Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR)

Floren, C.; Wiedemann, I.; Brenig, Bertram; Schütz, Ekkehard; Beck, Julia

doi:10.1016/j.foodchem.2014.10.138

Cited by 157 publications

(101 citation statements)

References 22 publications

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“…DNA characterized by more stability under intensive heating, pressures, and chemical processing, has conserved structure in whole body cells, has a great identification power since they are rely on the recognition of specific DNA segments sequence of a particular tissue or animal (Calvo et al, 2001;Frezza et al, 2003;Girish et al, 2004;Lanzilao et al, 2005;Akasaki et al, 2006;Arslan et al, 2006;Rashid et al, 2014). From DNAbased techniques, polymerase chain reaction (PCR) is the most employed, simple, time saving, sensitive and specific method that could identify the species of origin exposed to different processing conditions (Mafra et al, 2008;Bottero and Dalmasso, 2011;Floren et al, 2015). In addition, the use of PCR in food analysis has provided various analytical methods for rapid detection and identification at species and intra-species level; however DNA-based methods still face some important limitations especially for quantitative measurements of food composition (Woolfe and Primrose, 2004).…”

Section: Advances In Animal and Veterinary Sciencesmentioning

confidence: 99%

Identification of Different Animal Species in Meat and Meat Products: Trends and Advances

Farag¹,

Alagawany²,

El‐Hack³

et al. 2015

Adv. Anim. Vet. Sci.

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| Identification of animal species of origin in meat and meat products is a matter of great concerns such as religious, economical, legal as well as medical aspects. Thus, several analytical techniques have been suggested for the identification of meat species either in individual or in mixed samples to protect consumers from the fraudulent and bad habits of marketing. DNA-based techniques especially the techniques based on polymerase chain reaction (PCR) are recognized as the most appropriate methods employed for species identification in raw and processed meat. PCR techniques including randomly amplified polymorphic DNA (PCR-RAPD), restriction fragment length polymorphism (PCR-RFLP), PCR with species-specific primers, real-time PCR and PCR-nucleotide sequencing allow identification of meat species under different processing conditions. But the variability of DNA content on the level of species as well as target tissue make the DNA-based methods somewhat unsuitable for the quantification of exact percentages of different species in meat and meat products. For these reasons the proteomic approaches depending on identification of different peptide biomarkers has been developed and employed to give information on the different composition of food. To broad the knowledge about these technologies, this review is compiled in an attempt to provide an overview of the possible PCR-based analytical techniques that could help in identifying the meat species of origin in meat and meat products and threw the light on the identification of species specific peptide biomarkers by proteomic technologies as a new and attractive alternative that could overcome some of the limitations that faced DNAbased methods especially when used for meat exposed to intensive heating of processing as well as for meat mixtures.

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Section: Advances In Animal and Veterinary Sciencesmentioning

confidence: 99%

Identification of Different Animal Species in Meat and Meat Products: Trends and Advances

Farag¹,

Alagawany²,

El‐Hack³

et al. 2015

Adv. Anim. Vet. Sci.

View full text Add to dashboard Cite

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“…Digital PCR (dPCR) is a new technique for precise quantification that can eliminate the effects of matrixes, improve the sensitivity and precision, and provide an absolute measurement of nucleic acid concentration without the use of standard curves [22]. This technique has been extensively used in several areas, including quantitative gene expression analysis [23], bacterial abundance [24], identification of genetically modified organisms [25] and food authentication [26]. In the quantification of DNA from transgenic soy, dPCR may be more suitable for quantitative analysis, as it exhibits a measurement uncertainty of only 17% or below for a single reaction [27].…”

Section: Introductionmentioning

confidence: 99%

A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food

et al. 2017

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Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination. The method exhibited good repeatability and stability in different thermal treatments and at ultra-high pressure. The relative standard deviation (RSD) values of 5% chicken content was less than 5.4% for ultra-high pressure or heat treatment. Moreover, we confirmed that different parts of meat had no effect on quantification accuracy of the ddPCR method. In contrast to real-time PCR, we examined the performance of ddPCR as a more precise, sensitive and stable analytical strategy to overcome potential problems of discrepancies in amplification efficiency discrepancy and to obtain the copy numbers directly without standard curves. The method and strategy developed in this study can be applied to quantify the presence and to confirm the absence of adulterants not only to sheep but also to other kinds of meat and meat products.

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“…Given its advantages, the ddPCR system developed in the present study represents a new strategy to quantify pathogens directly in food samples, as described also by Floren et al (2015) and Verhaegen et al (2016). First, the ddPCR system optimized in the present study has increased the tolerance to inhibitors arising from cheese samples (e.g., fats, proteins, high concentration of Ca 2+ ) to improve the LoD compared with qPCR.…”

Section: Discussionmentioning

confidence: 92%

“…This approach partitions the sample into hundreds of millions of water-in-oil droplets before thermal cycling (McDermott et al, 2013). These droplets are monitored for positive amplification after endpoint PCR amplification using fluorescent target-specific hydrolysis probes (Floren et al, 2015). Until now, this method has been adopted for: routine analyses of genetically modified organisms in food and animal feed (Morisset et al, 2013; Gerdes et al, 2016); detection and quantification of pathogenic bacteria such as Salmonella spp., Campylobacter jejuni and Listeria monocytogenes in environmental water (Rothrock et al, 2013); exact quantification of different species in meat and processed meat products (Floren et al, 2015); monitoring the dynamics of microbial populations in soils with different population levels (Kim et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Development of a Droplet Digital Polymerase Chain Reaction for Rapid and Simultaneous Identification of Common Foodborne Pathogens in Soft Cheese

et al. 2016

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Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a “third-generation PCR”) provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4 × 106 to 4 × 101 CFU/g) of pure cultures from the American Type Culture Collection. Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay) without the necessity of a standard curve. In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.

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Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR)

Cited by 157 publications

References 22 publications

Identification of Different Animal Species in Meat and Meat Products: Trends and Advances

Identification of Different Animal Species in Meat and Meat Products: Trends and Advances

A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food

Development of a Droplet Digital Polymerase Chain Reaction for Rapid and Simultaneous Identification of Common Foodborne Pathogens in Soft Cheese

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