Detection of Alphitobius diaperinus by Real-Time Polymerase Chain Reaction With a Single-Copy Gene Target.

Use of edible insects as an alternative source of proteins in food and feed is increasing. These last years, numerous companies in Europe have started producing insects for food and feed purposes. In the European Union, the use of edible insects for human consumption falls within Regulation (EU) No. 2015/2283 on novel foods. For feed, Commission Regulation (EU) 2017/893 authorizes seven insect species as processed animal proteins for aquaculture. Methods of authentication are required to check the conformity of the products. In this study, we propose a real-time polymerase chain reaction (PCR) method for the specific detection of the lesser mealworm (Alphitobius diaperinus), one of the species included in the shortlist of authorized insects. The selected target is the cadherin gene with a single-copy (per haploid genome) illustrated by our experimental evidence. The PCR test amplified a 134-bp fragment of the cadherin gene. The qualitative method was assessed toward several performance criteria. Specificity was checked against 54 insect species next to other animal and plant species. The sensitivity, efficiency, robustness, and transferability of the PCR assay were also successfully tested. Finally, the applicability of the test was assessed on real-life processed samples (industrial meals) of A. diaperinus. The study also showed that there seems to be a huge confusion on the correct labeling of the marketed mealworms. We did not succeed to get Alphitobius laevigatus samples. They all appeared to belong to the A. diaperinus taxon.

Official Feed Control Linked to the Detection of Animal Byproducts: Past, Present, and Future.

In the context of the expansion of the human population, availability of food, and in extension of animal feed, is a big issue. Favoring a circular economy by the valorization of byproducts is a sustainable way to be more efficient. Animal byproducts are an interesting source of feed materials due to their richness in proteins of high nutritional value. Prevention and control efforts have allowed a gradual lifting of the feed ban regarding the use of animal byproducts. Nevertheless, the challenge remains the development of analytical methods enabling a distinction between authorized and unauthorized feed materials. This Review focuses on the historical and epidemiological context of the official control, the evaluation of current and foreseen legislation, and the available methods of analysis for the detection of constituents of animal origin in feedingstuffs. It also underlines the analytical limitations of the approach and discusses some prospects of novel methods to ensure food and feed safety.

Detection of ornamental transgenic fish by real-time PCR and fluorescence microscopy.

Numbers of ornamental transgenic fish are increasing, and some of them are illegally imported into Europe. The fish are modified to display different fluorescent colors under UV light. In this article, we propose real-time PCR methods to detect fish presenting green, yellow and red fluorescent coloring. The methods were tested with success and were able to detect illegally imported fish in two European countries. The article also discusses some practical information that can be useful for routine analysis. In addition, a real-time PCR test able to highlight the presence of fish DNA in general is proposed to check the amplifiability of the DNA extracted from common bony fish species of the teleost subclass. Finally, as the testing by PCR can take several days and rapid decisions must be taken with living organisms, we explored the potential of fluorescence microscopy as a screening test to determine whether animals are suspect or can be released.

Detection and identification of transgenic events by next generation sequencing combined with enrichment technologies.

Next generation sequencing (NGS) is a promising tool for analysing the quality and safety of food and feed products. The detection and identification of genetically modified organisms (GMOs) is complex, as the diversity of transgenic events and types of structural elements introduced in plants continue to increase. In this paper, we show how a strategy that combines enrichment technologies with NGS can be used to detect a large panel of structural elements and partially or completely reconstruct the new sequence inserted into the plant genome in a single analysis, even at low GMO percentages. The strategy of enriching sequences of interest makes the approach applicable even to mixed products, which was not possible before due to insufficient coverage of the different genomes present. This approach is also the first step towards a more complete characterisation of agrifood products in a single analysis.

Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

Development of real-time PCR tests for the detection of Tenebrio molitor in food and feed.

Insects are rich in proteins and could be an alternative source of proteins to feed animals and humans. Numerous companies have started the production of insects for feed purposes. In Europe, these processed animal proteins are not yet authorised by legislation as many questions still need to be answered concerning this 'novel food'. Authorisations will be possible when methods of authentication of the products are available. In this study we propose real-time PCR methods for the specific detection of the mealworm (Tenebriomolitor), one of the most widely used insects for food and feed production. Two PCR assays are proposed: the first based on the wingless gene and the second based on the cadherin gene. The PCR tests amplify fragments of 87 bp. These qualitative methods were tested according to several performance criteria. The specificity was tested on 34 insect species' DNA, but also on non-insect species including crustacean, mammals, birds and plants. The limit of detection was determined and was below 20 copies for the two PCR tests. The applicability of the tests was demonstrated by the analysis of real-life processed samples containing T. molitor.

Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs.

The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

Collaborative study on the effect of grinding on the detection of bones from processed animal proteins in feed by light microscopy.

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

The GMOseek matrix: a decision support tool for optimizing the detection of genetically modified plants.

BACKGROUND: Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs' molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information. DESCRIPTION: The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available. CONCLUSIONS: The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms.

Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Evaluation of different machines used to quantify genetic modification by real-time PCR.

Quantification of genetic modification (GM) is often undertaken to test for compliance with the European Union GM labeling threshold in food. Different control laboratories will often use common validated methods, but with different models of real-time PCR machines. We performed two separate ring trials to evaluate the relative precision and accuracy of different types of real-time PCR machines used to quantify the concentration of GM maize. Both trials used dual-labeled fluorogenic probes for quantification. The first ring trial used separate GM and reference assays (a single fluorescence channel), and the second used a combined duplex assay (two simultaneous fluorescence channels). Five manufacturers and seven models--including a 96-well microtiter-plate, rotary, and portable machines--were examined. In one trial, the machine used had a significant effect on precision, but in the other it did not. Overall, the degree of variation due to the machine model was lower than other factors. No significant repeatable difference in accuracy was observed between machine models. It was not possible to use sufficient replication of machine type in each laboratory to examine all sources of variation in this study, but the results strongly indicate that factors other than machine type or manufacturer (e.g., method or laboratory) contribute more to variation in a GM quantification result.

Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects.

Five double-target multiplex plasmids to be used as calibrants for GMO quantification were constructed. They were composed of two modified targets associated in tandem in the same plasmid: (1) a part of the soybean lectin gene and (2) a part of the transgenic construction of the GTS40-3-2 event. Modifications were performed in such a way that each target could be amplified with the same primers as those for the original target from which they were derived but such that each was specifically detected with an appropriate probe. Sequence modifications were done to keep the parameters of the new target as similar as possible to those of its original sequence. The plasmids were designed to be used either in separate reactions or in multiplex reactions. Evidence is given that with each of the five different plasmids used in separate wells as a calibrant for a different copy number, a calibration curve can be built. When the targets were amplified together (in multiplex) and at different concentrations inside the same well, the calibration curves showed that there was a competition effect between the targets and this limits the range of copy numbers for calibration over a maximum of 2 orders of magnitude. Another possible application of multiplex plasmids is discussed.

Novel approach for interlaboratory transfer of real-time PCR methods: detecting bovine meat and bone meal in feed.

The availability of robust methods for the species-specific detection of meat and bone meal (MBM) in compound feedingstuffs is an important prerequisite to enforce current and upcoming European legislation on the use of processed animal proteins in animal nutrition. Among possible methods, those based on DNA turned out to be a reliable tool for this aim, since DNA is a quite thermostable molecule able to resist severe heat treatments applied in the manufacturing of animal meals. The application of such methods by control laboratories implies that the method has been validated including an assessment of its robustness. Successful transferability between laboratories is considered an important robustness criterion of the method. However, corresponding guidelines regarding the design of such a study relevant to this field are missing. Here, we demonstrate the feasibility of an alternative concept that was applied to check for the transferability of a qualitative assay for the detection of banned MBM in feedingstuffs at trace level based on real-time PCR. The concept was based on an experimental nested design applying analysis of variance (ANOVA) that was conducted independently in two laboratories and which allows for establishing major factors influencing the result of analysis. Statistical assessment of the results confirmed the importance of the DNA extraction/purification step utilised, whereas the PCR step turned out to be a minor factor regarding the overall variability of the results. Furthermore, blind samples comprised of compound feed adulterated with MBM at 0.1% and blank compound feed were correctly classified as "positive" or "negative" samples, thus confirming fitness of purpose for the method. This approach can be of interest for other research groups working in the development of real-time PCR methods and in their use by control laboratories.

Identification of lactic acid bacteria within the consortium of a kefir grain by sequencing 16S rDNA variable regions.

The microflora of a kefir grain was identified using a polymerase chain reaction-based strategy combined with 16S rRNA gene sequencing. DNA was extracted from the kefir grain and amplified in its 16S rDNA V1 and V2 regions. To guarantee a good representation of the overall lactic acid bacteria populations, DNA amplification was performed separately with primers specific either to the dominant or to the less abundant bacterial groups. The amplified fragments were cloned in Escherichia coli and then sequenced. Sequences of the V1 region were gathered into 5 groups of similarity and identified by aligning with the sequences of a public library. The V1 region allowed the identification of Lactobacillus kefiranofaciens, L. kefir, L. parakefir, and Lactococcus lactis but was inappropriate for the identification of leuconostocs at species level. Among 16S rDNA variable regions, the V2 region showed the highest variability between Leuconostoc species. Nevertheless, even in the V2 region, differences were too tenuous for effective identification of L. mesenteroides. The methodology described here allowed detection of the dominant species within each targeted microbial group.

Detection of ruminant meat and bone meals in animal feed by real-time polymerase chain reaction: result of an interlaboratory study.

The commercialization of animal feeds infected by prions proved to be the main cause of transmission of bovine spongiform encephalopathy (BSE). Therefore, feed bans were enforced, initially for ruminant feeds, and later for all feeds for farmed animals. The development and validation of analytical methods for the species-specific detection of animal proteins in animal feed has been indicated in the TSE (Transmissible Spongiform Encephalopathies) Roadmap (European Commission. The TSE (Transmissible Spongiform Encephalopathy) roadmap. URL: http://europa.eu.int/comm/food/food/biosafety/bse/roadmap_en.pdf, 2005) as the main condition for lifting the extended feed ban. Methods based on polymerase chain reaction (PCR) seem to be a promising solution for this aim. The main objective of this study was to determine the applicability of four different real-time PCR methods, developed by three National expert laboratories from the European Union (EU), for the detection and identification of cattle or ruminant species in typical compound feeds, fortified with meat and bone meals (MBM) from different animal species at different concentration levels. The MBM samples utilized in this study have been treated using the sterilization condition mandatory within the European Union (steam pressure sterilization at 133 degrees C, 3 bar, and 20 min), which is an additional challenge to the PCR methods evaluated in this study. The results indicate that the three labs applying their PCR methods were able to detect 0.1% of cattle MBM, either alone or in mixtures with different materials such as fishmeal, which demonstrates the improvement made by this technique, especially when compared with results from former interlaboratory studies.

Effective PCR detection of animal species in highly processed animal byproducts and compound feeds.

In this paper we present a polymerase chain reaction (PCR)-based method for detecting meat and bone meal (MBM) in compound feedingstuffs. By choosing adequate DNA targets from an appropriate localisation in the genome, the real-time PCR method developed here proved to be robust to severe heat treatment of the MBM, showing high sensitivity in the detection of MBM. The method developed here permits the specific detection of processed pig and cattle materials treated at 134 degrees C in various feed matrices down to a limit of detection of about 0.1%. This technique has also been successfully applied to well-characterised MBM samples heated to as high as 141 degrees C, as well as to various blind feed samples with very low MBM contents. Finally, the method also passed several official European ring trials.

Quantitative determination of Roundup Ready soybean (Glycine max) extracted from highly processed flour.

Roundup Ready soybean powder has been subjected to different amounts of DNA fragmentation to assess the accuracy of real-time PCR on processed food. Certified reference material (CRM) containing 10 g kg(-1) of Roundup Ready soybean (ERM-BF410d) prepared by a dry-mixing processing method was exposed to water at two temperatures, using three different mixing devices, or to baking temperature (250 degrees C) for 30 min. The amount of DNA extracted from the different samples was quantified by fluorimetry. The amount of fragmentation of the extracted DNA was characterised by gel and capillary electrophoresis and the percentage of genetically modified (GM) soybean was determined by a double quantitative real-time PCR method. Measurement of the event GTS 40-3-2 (RUR) was possible in all the treated materials, because small amplicons were amplified. Correct RUR percentages could be measured for intact powders with little or no DNA fragmentation. For samples with a high level of DNA degradation, however, the accuracy of the measurement was found to depend on the method used for DNA extraction. Genomic DNA isolated by use of silica resin resulted in statistically significant overestimation of the amount of GM.

Discriminating animal fats and their origins: assessing the potentials of Fourier transform infrared spectroscopy, gas chromatography, immunoassay and polymerase chain reaction techniques.

The objective of the reported study was to assess the abilities of various methods to differentiate the sources of fats used in feedstuff formulations. The main target was the identification of tallow (ruminant fat) and its differentiation from non-ruminant fats. Four different techniques were compared in terms of their suitability for enforcing existing and upcoming legislation on animal by-products: (1) Fourier transform infrared spectroscopy (FT-IR) applied to fat samples, (2) gas chromatography coupled with mass spectrometry (GC-MS) to determine fatty acid profiles, (3) immunoassays focusing on the protein fraction included in the fat, and (4) polymerase chain reaction (PCR) for the detection of bovine-specific DNA. Samples of the different fats and oils as well as mixtures of these fats were probed using these analytical methods. FT-IR and GC-MS differentiated pure fat samples quite well but showed limited ability to identify the animal species or even the animal class the fat(s) belonged to; no single compound or spectral signal that could permit species identification could be found. However, immunoassays and PCR were both able to identify the species or groups of species that the fats originated from, and they were the only techniques able to identify low concentrations of tallow in a mixture of fats prepared by the rendering industry, even when the samples had been sterilised at temperatures >133 degrees C. Fats used in animal nutrition come mainly from the rendering industry, thereby confirming the suitability of PCR and immunoassays for their identification. However, neither of these latter techniques was able to detect "premier jus" tallow, representing the highest quality standard of fat with extremely low protein concentration.

Real-time polymerase chain reaction approach for quantitation of ruminant-specific DNA to indicate a correlation between DNA amount and meat and bone meal heat treatments.

The use of ruminant-derived proteins in ruminant feeds has been banned in both the European Union and the United States to prevent further spread of bovine spongiform encephalopathy. Enforcement of these regulations relies on the ability to identify the presence of prohibited proteins in feed. We developed a quantitative real-time polymerase chain reaction assay for the quantification of ruminant-specific DNA as index of protein content. The assay is based on the amplification of a 117 base pair mitochondrial 16S rRNA DNA gene fragment and an internal positive control (IPC). The use of an IPC permits compensation for differences in DNA extraction efficiency and avoids the occurrence of false-negative results. We demonstrated a decrease in target DNA amount with a difference of 2 logs between meat and bone meal (MBM) treated at 133 degrees and 145 degrees C. Such a difference indicates that bias could occur when DNA-based methods are used for quantitation purposes. Risk management could benefit from future efforts concerning validation of the method for MBM detection in feedstuff and safety evaluation of the use of animal-derived proteins in animal nutrition.

Determination of processed animal proteins, including meat and bone meal, in animal feed.

An intercomparison study was conducted to determine the presence of processed animal proteins (PAPs), including meat and bone meal (MBM) from various species, in animal feed. The performances of different methods, such as microscopy, polymerase chain reaction (PCR), immunoassays, and a protocol based on iquid chromatography (LC), were compared. Laboratories were asked to analyze for PAPs from all terrestrial animals and fish (total PAPs); mammalian PAPs; ruminant PAPs; and porcine PAPs. They were free to use their method of choice. In addition, laboratories using microscopy were asked to determine the presence of PAPs from terrestrial animals, which is applicable only to microscopy. For total PAPs microscopy, LC and some immunoassays showed sufficient results at a concentration as low as 0.1% MBM in the feed. In contrast, PCR was not fit for purpose. In differentiating between MBM from terrestrial animals and fishmeal, microscopy detected 0.5% of terrestrial MBM in feed in the presence of 5% fishmeal, but was less successful when the concentration of MBM from terrestrial animals was 0.1%. The animal-specific determination of MBM from mammals or, more specifically from either ruminants or pigs, by PCR showed poor results, as indicated by a high number of false-positive and false-negative results. The only PCR method that scored quite well was applied by a member of the organizer team of the study. Immunoassays scored much better than PCR, showing sufficient sensitivity but some deficiency in terms of specificity. The results also demonstrated that the reliable determination of MBM from ruminants has not been resolved, especially for low concentrations of MBM (0.1%) in feed. Comparison of the results for mammalian MBM from all methods indicated that, for control purposes, the immunoassay method, especially when applied as dipsticks, could be used as a rapid screening method combined with microscopy to confirm the positive samples. However, implementation of such a system would require that the immunoassays were previously validated to demonstrate that this approach is fit for purpose. The determination of ruminant or porcine PAPs by immunoassays was more difficult, partly because the MBM in this study contained about 50% bovine and porcine material, thereby reducing the target concentration level to 0.05%.