Filling Analytical Gaps in Allergen Detection─Real-Time PCR for the Detection of Commercially Relevant Cephalopods and Gastropods in Food.

Mollusks belong to the group of shellfish, which are considered to be among the elicitors of severe food allergies worldwide. In recent years, numerous PCR detection methods have been developed for other shellfish such as crustaceans. However, cephalopods and gastropods were not considered in the development of these shellfish detection systems. In this study, we have developed highly specific real-time PCR methods for the comprehensive detection of all commercially relevant cephalopod species and the gastropod families Helicidae, Buccinidae, and Muricidae in food matrices. In total, we cross-tested over 100 animal and plant species to show the specificity of our systems. The limit of detection (LOD12) was set at 1 pg of cephalopod and gastropod DNA or 10 ppm (mg/kg) spiked in a vegetarian food product. The robustness of the protocol was confirmed by testing multiple parameters while cooking and autoclaving of samples ensured the practical applicability of the systems.

Application of Mass Spectrometry-Based Immunoassays for the Species- and Tissue-Specific Quantification of Banned Processed Animal Proteins in Feeds.

Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of nonruminant PAPs for use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, because the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here, we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides, and LC-MS/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of six ring trial samples obtained in former PCR and microscopy proficiency tests. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.

Insects in food and feed - allergenicity risk assessment and analytical detection.

Insects and insect-based food products have entered in the European market, carrying along issues of safety and the need of establishing a new legal framework. The consumption of massively reared insects can pose chemical and microbiological risks, and insect proteins are likely to represent a hazard for a subpopulation of allergic individuals. All insect-based products are considered 'Novel Food' and fall under EU regulation 2015/2283, according to which a specific application to the European Commission, followed by EFSA scientific evaluation, is needed before the product is put on the market. The recent EU Regulation 2017/893, entered into force on 1 July 2017, allowed a shortlist of seven insect species to be included in the formulation of feeds for aquaculture. Previously, the addition of any insect to any feed for farmed animals was not allowed, due to the risk of prion-derived diseases. The introduction of this new Regulation raises the issue to switch from a classical detection method based on microscopy to a more sophisticated and species-specific method. The overall aims of this EU-FORA project were (i) to set up a new next generation sequencing (NGS)-based molecular method for the identification of insect DNA in feeds for aquaculture; and (ii) to carry out a conceptual work on a probabilistic quantitative risk assessment focused on the allergenicity of yellow mealworm (Tenebrio molitor) employed in foods.

Species Differentiation and Quantification of Processed Animal Proteins and Blood Products in Fish Feed Using an 8-Plex Mass Spectrometry-Based Immunoassay.

With the reintroduction of nonruminant processed animal proteins (PAPs) for use in aquaculture in 2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date, light microscopy and polymerase chain reaction are the official methods for proving the absence of illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species differentiation are needed. To address this issue, we developed and validated an 8-plex mass spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-macroglobulin peptides using a group-specific antibody, and a subsequent analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry for species identification and quantification. This workflow can be used to quantitatively determine the species origin in future feed authentication studies.

Mass Spectrometry-Based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds.

The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR) are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of nonruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides, and a LC-MS/MS-based analysis for peptide quantification using isotope labeled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of 4 orders of magnitude with a detection limit in the range of 0.1% to 1% (w/w).

Determination of osteocalcin in meat and bone meal of bovine and porcine origin using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and high-resolution hybrid mass spectrometry.

A method has been developed for determining the origin of meat and bone meal (MBM) by detecting species-specific osteocalcin (OC) using matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) and high-resolution hybrid mass spectrometry (HR-Q/TOF MS). The analysis is based on the detection of typical species-specific OC and its tryptic peptide fragments which differ in mass due to differences in the amino-acid sequences between species. After dissolving the MBM samples in EDTA buffer, purification after ultrafiltration was performed using two methods: solid-phase extraction using Zip-Tip C(18) or size exclusion coupled with reverse-phase chromatography. Fractions containing partially purified intact OC were analyzed using LC-Q/TOF and MALDI/TOF mass spectrometry. Species-specific OC was detected at the typical protonated and doubly protonated molecular ions. Furthermore, typical porcine- and bovine-derived tryptic fragments from MBM were detected after enzymatic digestion. In order to determine the underlying amino-acid sequences and to confirm the assignment to OC-derived peptides, MS/MS analysis was carried out. In conclusion, we were able to detect OC in bovine and porcine MBM with high sensitivity and the MS-based method described here by which total OC mass and marker peptides of digested OC are recorded can be used as an alternative approach to detect genus-specific differences in MBM and can be applied as a confirmatory method to mainly immunological osteocalcin screening methods.

Gene transcription analysis of carrot allergens by relative quantification with single and duplex reverse transcription real-time PCR.

Single and duplex real-time polymerase chain reaction (PCR) systems have been developed to quantify specific mRNA transcription of genes coding for the major Daucus carota allergen isoforms Dau c 1.01 and Dau c 1.02. Methods were tested with samples from the local market. Whereas the gene transcription levels for Dau c 1.01 were consistently high in all investigated samples, significant differences for the Dau c 1.02 transcription could be demonstrated in randomly collected market samples. The gene transcription level for the minor Dau c 1.02 variant is about one log below Dau c 1.01. Both formats, single or duplex real-time methods, exhibit ideal cycle threshold (CT) ranges and good reproducibility. In particular, the easily performed duplex real-time PCR system is potentially suited for the selection of hypoallergenic varieties and studying the impact of post-harvesting or environmental conditions.

A novel approach for the detection of DNA using immobilized peptide nucleic acid (PNA) probes and signal enhancement by real-time immuno-polymerase chain reaction (RT-iPCR).

A new approach for the detection of DNA target molecules is described, using capture probes and subsequent signal enhancement by a uniform polymerase chain reaction (PCR). Peptide nucleic acid probes were immobilized in real-time PCR-compatible microtiter plates. After hybridization of biotinylated DNA targets, detection was performed by real-time immuno-PCR, a method formerly used for protein detection. We demonstrate the feasibility of this strategy for the qualitative detection of DNA oligonucleotides with a detection limit (LOD) of 6 attomol. Furthermore, the method was applied to PCR-amplified samples from genetically modified maize DNA (Mon810). A 483-bp DNA fragment was detected in mixture with 99.9% of noncomplementary DNA with a sensitivity down to the level of attomole.