A Large-Scale Outbreak of Trichinellosis from Infected Wild Boar Meat in Croatia and the Role of Real-Time PCR Assays in Confirming the Source of the Disease.

BACKGROUND: Trichinellosis in Croatia posed a significant health concern during the 1990s, followed by a notable improvement in the epidemiological situation. However, in 2017, there was a resurgence, with 37 recorded cases in 3 outbreaks and 3 sporadic cases. The source of this epidemic was homemade meat products derived from wild boar meat, leading to 26 infections. METHODS: At the beginning of the outbreak and during the treatment of the patients, the medical and epidemiological records prepared throughout the investigation and over the course of patient treatment were reviewed. The recovery of the first-stage (L1) larvae from suspect meat products was achieved by artificial digestion. The molecular identification of the isolated larvae was performed by multiplex PCR. The molecular identification of the meat used to prepare the meat products was performed by real-time PCR assays. RESULTS: The epidemic started in early 2017. In total, 71 exposed persons were documented: 26 with clinical symptoms and 3 hospitalised in two cities in different counties. The L1 burden in three different meat products was from 5.25 to 7.08 larvae per gram (LPG), and T. spiralis was determined as the aetiological agent of the outbreak. The molecular and biological identification confirmed that implicated meat products were made solely from wild boar meat. CONCLUSIONS: Although trichinellosis is no longer a frequent occurrence in Croatia, several cases are still registered nearly every year. Wild boar meat poses an important risk factor for human health if compulsory testing is not conducted before consumption, especially if the meat products are consumed without proper thermal processing.

Development of a DNA Metabarcoding Method for the Identification of Insects in Food.

Insects have the potential to become an efficient and reliable food source for humans in the future and could contribute to solving problems with the current food chain. Analytical methods to verify the authenticity of foods are essential for consumer acceptance. We present a DNA metabarcoding method that enables the identification and differentiation of insects in food. The method, developed on Illumina platforms, is targeting a 200 bp mitochondrial 16S rDNA fragment, which we found to be suitable for distinguishing more than 1000 insect species. We designed a novel universal primer pair for a singleplex PCR assay. Individual DNA extracts from reference samples, DNA extracts from model foods and food products commercially available were investigated. In all of the samples investigated, the insect species were correctly identified. The developed DNA metabarcoding method has a high potential to identify and differentiate insect DNA in the context of food authentication in routine analysis.

Interlaboratory Validation of a DNA Metabarcoding Assay for Mammalian and Poultry Species to Detect Food Adulteration.

Meat species authentication in food is most commonly based on the detection of genetic variations. Official food control laboratories frequently apply single and multiplex real-time polymerase chain reaction (PCR) assays and/or DNA arrays. However, in the near future, DNA metabarcoding, the generation of PCR products for DNA barcodes, followed by massively parallel sequencing by next generation sequencing (NGS) technologies, could be an attractive alternative. DNA metabarcoding is superior to well-established methodologies since it allows simultaneous identification of a wide variety of species not only in individual foodstuffs but even in complex mixtures. We have recently published a DNA metabarcoding assay for the identification and differentiation of 15 mammalian species and six poultry species. With the aim to harmonize analytical methods for food authentication across EU Member States, the DNA metabarcoding assay has been tested in an interlaboratory ring trial including 15 laboratories. Each laboratory analyzed 16 anonymously labelled samples (eight samples, two subsamples each), comprising six DNA extract mixtures, one DNA extract from a model sausage, and one DNA extract from maize (negative control). Evaluation of data on repeatability, reproducibility, robustness, and measurement uncertainty indicated that the DNA metabarcoding method is applicable for meat species authentication in routine analysis.

Design of Mismatch Primers to Identify and Differentiate Closely Related (Sub)Species: Application to the Authentication of Meat Products.

Single-nucleotide polymorphisms (SNPs) are powerful molecular markers for the identification and differentiation of closely related organisms. A variety of methods can be used to determine the allele that is present at a specific locus in the genome, including real-time PCR by using an allele-specific primer. In order to increase the selectivity for the target allele, deliberate mismatch bases at the 3' end of the allele-specific primer may be introduced. This strategy has already been used for the identification and differentiation of microorganisms and plants. We have recently developed real-time PCR assays involving mismatch primers for the identification and differentiation of closely related deer species (red deer, fallow deer, sika deer) or the discrimination of wild boar and domestic pig in game meat products. These methods are applicable to detect meat species adulteration in food products.In this chapter, we offer a protocol for the design of PCR primer/probe systems suitable for meat species authentication in food. We address the retrieval and alignment of sequences, primer design by using a commercial software and the introduction of deliberate mismatch bases. In addition, we describe how the suitability of primer/probe systems can be tested in silico and in practice. We use the design of PCR primer/probe systems for wild boar and domestic pig as example.

Development of a DNA Metabarcoding Method for the Identification of Bivalve Species in Seafood Products.

The production of bivalve species has been increasing in the last decades. In spite of strict requirements for species declaration, incorrect labelling of bivalve products has repeatedly been detected. We present a DNA metabarcoding method allowing the identification of bivalve species belonging to the bivalve families Mytilidae (mussels), Pectinidae (scallops), and Ostreidae (oysters) in foodstuffs. The method, developed on Illumina instruments, targets a 150 bp fragment of mitochondrial 16S rDNA. We designed seven primers (three primers for mussel species, two primers for scallop species and a primer pair for oyster species) and combined them in a triplex PCR assay. In each of eleven reference samples, the bivalve species was identified correctly. In ten DNA extract mixtures, not only the main component (97.0-98.0%) but also the minor components (0.5-1.5%) were detected correctly, with only a few exceptions. The DNA metabarcoding method was found to be applicable to complex and processed foodstuffs, allowing the identification of bivalves in, e.g., marinated form, in sauces, in seafood mixes and even in instant noodle seafood. The method is highly suitable for food authentication in routine analysis, in particular in combination with a DNA metabarcoding method for mammalian and poultry species published recently.

Real-Time PCR Assay for the Detection and Quantification of Roe Deer to Detect Food Adulteration-Interlaboratory Validation Involving Laboratories in Austria, Germany, and Switzerland.

Game meat products are particularly prone to be adulterated by replacing game meat with cheaper meat species. Recently, we have presented a real-time polymerase chain reaction (PCR) assay for the identification and quantification of roe deer in food. Quantification of the roe deer content in % (w/w) was achieved relatively by subjecting the DNA isolates to a reference real-time PCR assay in addition to the real-time PCR assay for roe deer. Aiming at harmonizing analytical methods for food authentication across EU Member States, the real-time PCR assay for roe deer has been tested in an interlaboratory ring trial including 14 laboratories from Austria, Germany, and Switzerland. Participating laboratories obtained aliquots of DNA isolates from a meat mixture containing 24.8% (w/w) roe deer in pork, roe deer meat, and 12 meat samples whose roe deer content was not disclosed. Performance characteristics included amplification efficiency, level of detection (LOD95%), repeatability, reproducibility, and accuracy of quantitative results. With a relative reproducibility standard deviation ranging from 13.35 to 25.08% (after outlier removal) and recoveries ranging from 84.4 to 114.3%, the real-time PCR assay was found to be applicable for the detection and quantification of roe deer in raw meat samples to detect food adulteration.

Identification of Mammalian and Poultry Species in Food and Pet Food Samples Using 16S rDNA Metabarcoding.

The substitution of more appreciated animal species by animal species of lower commercial value is a common type of meat product adulteration. DNA metabarcoding, the combination of DNA barcoding with next-generation sequencing (NGS), plays an increasing role in food authentication. In the present study, we investigated the applicability of a DNA metabarcoding method for routine analysis of mammalian and poultry species in food and pet food products. We analyzed a total of 104 samples (25 reference samples, 56 food products and 23 pet food products) by DNA metabarcoding and by using a commercial DNA array and/or by real-time PCR. The qualitative and quantitative results obtained by the DNA metabarcoding method were in line with those obtained by PCR. Results from the independent analysis of a subset of seven reference samples in two laboratories demonstrate the robustness and reproducibility of the DNA metabarcoding method. DNA metabarcoding is particularly suitable for detecting unexpected species ignored by targeted methods such as real-time PCR and can also be an attractive alternative with respect to the expenses as indicated by current data from the cost accounting of the AGES laboratory. Our results for the commercial samples show that in addition to food products, DNA metabarcoding is particularly applicable to pet food products, which frequently contain multiple animal species and are also highly prone to adulteration as indicated by the high portion of analyzed pet food products containing undeclared species.

Development and validation of a real-time PCR assay to detect Cannabis sativa in food.

Regarding the prospective investigation of food authenticity and adulteration the aim of the present study was the development and validation of a real-time PCR assay to identify hemp (Cannabis sativa) which has gained increasing importance as a valuable food ingredient. The assay targets a specific spacer DNA sequence in Cannabis sativa chloroplasts and detects 1.5 pg hemp DNA, which is equivalent to 18 copies/µL. Corresponding to the very low LOD (0.00031 ng/µL) the method allows the detection of hemp even in the infinitesimal concentration of contaminants. Due to a SNP in position 603, hemp can be identified unequivocally and discriminated from its closest relative hops (Humulus lupulus). The PCR method shows no cross-reactivity with 39 of 46 tested plant species. Low cross-reactivity with mulberry, stinging nettle, lavender, cornflower, wine, figs and hops can be neglected, because the Δ Ct-values are > 14, and the obtained Ct-values are beyond the cut-off for a positive assessment (Ct-values ≤ 33). Moreover, the suitability of the method to identify hemp as a food ingredient was proved by analysing diverse food products such as chocolate or cookies.

Insights into the Potential of Sourdough-Related Lactic Acid Bacteria to Degrade Proteins in Wheat.

Sourdough processing contributes to better digestible wheat-based bakery products, especially due to the proteolytic activity of lactic acid bacteria (LAB). Therefore, sourdough-related LAB were screened for their capacity to degrade immunogenic proteins like gluten and alpha-amylase-trypsin inhibitors (ATIs). Firstly, the growth of 87 isolates was evaluated on a gluten-based medium. Further, the breakdown capacity of selected isolates was determined for gluten with a focus on gliadins by measuring acidification parameters and MALDI-TOF MS protein profiles. ATI degradation after 72 h of incubation within an ATI-based medium was investigated by means of acidification, HPLC, and competitive ELISA. All isolates exhibited the potential to degrade ATIs to a high degree, whereas the gliadin degradation capacity varied more greatly among tested LAB, with Lacticaseibacillus paracasei Lpa4 exhibiting the strongest alterations of the gliadin pattern, followed by Lactiplantibacillus plantarum Lpl5. ATI degradation capacities ranged from 52.3% to 85.0% by HPLC and 22.2% to 70.2% by ELISA, with Lacticaseibacillus paracasei Lpa4 showing superior breakdown properties. Hence, a selection of specific starter cultures can be used in sourdough processing for wheat-based bakery products with reduced gluten and ATI content and, further, better tolerated products for patients suffering from non-celiac wheat sensitivity (NCWS).

Applicability of a duplex and four singleplex real-time PCR assays for the qualitative and quantitative determination of wild boar and domestic pig meat in processed food products.

Appropriate analytical methods are needed for the detection of food authentication. We investigated the applicability of a duplex real-time PCR assay targeting chromosome 1 and two singleplex real-time PCR assays targeting chromosome 9, both published recently, for the qualitative and quantitative determination of wild boar and domestic pig in processed food products. In addition, two singleplex real-time PCR assays targeting chromosome 7 were tested for their suitability to differentiate the two subspecies. Even by targeting the three genome loci, the probability of misclassification was not completely eliminated. Application of the real-time PCR assays to a total of 35 commercial meat products, including 22 goulash products, revealed that domestic pig DNA was frequently present, even in 14 out of 15 products declared to consist of 100% wild boar. Quantitative results obtained with the real-time PCR assays for wild boar (p < 0.001) and those for domestic pig (p < 0.001) were significantly different. However, the results obtained with the real-time PCR assays for wild boar (r = 0.673; p < 0.001) and those for domestic pig (r = 0.505; p = 0.002) were found to be significantly correlated. If the rules given in the paper are followed, the real-time PCR assays are applicable for routine analysis.

Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR.

Studies indicate that many meat products are not authentic, most frequently because the meat species differ from those given on the food labels. At present, DNA based methods play the most important role in meat species authentication. Discrimination of wild boar and domestic pig meat in food is challenging because it is differentiation on the subspecies level. We developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and a duplex real-time PCR assay targeting SNP g.299084751 C > T in the NR6A1 gene located on chromosome 1. The singleplex real-time PCR assays led to some ambiguous results for Mangalica and Krskopolje pig breeds and wild boar individuals from Germany, the duplex real-time PCR assay particularly for the Turopolje pig breed. We demonstrate that the probability of misclassification can be substantially reduced if the results of both the singleplex real-time PCR assays and the duplex real-time PCR assay are taken into consideration. 86 (91.5%) of a total of 94 individuals, comprising 64 domestic pigs (14 different breeds and 6 cross-breeds) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were classified correctly.

Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food.

Meat products are prone to adulteration by the replacement of meat from more expensive animal species with meat from cheaper sources. We present a DNA metabarcoding method allowing the identification and differentiation of 15 mammalian and six poultry species in foodstuffs. The method, developed on the MiSeq® platform, targets a mitochondrial 16S rDNA region recently found to be suitable for the differentiation of 300 mammalian species. We designed a novel primer pair for poultry and applied it in combination with the primer pair for mammalian species in a duplex assay. The applicability of the method was investigated by analysing DNA extracts from muscle, DNA extract mixtures and extracts from model sausages. Our results indicated that the species of interest can be identified, differentiated and detected down to a proportion of 0.1%. Since 96 samples can be sequenced in one run, the method has high potential for application in routine analysis.

Tetraplex real-time PCR assay for the simultaneous identification and quantification of roe deer, red deer, fallow deer and sika deer for deer meat authentication.

Analytical methods are needed for the identification and quantification of meat species to detect food adulteration. Since game meat is more expensive than meat from domesticated animal species, it is a potential target for adulteration. We present a tetraplex real-time PCR assay that allows the simultaneous determination of the content of roe deer, red deer, fallow deer and sika deer. The tetraplex assay showed only moderate cross-reactivity with closely related species. After optimization the tetraplex assay had a limit of detection of 0.1% (w/w) and a limit of quantification of 0.5% (w/w) for each of the four deer species. The tetraplex assay was found to be robust, slight modifications of the experimental setup did not lower its performance. Recoveries obtained by analyzing DNA mixtures and DNA isolates from model game sausages were similar to those obtained with the singleplex assays.

Sika deer (Cervus nippon)-specific real-time PCR method to detect fraudulent labelling of meat and meat products.

Since game meat is more valuable and expensive than meat from domesticated animal species it is a potential target for adulteration. Analytical methods must allow the identification and quantification of meat species to be applicable for the detection of fraudulent labelling. We developed a real-time PCR assay for the authentication of sika deer (Cervus nippon) and products thereof. The primer/probe system amplifies a 71 bp fragment of the kappa-casein precursor gene. Since the target sequence contained only one sika deer-specific base, we introduced a deliberate base mismatch in the forward primer. The real-time PCR assay did not show cross-reactivity with 19 animal and 49 plant species tested. Low cross-reactivity was observed with red deer, fallow deer, reindeer and moose. However, with a ΔCt value of ≥11.79 between sika deer and the cross-reacting species, cross-reactivity will not affect the accuracy of the method. LOD and LOQ, determined by analysing serial dilutions of a DNA extract containing 1% (w/w) sika deer DNA in pig DNA, were 0.3% and 0.5%, respectively. The accuracy was evaluated by analysing DNA mixtures and DNA isolates from meat extract mixtures and meat mixtures. In general, recoveries were in the range from 70 to 130%.

Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration.

The aim of the present study was to develop a real-time PCR assay for the identification and quantification of fallow deer (Dama dama) in food to detect food adulteration. Despite high sequence homology among different deer species, a fallow deer-specific primer/probe system targeting a fragment of the nuclear MC1-R gene was designed. This primer/probe system did not amplify DNA from 19 other animals and 50 edible plant species. Moderate cross-reactivity was observed for sika deer, red deer, roe deer, reindeer and wild boar. The LOD and LOQ of the real-time PCR assay were 0.1% and 0.4%, respectively. To validate the assay, DNA mixtures, meat extract mixtures, meat mixtures and model game sausages were analyzed. Satisfactory quantitative results were obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest.

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

Development and validation of a triplex real-time PCR assay for the simultaneous detection of three mustard species and three celery varieties in food.

The paper presents a triplex real-time PCR assay allowing the simultaneous detection of three mustard species (white, black and brown mustard) and three celery varieties (celery roots, celery stalks and leaf celery) in foodstuffs. The triplex assay does not show cross-reactivity with other Brassicaceae. Low cross-reactivities were observed with fenugreek, cumin, ginger, caraway, turmeric, lovage and rye, the ΔCt values were, however, ⩾ 12 compared to positive controls. The triplex assay allows the detection of traces of DNA of the allergenic components in spite of an excess of the other DNA templates. Analysis of extracts from model sausages containing defined concentrations of mustard and celery showed that the triplex assay is applicable to both raw and processed foods. It was found to allow the detection of 1 ppm black/brown mustard and 50 ppm white mustard and celery in raw and brewed sausages with a probability ⩾ 95%.

Development and validation of a TaqMan real-time PCR assay for the identification and quantification of roe deer (Capreolus capreolus) in food to detect food adulteration.

In order to protect the consumer from meat adulteration it is necessary to identify and quantify the meat content in foodstuffs. Game meat is particularly susceptible for fraudulent labeling since it is more valuable than meat from domestic animals. The paper presents a TaqMan real-time PCR assay for the quantitative determination of roe deer in meat products. The assay developed does not show cross-reactivity with 23 animal and 43 plant species tested and is therefore specific for roe deer. The amplification efficiency determined by analyzing serially diluted roe deer DNA extracts was found to be 93.9%. For quantifying the roe deer content in % (w/w), a reference system based on the myostatin gene was used. The quantification strategy was validated by determining the roe deer content in model meat mixtures and a model sausage. In addition, the real-time PCR assay was applied to the analysis of commercially available meat products.

Comparison of R5 and G12 Antibody-Based ELISA Used for the Determination of the Gluten Content in Official Food Samples.

Celiac Disease (CD) is one of the most common food intolerances. It comes along with serious damage of the mucosa in the small intestine and is caused by the storage proteins-termed "gluten"-of wheat, rye, barley and possibly oats. Sensitive individuals need to stick to a strict gluten-free diet. The gluten level in food products labeled as "gluten-free", must not exceed 20 mg/kg. It is obvious that effective test methods are needed to accurately determine the gluten concentration in foods. The determination of the presence of gluten in foodstuffs is mainly done by means of an immunochemical method called ELISA (enzyme-linked immunosorbent assay). To check the suitability of a G12 antibody-based gluten detection kit for its use in official control systems a number of routine samples were tested in parallel with two different test kits, as would be done in a routine lab. The determination of the gluten content was performed on samples entering the official laboratory including samples from official control plans, commercially available and private samples to request gluten-free labels. The results obtained with the G12 antibody ELISA assay were comparable to the official R5 method. A validation of the two different methods was not part of this study.