Development and validation of the modular Feed-code method for qualitative and quantitative determination of feed botanical composition

Abstract

The analysis of feed composition in terms of ingredients is addressed by Regulation (EC) 767/2009 and is important for detecting economic fraud and for monitoring feed safety. Within the framework of the EU project Feed-code, we developed and internally validated a modular assay, relying on intron polymorphism, for the complete qualitative analysis of the botanical composition of feed and the quantitative determination of six target plant species. Main performance parameters of each module, such as applicability, repeatability, specificity, and limit of detection, were evaluated. The whole assay was applied to a set of feed-like samples and results were in agreement with the expected composition. Application to a large set of compound feed and individual raw materials revealed the occurrence of botanical impurities. When compared with microscopic analysis, the proposed method gave more reliable results. We conclude that the Feed-code prototype, readily upgradable to include more plant species, is worthy of consideration for a full validation through a collaborative trial. Graphical Abstract The modular Feed-code method for the authentication of feed botanical composition.

A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples

“…On general terms and with reference to the species studied, noncompliance to the declared composition was found for almost all feed samples, independently of the methodology used (end-point PCR/ Taqman assays). Unconformities to the label were also detected using TBP methodology applied to feed analysis [ 11 ]. Errors or counterfeits related either to missing ingredients, declared in the label but actually absent (flax in CPR-X10 and sunflower in CPR-X13) or, most often, to the presence of non-declared species.…”

“…The Feed-Code project, “Animal feed certification instrument and procedure to guarantee the quality of meat and dairy products through automatic, simple and rapid DNA barcode method based on tubulin-based polymorphism (TBP)", financed under the EU FP7 Capacities program, took up the challenge and worked out an effective control system that can properly address the issues raised by the different EC regulations reported above. The TBP (Tubulin-Based-Polymorphism) molecular marker is applied to feed analysis resulting in an easy and efficient DNA profiling of the different botanical species that are actually present in any given feed sample [ 11 ]. Here it is shown that AOX-based methodology, initially proposed for validation of the TBP method in the framework of the Feed-Code project, defines an additional suitable molecular marker approach to ascertain plant species composition of animal feed samples.…”

“…Efficient methodologies for species authentication in processed foods and feeds, following different molecular approaches have been developed in the last few years [ 8 ]. However, very few reports, relying on both end-point and Real-time PCR, describe the use of those techniques to detect plant species in feed samples [ 9 – 11 ]. Real-time PCR instrumentation requires considerably short hands-on time, and detection of amplified products is automated, simple and reproducible [ 12 ].…”

“…When combined with the chemistry of TaqMan MGB probes, this technique represents the most specific and sensitive detection system if few target DNA is present. Braglia and co-authors [ 11 ] reported the most accurate known method that allowed plant species detection in feed samples, based on species-specific TaqMan MGB assays designed on Tubulin gene family members. This method, called Tubulin-Based-Polymorphism (TBP), initially based in amplicons separation in acrylamide gels [ 10 ] has been improved in the frame of an EU funded project named Feed-Code ( http://www.feedcode-project.eu ).…”

On the applicability of the Tubulin-Based Polymorphism (TBP) genotyping method: a comprehensive guide illustrated through the application on different genetic resources in the legume family

“…Successful applications of TBP have been demonstrated in fields as diverse as genomic profiling within diverse botanical groups, such as grasses (Eleusine indica, Phalaris arundinacea, Poa trivialis, etc. ), ornamentals (Rosa spp) and trees [21,22]; parental identification, hybrid origin recognition and genetic relationships reconstruction [23]; ploidy level characterization [24]; qualitative and quantitative determinations of plant ingredients in feed [25]. Nevertheless, TBP as method for plant genotyping remains only occasionally utilized by the scientific community [26][27][28].…”