Species identification of commercial jerky products in food and feed using direct pentaplex PCR assay

The direct pentaplex PCR assay was developed for simultaneous identification using species-specific primer sets and a universal eukaryotic primer set in processed jerky products without DNA extraction. The specific primer sets of target meat species amplified the expected 83-, 133-, 166-, and 204-bp PCR products for pork, chicken, beef, and duck, respectively, and obtained no cross-reactivity against a total of sixteen animal species. A universal eukaryotic primer set amplified a 99-bp conserved fragment in all meat species. To evaluate the sensitivity of this assay, the different percentages of jerky samples were prepared with the meat species having the possibility to be mixed. Adulterated beef jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% pork and adulterated duck jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% chicken were prepared in a laboratory. The detection level of direct pentaplex PCR was below 0.1% pork in adulterated beef jerky and 0.1% chicken in adulterated duck jerky. The optimized assay was also applied to the analysis of commercial food and feed jerky products. The meat species in commercial jerky products were successfully identified without DNA extraction.     

Applicability assessment of a real-time PCR assay for the specific detection of bovine,ovine and caprine material in feedstuffs

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of bovine, ovine and caprine processed animal protein (PAP) in industrial feedstuffs. The method uses species-specific primers and probes targeting short mitochondrial D-loop sequences, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 126 industrial feed samples that were manufactured to reproduce rendering processes of commercial feeds destined for farmed animals. The assay successfully classified samples as positive or negative according to the ruminant composition, enabling qualitative detection of banned material in feeds at levels as low as 0.1%. Although the method provides quantitative potential, results suggest that the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing treatments of the feeds, which affect the amount and quality of amplifiable DNA.     

A multiplex PCR assay for species identification of raw and cooked bonito

Attempts are made to establish one-step multiplex PCR assay for distinguishing five species of raw and cooked bonito including Euthynnus pelamis, Euthynnus affinis, Auxis rochei, Auxis thazard, and Sarda orientalis. After constructing the 1141 bp complete mitochondrial cytochrome b genes of five bonito species and other five contrastive Scombridae species, five sets of species-specific primer were designed to amplify different cytochrome b gene fragments in each species individually. The amplified lengths of fragments were respectively 143 bp for A. rochei, 236 bp for E. pelamis, 318 bp for A. thazard, 398 bp for E. affinis and 506 bp for S. orientalis, which could be obviously differentiated from each other on DNA electrophoresis. The five sets of species-specific primer were mixed and applied to simultaneously detect bonito species. All species from 12 commercial raw fish and five species out of eight cooked bonito fillets were successfully identified by the multiplex PCR assay. Experiments carried out demonstrate that the multiplex PCR assay was useful for identifying species of non-overheating fish product.     

Development of triplex PCR for simultaneous detection of maize,wheat and soybean

A reliable and fast detection of important food plants, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and soybean (Glycine max L.) is of particular interest for food authenticity and safety assessment. In this study, the novel multiplex polymerase chain reaction (PCR) method was developed for the rapid qualitative detection of soybean, maize and wheat. To this purpose, new soybean-specific and maize-specific PCR primers were designed. Their specificity was assayed by uniplex PCRs with different plant species, namely maize, soybean, wheat, oats (Avena sativa), and barley (Hordeum vulgare L). Gel electrophoresis of the amplification products demonstrated high specificity of both primer pairs for identification of relevant species. Subsequently, based on the developed DNA markers, the species-specific triplex PCR targeting maize invertase gene, soybean lectin gene and wheat low-molecular-weight glutenin subunit was developed and optimized for simultaneous identification of these three plant species. The developed PCR method enables specific, effective and rapid detection of maize, wheat and soybean and may be used for food analysis.     

Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures

Five species-specific Real-Time PCR protocols were developed, standardised and validated for the identification of turkey, chicken, beef, pork and sheep meat in complex food products. Specific primers and probes were designed for each assay. 16S rRNA and cyt-b target genes located in the mitochondrial DNA were used for this purpose. The limit of detection of the Real-Time PCR methods ranged between 0.02 pg and 0.80 pg of template DNA, with an efficiency between 95% and 100%. All methods were able to detect the target species when spiked at 1% in any other species and no relevant difference was observed between the Ct values of raw and cooked samples. An Internal Amplification Control was used to detect possible false negatives due to inhibitory substances eventually present in the sample matrix. The assays were tested on meat mixtures to evaluate the diagnostic sensitivity and specificity.     

Targeting double genes in multiplex PCR for discriminating bovine,buffalo and porcine materials in food chain

Beef, buffalo and pork are the major meat of economic, religious and health concern. Current methods to authenticate these materials in food chain are based on mainly single gene targets which are susceptible to break down by food processing treatments. We, for the first time, described here a double gene targeting short-amplicon length multiplex polymerase chain reaction assay for discriminating bovine, buffalo and porcine materials in a single assay platform. The advantage of the assay is evidenced in terms of fidelity, cost and time since it is highly unlikely that two different targets would be missing even in a decomposed specimen. Detection of multiple targets in a single assay definitely saves analytical cost and time. Mitochondrial cytochrome b (cytb) and ND5 genes were targeted and six different targets (length: 90–146 bp), two for each of cow (120 and 106bp), buffalo (90 and 138bp) and pig (73 and 146bp), were amplified from raw, boiled, autoclaved and microwaved cooked meat under pure and mixed matrices. The detection limit was 0.02 ng DNA under pure states and 0.1% meat in binary mixtures and meatball products. Screening of Malaysian meatball products revealed all beef products were buffalo positive in which 35% were totally replaced. In contrast, all pork products were found uncontaminated from beef and buffalo.     

Development and evaluation of a real-time PCR assay for detection of lobster,a crustacean shellfish allergen

Crustacean shellfish are a leading cause of food allergy in American adults, and the Food Allergen Labeling and Consumer Protection Act requires that different types of crustacean shellfish be distinguished from each other. In general ELISA assays are not capable of differentiating crustacean type, but PCR assays are. In this work, a real-time PCR assay for lobster, a crustacean shellfish allergen, was developed and evaluated. Food matrices were spiked with lobster meat at 0.1, 1, 10, 100, 1000, 10⁴, and 10⁵ parts per million (ppm). In addition to testing of several different food matrices, method performance was determined using conditions which have historically proven challenging for PCR analyses, specifically food matrices with low DNA content and acidic pH levels, as well as foods that were treated with combined high temperature and pressure. Real-time PCR standard curves were generated from spiked, treated foods and analyzed with respect to linear range and reaction efficiency. In most cases, the assay was linear over 6–8 orders of magnitude; lower limits of detection were 0.1–1 ppm and reaction efficiencies were within the preferred range of 100 ± 10%. A notable exception occurred in the case of heat treatment at acidic pH, which resulted in severe delay or complete loss of amplification signals.     

A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods

Meat adulteration has posed considerable risks to public health. In this study, we developed a novel real-time quantitative PCR method for the detection of some mammalian and poultry species that are used as meat products or meat adulterants. The method was based on the detection of the single-copy nuclear gene myostatin. The specificity, heterogeneity, and copy number of myostatin were evaluated. Additionally, we determined the sensitivity and precision of the method. The results revealed that myostatin had high specificity and low heterogeneity among different mammalian and poultry species. The limit of detection was 5 pg of animal genomic DNA or 0.001% meat ingredient, and the limit of quantification was 10 pg of animal genomic DNA or 0.01% meat ingredient. The quantification results of 12 blind samples showed that the biases between the measured and true values were <25%. Therefore, the developed quantitative real-time PCR method for mammalian and poultry species is suitable for identification and quantification of different meat ingredients as a reference gene.     

A novel reference real-time PCR assay for the relative quantification of (game) meat species in raw and heat-processed food

In order to increase their profit, food producers may be tempted to replace expensive meat species by cheaper ones. Due to their selectivity and sensitivity, PCR based methods are frequently applied to identify and quantify meat species to detect food adulteration. Quantification of meat species in highly processed food products is, however, a difficult task. When we used a previously published reference system to relatively quantify the deer content in heat treated foodstuffs, we obtained recoveries that were substantially >100%. In the present study we aimed to improve the applicability of the reference system, targeting a 97 bp fragment of the myostatin gene, to heat treated products by reducing the length of the target sequence. The novel reference system, targeting a 70 bp fragment of the myostatin gene, was found to amplify the target region in 27 mammals and poultry species. Meat mixtures as well as raw and heat treated model sausages were analysed to demonstrate the applicability of the novel reference system for relatively quantifying the game meat content in processed food products. With the novel reference system, the bias introduced by heat treatment was largely eliminated.     

Duplex real-time PCR assay for the simultaneous determination of the roe deer (Capreolus capreolus) and deer (sum of fallow deer,red deer and sika deer) content in game meat products

Due to the high price of game meat, food producers may be tempted to adulterate their products with cheaper meat. This paper presents a duplex real-time PCR assay which allows the simultaneous determination of the content of roe deer (Capreolus capreolus) and deer* (the sum of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon)) in food products to detect food adulteration. Relative quantification is carried out by using a reference (“all meat”) PCR assay based on the myostatin gene. The quantification approach was validated by analyzing binary meat mixtures with pork, “all game” meat mixtures containing each of the four game species in pork and a model game sausage. Compared to singleplex assays the duplex assay is time and cost saving. Thus, it is highly applicable to routine analysis in order to verify the authenticity of game meat products.     

Sextuplex PCR combined with immunomagnetic separation and PMA treatment for rapid detection and specific identification of viable Salmonella spp., Salmonella enterica serovars Paratyphi B,Salmonella Typhimurium,and Salmonella Enteritidis in raw meat

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 10¹ CFU/mL in pure culture and 10² CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.