, decreased use of biological reagents, and improved confidence in the detection of the toxin, along with the ability to characterize the antibody/toxin interaction. The analytical method introduced in this paper could be applied to other seafood toxins, as well as to a wide range of low molecular weight targets. [ABSTRACT FROM AUTHOR]

Abstract: The human noroviruses are the most common non-bacterial cause of gastroenteritis and are responsible for as much as 50% of all gastroenteritis outbreaks worldwide. Norovirus (NoV), a single stranded RNA virus, is highly contagious with an infectious dose of less than 100 viral particles. While techniques exist for the identification of NoV, the lack of a reliable cell culture system, NoV genetic variability, and time-consuming sample preparation steps required to isolate the virus (or its genome) prior to molecular based methods has hindered rapid virus detection. To better protect the public from virus-contaminated food and enable better detection in clinical and environmental samples, sensitive and selective methods with simple sample preparation are needed. Surface plasmon resonance (SPR) biosensors represent an emerging detection platform, and this approach has been applied to the rapid detection of foodborne small molecule toxins, protein toxins, and bacteria. This analytical technique, however, has yet to be fully investigated for rapid virus detection, especially for intact viral particles extracted from food matrices. For this study, the culturable, non-human pathogen feline calicivirus (FCV), which has similar morphology and is genetically related to NoV, was chosen as a surrogate virus for designing and evaluating an SPR assay. An antibody-based assay was performed by first immobilizing anti-FCV to an SPR chip surface and then directly measuring virus binding and subsequent secondary antibody binding. The resulting biosensor directly detected intact FCV particles with limits of detection of approximately 104 TCID50 FCV/mL from purified cell culture lysates. In addition, intact virus detection in FCV-spiked oyster matrix was possible when using a simple extraction procedure and employing a secondary antibody to FCV for quantitation. The results from these preliminary studies show promise for the development of a rapid assay for detecting intact viruses, such as NoV, using an SPR biosensor. While the current level of sensitivity achieved with this SPR biosensor may be more applicable to virus detection in clinical specimens, broader application and increased sensitivity of this method for foodborne viruses may be achieved when performed in conjunction with efficient virus extraction and concentration methods. [Copyright &y& Elsevier]

Domoic acid (DA)-producing harmful algal blooms (HABs) have been present at unprecedented geographic extent and duration in recent years causing an increase in contamination of seafood by this common environmental neurotoxin. The toxin is responsible for the neurotoxic illness, amnesic shellfish poisoning (ASP), that is characterized by gastro-intestinal distress, seizures, memory loss, and death. Established seafood safety regulatory limits of 20 μg DA/g shellfish have been relatively successful at protecting human seafood consumers from short-term high-level exposures and episodes of acute ASP. Significant concerns, however, remain regarding the potential impact of repetitive low-level or chronic DA exposure for which there are no protections. Here, we report the novel discovery of a DA-specific antibody in the serum of chronically-exposed tribal shellfish harvesters from a region where DA is commonly detected at low levels in razor clams year-round. The toxin was also detected in tribal shellfish consumers' urine samples confirming systemic DA exposure via consumption of legally-harvested razor clams. The presence of a DA-specific antibody in the serum of human shellfish consumers confirms long-term chronic DA exposure and may be useful as a diagnostic biomarker in a clinical setting. Adverse effects of chronic low-level DA exposure have been previously documented in laboratory animal studies and tribal razor clam consumers, underscoring the potential clinical impact of such a diagnostic biomarker for protecting human health. The discovery of this type of antibody response to chronic DA exposure has broader implications for other environmental neurotoxins of concern. [ABSTRACT FROM AUTHOR]

Several families of catfish species are extensively aquacultured around the world; however, only those from the family Ictaluridae can be labeled as catfish in the United States. Non-Ictalurid catfish species that are marketed as “catfish” in the USA are considered misbranded. Misbranding in general has led to an increased interest in developing deoxyribonucleic acid (DNA)-based methods such as DNA barcoding, polymerase chain reaction restriction fragment length polymorphism, and DNA microarrays with fluorescence detection for the identification of fish species. In this proof-of-concept study, DNA microarrays coupled with a newly developed mid-infrared imaging detection method were applied to the identification of seven species of catfish for the first time. Species-specific DNA probes targeting three regions per species of the cytochrome c oxidase 1 (barcoding) gene were developed and printed as microarrays on glass slides. Deoxyribonucleic acid targets labeled with biotin were hybridized to their complementary probes using a strategy that allowed the selective formation of a silver layer on hybridized spots needed for detection. Using this three-probe format, the seven species were all identified correctly, even when a limited number of false positive spots were observed. Raman spectroscopy was employed to further characterize the arrays. [ABSTRACT FROM AUTHOR]

Highlights • Novel method was developed for classifying marine oils based on their lipid matrices. • Mid-infrared was more sensitive than near-infrared for the classification of oils. • Ethyl esters in triacylglycerols were correctly classified as mixtures at 11% (w/w). • Six quantitative models were developed for major fatty acids/fatty acid classes. • Procedures show promise for verifying accuracy of marine oil label declarations. Abstract The increased demand for marine oil dietary supplements requires rapid and accurate screening to verify product quality and accuracy of label declarations. Simple and rapid procedures, based on spectra from a portable Fourier-transform infrared (FTIR) device and a benchtop FT-near infrared (FT-NIR) spectrometer combined with chemometrics, were developed to: (1) differentiate sources of omega-3 polyunsaturated fatty acids (PUFA) in their natural triacylglycerol (TAG) or concentrated ethyl ester (EE) forms, and (2) quantify six main fatty acids (FA)/FA classes. A partial least squares discriminant analysis (PLS-DA) model developed for ATR-FTIR data was successfully used to classify a large number of representative products (n = 95) as belonging to the TAG or EE classes, or as mixtures of such. Broad-based partial least squares regression (PLSR) models for each of six fatty acid (FA)/FA classes were successfully validated by cross-validation and by using an independent validation test set. For both complementary procedures, PLSR model predictions showed good linear correlations with a reference gas chromatographic method (R2 > 0.91) and excellent predictive qualities for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These simple, rapid, and nondestructive spectroscopic procedures have the potential for differentiating between natural and concentrated forms of omega-3 PUFA and verifying the accuracy of label declarations. [ABSTRACT FROM AUTHOR]

A nanoarray planar waveguide biosensor was developed for the detection of tetrodotoxin (TTX). This technique, specifically designed for the first time for TTX, provided a compact versatile user friendly device that obtained a test result in ten minutes. The device consisted of nanoprinted toxin-conjugate arrays constructed in the manner of an indirect competitive immunoassay, for the analysis of puffer fish samples under high flow conditions. The applicability to natural samples was investigated through the study of matrix effects and toxin recovery. The biosensor enabled the detection of TTX from 0.4 to 3.29 μg g −1 puffer fish tissue. The sensitivity attained demonstrates this assay as a rapid and sensitive screening method to detect TTX in different species of puffer fish, well below the Japanese maximum permitted level (2 μg g −1 ) and the estimated safety level used in the EU and the US (0.8 μg g −1 ). Assay repeatability and reproducibility were assessed at 0.4 and 0.8 μg g −1 , showing relative standard deviation (RSD) values below 15% and toxin recovery within 85–115%. The appropriate correlation of data obtained from the biosensor compared to that reported by ELISA, RBA, SPR biosensor and LC–MS/MS for the analysis of 12 puffer fish samples, proved the feasibility and reliability of this immunosensor to support monitoring programs and research activities. [ABSTRACT FROM AUTHOR]

To date most mid-infrared spectroscopic studies have been limited, due to lack of sensitivity, to the structural characterization of a single oligonucleotide probe immobilized over the entire surface of a gold-coated slide or other infrared substrate. By contrast, widely used and commercially available glass slides and a microarray spotter that prints approximately 120-μm-diameter DNA spots were employed in the present work. To our knowledge, mid-infrared chemical imaging (IRCI) in the external reflection mode has been applied in the present study for the first time to the detection of nanostructure-based DNA microarrays spotted on glass slides. Alkyl amine-modified oligonucleotide probes were immobilized on glass slides that had been prefunctionalized with succinimidyl ester groups. This molecular fluorophore-free method entailed the binding of gold-nanoparticle–streptavidin conjugates to biotinylated DNA targets. Hybridization was visualized by the silver enhancement of gold nanoparticles. The adlayer of silver, selectively bound only to hybridized spots in a microarray, formed the external reflective infrared substrate that was necessary for the detection of DNA hybridization by IRCI in the present proof-of-concept study. IRCI made it possible to discriminate between diffuse and specular external reflection modes. The promising qualitative results are presented herein, and the implications for quantitative determination of DNA microarrays are discussed. [ABSTRACT FROM AUTHOR]

Paralytic shellfish toxins (PSTs) in bivalve molluscs represent a public health risk and are controlled via compliance with a regulatory limit of 0.8 mg saxitoxin (STX)⋅2HCl equivalents per kilogram of shellfish meat (eq/kg). Shellfish industries would benefit from the use of rapid immunological screening tests for PSTs to be used for regulation, but to date none have been fully validated. An interlaboratory study involving 16 laboratories was performed to determine the suitability of the Neogen test to detect PSTs in mussels and oysters. Participants performed the standard protocol recommended by the manufacturer and a modified protocol with a conversion step to improve detection of gonyautoxin 1&4. The statistical analysis showed that the protocols had good homogeneity across all laboratories, with satisfactory repeatability, laboratory, and reproducibility variation near the regulatory level. The mean probability of detection (POD) at 0.8 mg STX⋅2HCl eq/kg using the standard protocol in mussels and oysters was 0.966 and 0.997, respectively, and 0.968 and 0.966 using the modified protocol. The estimated LOD in mussels was 0.316 mg STX⋅2HCl eq/kg with the standard and 0.682 mg STX⋅2HCl eq/kg with the modified protocol, and 0.710 and 0.734 mg STX⋅2HCl eq/kg for oysters, respectively. The Neogen test may be acceptable for regulatory purposes for oysters in accordance with European Commission directives in which the standard protocol provides, at the regulatory level, a probability of a negative response of 0.033 on 95% of occasions. Its use for mussels is less consistent at the regulatory level due to the wide prediction interval around the POD.

A label-free surface plasmon resonance biosensor method was applied to determine tetrodotoxin (TTX) in pufferfish matrixes using an antibody inhibition assay format. A prevalidation study was conducted to demonstrate the assay performance characteristics, such as selectivity, LOD, LOQ, repeatability, reproducibility, and accuracy. Three participating laboratories reported standard curves in buffer and pufferfish matrix. A set of blind samples with TTX spiked into buffer as well as in 10% pufferfish extract were analyzed. Additionally, three blind naturally contaminated samples were analyzed, and the results were compared to those obtained using a reference method (HPLC/electrospray ionization-selected reaction monitoring-MS). The developed method was demonstrated to be capable of detecting TTX in pufferfish matrix standard samples in a broad concentration range (2-9000 ng/mL) with an LOD of 1.5 ng/mL. Between-laboratory recovery values were in the range of 51-190% with a mean of 107%, and 64-180% with a mean of 103% for TTX-spiked samples in buffer and pufferfish matrix, respectively. Between-laboratory recoveries were in the satisfactory range of 101-119% for naturally contaminated samples. This robust, rapid, and noninvasive method may serve as an attractive alternative to established methods for detection of TTX in pufferfish extracts.

Tetrodotoxin (TTX) is a low molecular weight neurotoxin found in a number of animal species, including pufferfish. One emerging method for TTX detection employs surface plasmon resonance (SPR) immunosensors. SPR, an optical technique that allows for label-free, real-time, multiplexed analysis, can have detection limits that rival many of the conventional transduction methods. Preliminary SPR approaches for TTX were successful, yet suffered from low throughput and used noncommercial instrumentation. To advance this method for broader use, the immunoassay was transferred to a commercial instrument and optimized for improved detection. This manuscript outlines the assay development and results for complex matrices relevant to seafood safety (pufferfish) and food adulteration (milk, apple juice). In addition, results are compared to those obtained using receptor binding assay, ELISA, HPLC-FD, and LC/MS/MS detection techniques. Results highlight the advantages of SPR assays, including rapid screening capability with low reagent consumption and low- to subppb detection limits.

Tetrodotoxin (TTX) is a low molecular weight (approximately 319 Da) neurotoxin found in a number of animal species, including pufferfish. Protection from toxin tainted food stuffs requires rapid, sensitive, and specific diagnostic tests. An emerging technique for the detection of both proteins and nucleic acids is Fluidic Force Discrimination (FFD) assays. This simple and rapid method typically uses a sandwich immunoassay format labeled with micrometer-diameter beads and has the novel capability of removing nonspecifically attached beads under controlled, fluidic conditions. This technique allows for near real-time, multiplexed analysis at levels of detection that exceed many of the conventional transduction methods (e.g., ELISAs). In addition, the large linear dynamic range afforded by FFD should decrease the need to perform multiple sample dilutions, a common challenge for food testing. By applying FFD assays to an inhibition immunoassay platform specific for TTX and transduction via low magnification microscopy, levels of detection of approximately 15 ng/mL and linear dynamic ranges of 4 to 5 orders of magnitude were achieved. The results from these studies on the first small molecule FFD assay, along with the impact to detection of seafood toxins, will be discussed in this manuscript.

A sandwich immunoassay for the rapid, low-level detection of Mycobacterium avium subsp. paratuberculosis has been developed. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and one of the major obstacles in controlling the spread of this disease is the inability to rapidly detect small amounts of bacteria or other diagnostic markers shed during the subclinical stage of infection. This paper details the development and performance of an assay for sonicated M. avium subsp. paratuberculosis lysate that is based on surface-enhanced Raman scattering (SERS). There are two key components of the assay: (i) an immobilized layer of monoclonal antibodies that target a surface protein on the microorganism; and (ii) extrinsic Raman labels (ERLs) that are designed to selectively bind to captured proteins and produce large SERS signals. By correlating the number of M. avium subsp. paratuberculosis bacilli present prior to sonication to the amount of total protein in the resulting sonicate, the detection limit determined for total protein can be translated to the microorganism concentration. These findings yield detection limits of 100 and 200 ng/ml (estimated to be 500 and 1,000 M. avium subsp. paratuberculosis bacilli/ml) for sonicate spiked in phosphate buffer and sonicate spiked in whole milk, respectively. Moreover, the time required to complete the assay, which includes sample preparation, antigen extraction, ERL incubation, and readout, is less than 24 h. The potential for incorporation of this novel assay into diagnostic laboratories is also briefly discussed.

The etiological agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis. Controlling the spread of this disease is hindered by the lack of sensitive, selective, and rapid detection methods for M. avium subsp. paratuberculosis. By using a recently optimized sandwich immunoassay (B. J. Yakes, R. J. Lipert, J. P. Bannantine, and M. D. Porter, Clin. Vaccine Immunol. 15:227-234, 2008), which incorporates a new monoclonal antibody for the selective capture and labeling of M. avium subsp. paratuberculosis and surface-enhanced Raman scattering for sensitive readout, detection limits of approximately 630 and approximately 740 M. avium subsp. paratuberculosis cells/ml are achieved in phosphate-buffered saline and whole milk samples, respectively, after spiking with heat-treated M. avium subsp. paratuberculosis. Surprisingly, these detection limits are 3 orders of magnitude lower than expected based on theoretical predictions. Experiments designed to determine the origin of the improvement revealed that the major membrane protein targeted by the monoclonal antibody was present in the sample suspensions as shed protein. This finding indicates that the capture and labeling of shed protein function as a facile amplification strategy for lowering the limit of detection for M. avium subsp. paratuberculosis that may also be applicable to the design of a wide range of highly sensitive assays for other cells and viruses.