Bovine blood biomarkers as a way of processed animal proteins detection in feedingstuffs

peer reviewedThe prohibition of using animal by-products in feedingstuffs depends on two factors: their nature defined by the tissue/cell type and the species of origin, and on their destination (pets, fur animals or other farmed animals). Proteomics is particularly well-suited to the purpose of PAPs detection as it is a tissue and species-specific method. The aim of this study was the identification and the selection of specific peptide biomarkers using tandem mass spectrometry for the detection of bovine blood products and blood meals in animal feed. Twenty-nine samples of blood meals and blood products (plasma or haemoglobin powder) of porcine, poultry and bovine origin as well as three milk products and two fish meals were analysed using a Q TOF mass spectrometer. Vegetal feed samples adulterated with 1% or 10% of bovine plasma powder, haemoglobin powder or blood meal were also analysed to evaluate the applicability of the method. Four proteins of interest were highlighted: Alpha-2-macroglobulin, apolipoprotein A-1, serotransferrin and haemoglobin (α and β chains). From these proteins, sixteen peptides were identified as potential bovine blood biomarkers in feedingstuffs. Nine of them could be used for the detection of plasma powder and seven of them for haemoglobin powder or blood meal. The evaluation of these peptides by a search against NCBInr database revealed that some of them could also be used to detect other ruminant bloods such as ovine or caprine ones. These preliminary results are promising. Efforts are now focused to improve the protocol in order to increase the sensitivity of the method as regards the selected proteins

Guidelines for sample preparation procedures in GMO analysis

This document aims at providing guidelines for a correct sample preparation in GMO analysis on food, feed, seed, plants and propagating material samples. It has been developed by the European Network of GMO Laboratories (ENGL), specifically by the ENGL working group “sample preparation procedures” upon a mandate of the ENGL steering committee. The structure of the document is based on that of an existing ISO standard (ISO 6498:2012), dealing with sample preparation for feed; starting from this, the procedures were adapted to the needs of GMO detection and the scope concerning matrices was widen to food, feed and seeds. These guidelines cover the key steps of the sample preparation, i.e. the size of the laboratory sample, the mass reduction techniques as well as techniques for comminution and mixing; considerations about the test portion are included and some performance tests to be applied at different steps of the process are provided. While sample preparation, falling after sampling and before analytical testing for GMOs, has suffered from a lack of guidance and harmonization, this document provides detailed and practical hints on this important step, thus contributing to foster testing harmonisation.JRC.I.3-Molecular Biology and Genomic

Kernel Lot Distribution Assessment (KeLDA): a Comparative Study of Protein and DNA-Based Detection Methods for GMO Testing

Monitoring of market products for detection of genetically modified organisms (GMO) is needed to comply with legislation in force in many regions of the world, to enforce traceability and to allow official control along the production and the distribution chains. This objective can be more easily achieved if reliable, time and cost-effective analytical methods are available. A GMO can be detected using either DNA-based or protein-based methods; both present advantages and disadvantages. The objective of this work was to assess the performance of a protein-based (lateral flow strips—LFT) and of a DNA-based (polymerase chain reaction—PCR) detection method for GMO analysis. One thousand five hundred samples of soybean, deriving from the sampling of 15 independent bulk lots in large shipments, were analysed to assess and compare the performance of the analytical methods and evaluate their suitability for GMO testing. Several indicators were used to compare the performance of the methods, including the percentage correlation between the PCR and LFT results. The GMO content of the samples ranged from 0 up to 100 %, allowing a full assessment of both analytical approaches with respect to all possible GMO content scenarios. The study revealed a very similar performance of the two methodologies, with low false-negative and false-positive results, and a very satisfactory capacity of both methods in detecting low amounts of target. While determining the fitness for purpose of both analytical approaches, this study also underlines the importance of alternative method characteristics, like costs and time.JRC.I.3-Molecular Biology and Genomic

Kernel Lot Distribution Assessment (KeLDA): a Comparative Study of Protein and DNA-Based Detection Methods for GMO Testing

Monitoring of market products for detection of genetically modified organisms (GMO) is needed to comply with legislation in force in many regions of the world, to enforce traceability and to allow official control along the production and the distribution chains. This objective can be more easily achieved if reliable, time and cost-effective analytical methods are available. A GMO can be detected using either DNA-based or protein-based methods; both present advantages and disadvantages. The objective of this work was to assess the performance of a protein-based (lateral flow strips—LFT) and of a DNA-based (polymerase chain reaction—PCR) detection method for GMO analysis. One thousand five hundred samples of soybean, deriving from the sampling of 15 independent bulk lots in large shipments, were analysed to assess and compare the performance of the analytical methods and evaluate their suitability for GMO testing. Several indicators were used to compare the performance of the methods, including the percentage correlation between the PCR and LFT results. The GMO content of the samples ranged from 0 up to 100 %, allowing a full assessment of both analytical approaches with respect to all possible GMO content scenarios. The study revealed a very similar performance of the two methodologies, with low false-negative and false-positive results, and a very satisfactory capacity of both methods in detecting low amounts of target. While determining the fitness for purpose of both analytical approaches, this study also underlines the importance of alternative method characteristics, like costs and time

A PCR-microarray method for the screening of genetically modified organisms

A new method to screen and to identify genetically modified organisms (GMO) is presented in this paper. It is based on the detection of multiple genetic elements common to GMO by their amplification via PCR followed by direct hybridisation of the amplicons on microarray. The pattern of the elements is then compared to a database of the composition of EU-approved GMO and an identification of the GMO is then proposed. The limit of detection of the method was ≤0.1% GMO content (w/w) expressed as the amount of target DNA present in the template for single unprocessed material. The DNA targets were detected both in reference materials and in mixtures with the same detection limit. The specificity for the detection of the different elements was found to be very good with no cross-reaction even in samples with two GMO present at different concentrations. The paper presents examples of GMO identification and discusses the potential and limitation of such approaches and how they can facilitate the work of private and enforcement detection laboratories.This study was supported by the European Commission through the FP5 program “GMOchips” (contract G6RD-CT2000-00419 2000-2003), the Belgian SSCT program and the Integrated Project “Co-Extra”, contract no. 007158 2005-2009, under the 6th Framework Programme, priority 5, food quality and safety.Peer reviewe

Overview and recommendations for the application of digital PCR

The digital Polymerase Chain Reaction (dPCR), for the detection and absolute quantification of DNA, is a relatively new technique but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified without the need for standard curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picoliters. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or these compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Common to both approaches, once PCR has been carried out simultaneously in all compartments/droplets, the number of positive and negative signals for each partition is counted by fluorescence measurement. With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that can be co-extracted during DNA extraction from different sources. Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human diseases, food viruses and bacteria including pathogens. When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format.JRC.F.5-Food and Feed Complianc

Developing a scientific journal in a changing world

After a brief historical overview, this article presents the current situation of the journal BASE, its objectives, its challenges, its functioning and its limitations. BASE is a journal that publishes articles in open access in the field of agricultural sciences in the broad sense and offers a free service to authors. The editorial board pays particular attention to the transparency of its validation and editing processes. The data presented allow BASE to be compared with other international scientific journals. At the end of the article, after outlining some of the difficulties encountered, several prospects are described that should address the disadvantages arising from the multidisciplinary nature of BASE

Developing a scientific journal in a changing world

After a brief historical review, this article presents the current situation of the journal BASE, its objectives, its challenges, its functioning and its limitations. BASE is a journal that publishes articles in Open Access in the field of agronomic sciences in the broad sense and offers a free service to authors. The editorial board pays particular attention to the transparency of its validation and editing processes. The figures presented allow BASE to find its place alongside all international scientific journals. At the end of the article, after describing the difficulties encountered, several perspectives are described. These should address the possible disadvantages of the multidisciplinary nature of BASE

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application