from Patricia Elízaquível, Gloria Sánchez and Rosa Aznar writing in Real-Time PCR in Food Science: Current Technology and Applications:

Foodborne diarrheagenic Escherichia coli strains belong to a minor number of O:H serotypes. Of them the shiga toxin-producing E. coli O157:H7 is the most frequently reported. Besides, non-O157 strains are increasingly being isolated from a variety of food products. E. coli infections are typically associated with transmission through animal products. However, in the last years, contaminated fresh produce is increasingly being implicated in E. coli O157:H7 outbreaks. Currently real-time PCR (qPCR) is considered as an alternative to standard culture methods for E. coli detection in food due to its high speed, specificity, sensitivity, reproducibility and minimization of cross-contamination. Moreover, quantification is possible when an enrichment step is omitted. Although qPCR is a very promising technique for pathogen detection in food, food laboratories and industries are still reluctant to extensively apply it. Real-time qPCR presents some challenges when applying in food, e.g. presence of inhibitors, low levels of cells, detection of dead cells. Besides, the selection of appropriate target regions is another challenge in E. coli detection because of their high genetic heterogeneity. In this review the different approaches proposed to circumvent the difficulties to detect pathogenic E. coli in food and the most frequently targeted genes are presented.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Kathie Grant and Corinne Amar writing in Real-Time PCR in Food Science: Current Technology and Applications:

The principle Clostridal foodborne pathogens, Clostridium botulinum and Clostridium perfringens are responsible, respectively, for two different toxin mediated foodborne diseases, namely botulism and C. perfringens food poisoning. Foodborne botulism is a severe, life-threatening disease which can affect a large number of people and although incidence is rare, it is considered a public health emergency. Whilst C. perfringens type A food poisoning is far less severe, it is one of the most common causes of bacterial food poisoning in both the UK and US. It is important to have rapid, accurate methods to detect these two clostridial pathogens and their toxins in order to confirm the cause of illness and identify the food source so that appropriate control and preventative interventions can be implemented. However, conventional laboratory methods to detect C. botulinum and C. perfringens in foods and clinical samples are lengthy, complex, may involve the use of animals and are not always very informative. Real-time PCR assays have been developed to rapidly detect the toxin genes of both pathogens and have been used, in conjunction with culture techniques, to: improve the diagnostic procedure; enhance incident and outbreak investigations and provide information on the pathogenicity of isolates. Real-time PCR detection assays for clostridial foodborne pathogens are also highly valuable to food producers providing faster methods for monitoring growth in food enabling the safety of food products to be assessed more rapidly and effectively. The reliability of real-time PCR detection assays depends upon a range of factors from the bacterial pathogen being detected and the sample matrix to the effective use of controls to ensure the efficiency of the nucleic acid extraction and accuracy of the amplification procedure. This review focuses on the practical application of real-time PCR detection assays for these two clostridial foodborne pathogens.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from George D. Di Giovanni, Gregory D. Sturbaum, and Huw V. Smith writing in Real-Time PCR in Food Science: Current Technology and Applications:

Many parasites are capable of infecting humans, with zoonotic and environmental transmission pathways having significant roles. Of particular significance are pathways involving contaminated food and water. Despite increasing risks posed by food and waterborne parasites due to global sourcing of food, cosmopolitan eating habits, and increased international travel; detection and epidemiologic methods for many of these parasites remains underdeveloped. Microscopy-based detection and diagnostic techniques are still revered as the gold standard for the detection of many food and waterborne parasites. However, the advent and employment of molecular methodologies has proven to surpass microscopy in three major aspects: sensitivity, specificity and the ability to speciate. While molecular methodologies have clear advantages over many traditional detection techniques, standardized PCR methods for the detection of food and waterborne parasites are lacking. This is largely due to multiple obstacles, such as: the diversity of test matrices (e.g. fruits, vegetables, meat products, shellfish, and water); different approaches needed for recovery, concentration, and DNA extraction for different parasites; intrinsically low levels of parasites present in samples; and a lack of multi-laboratory validation of promising methods. Although the PCR detection of food and waterborne parasites may be complex and challenging, recent advances in sample processing techniques and the development of real-time PCR assays are bringing the goal of standardized methodology within view. This review covers an overview of some important food and waterborne parasites, a description of conventional detection methodology, and advances in sample processing and real-time PCR assays. Research needs are discussed along with the benefits of real-time PCR detection and typing.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Doris Helen D'Souza, Marta Hernández, Nigel Cook and David Rodríguez-Lázaro writing in Real-Time PCR in Food Science: Current Technology and Applications:

Analysis of foodstuffs for virus contamination requires profoundly sensitive and accurate methods, due to the potentially low number of viruses and the complexity of the sample matrix. In view of these criteria, the polymerase chain reaction is the assay type of choice, with its rapidity being another useful factor. Real-time PCR (qPCR) is superceding conventional PCR in several areas of molecular diagnostics, and a large variety of published qPCR-based methods for foodborne pathogen detection is available in the scientific literature. In common with other molecular-based methods, qPCR-based analysis of foodstuffs for viruses requires effective controls to ensure that issues associated with low virus numbers and the complexity of the matrix do not result in false negative or positive interpretations of results. These controls are essential for implementation of qPCR-based methods for foodborne virus detection, but in most cases are not included in those which have been published hitherto. Alternative molecular techniques, such as nucleic acid sequence-based amplification (NASBA) and loop-mediated amplification (LAMP) are also suitable for utilization in detection methods for viruses in foods, the same requirements regarding controls pertaining. All molecular-based methods for foodborne virus detection must of necessity contain sample treatment procedures to extract the virus or its nucleic acid out of the food matrix, and these procedures can be elaborate due to matrix complexity. Nonetheless efficient sample treatment methods have been devised, and in combination with molecular assays effective methods for virus analysis are now available for foods. Implementation of these methods in routine diagnostics will support food safety management programs and assist in outbreak investigation, and help to ensure a safe food supply.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Arne Holst-Jensen writing in Real-Time PCR in Food Science: Current Technology and Applications:

Genetic modification (GM) alters the phenotype of the GM organism (GMO). This is achieved through application of gene technology and modification of genetic information stored in nucleic acids. The logical choice of methodology to detect and characterise GM is therefore analytical methods targeting nucleic acids. The polymerase chain reaction (PCR) methodology has been the preferred methodology of this type for two decades, and the following paper will review its applications and derivatives in relation to detection and characterisation of GM organisms (GMOs). The need for detection, identification, characterization and quantitation of GMOs depends on issues such as the legal status of the GMOs in question (authorized or not), labeling or contractual requirements, authentication, traceability and co-existence, environmental monitoring and risk assessments. The fitness for purpose of a specific analytical method is often limited to certain applications. Guidelines to establishment of analytical strategy and method selection can be very useful to those who order as well as to those who provide GMO analyses. A fundamental distinction can be made between screening and identification methods, respectively. The former may be used to group and separate putatively GMO-free samples from samples containing GMO. Both classes of methods may provide qualitative and quantitative information, but only the identification methods can provide accurate quantitation. GMO quantification is achieved almost exclusively with real-time PCR methods, but other alternatives are also available. PCR is also commonly used in combination with other techniques such as Southern blot analyses and DNA sequencing to characterize the genetic constitution of GMOs. Over the last decade extensive resources have been put into validation and critical assessment of performance characteristics and requirements for real-time PCR based GMO detection methods. GMO analyses can be particularly challenging because quantitation is required at very low concentrations, in products of highly variable nature, and where the introduced novel sequences of different GMOs belonging to the same or different species may result in misinterpretation and analytical interference. Consequently, there is a lot to learn from this field of science also for others working with real-time PCR methods. This review will provide several examples.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Carmen Diaz-Amigo and Bert Popping writing in Real-Time PCR in Food Science: Current Technology and Applications:

Food allergens, responsible for IgE-mediated allergic responses and listed in European, North American and Japanese regulations, are exclusively proteins and are ideally detected by analytical methods targeting either peptides or proteins. However, in some cases where no suitable methods for proteins exist or as an alternative method to substantiate results from protein-based methods, DNA-targeting methods can be used as indicators of the presence of potentially allergenic proteins. The advantage of DNA-targeting methods like PCR, real-time PCR is presently the lower cost and availability of free literature on several detection systems, including a certain degree of multiplexing. Clear disadvantages include the poor sensitivity for egg, milk and samples containing inhibitors (like polyphenols in chocolate) as well as its limited applicability in some industrial protein concentrates. In addition, if quantitative results need to be obtained, the DNA-based system needs to be calibrated for each matrix tested as protein-to-DNA composition is typically matrix specific. However, PCR based methods are well established in many laboratories and still regularly used. This review discusses suitable systems for detection of DNA of ingredients and foods containing allergenic proteins, potential pitfalls and multiplex capabilities of such systems.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from David Rodríguez-Lázaro and Marta Hernández writing in Real-Time PCR in Food Science: Current Technology and Applications:

Food safety and quality control programs are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostic have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Nigel Cook, Gabriel A de Ridder, Martin D'Agostino and Maureen B Taylor writing in Real-Time PCR in Food Science: Current Technology and Applications:

Assays based on nucleic acid amplification are highly efficient, but they can be affected by the presence of matrix-derived substances which can interfere or prevent the reaction from performing correctly. Careful sample treatment must be applied/used to remove these inhibitory substances. However no sample treatment can be relied on completely, thus an amplification control should be employed to be able to verify that the assay has performed correctly. An internal amplification control (IAC) is a non-target DNA sequence present in the very same reaction as the sample or target nucleic acid extract. If it is successfully amplified to produce a signal, any non-production of a target signal in the reaction is considered to signify that the sample did not contain the target pathogen or organism. If however the reaction produces neither a signal from the target nor the IAC, it signifies that the reaction has failed.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Dietrich Mäde writing in Real-Time PCR in Food Science: Current Technology and Applications:

Yersinia enterocolitica ranks as the third bacterial food pathogen in Europe. Because cultural assays are labour and time consuming, the routine analyses of food samples need to be improved. The domestic pig is considered as the moost important carrier of the zoontic strains but the data set for food samples is limited due to the limitations of the labour intensive cultural method. Duplex real-time PCR systems targeting the chromosomally encoded ail-gene allow a sensitive and specific detection. A heterologous internal amplification control based on the plasmid pUC18 or pUC19 is applied to monitor for PCR inhibitions. The duplex real-time PCR including the heterologous IAC is a robust method for screening food samples. The combination with the cultural standard method allows the detection and cultural confirmation of a high percentage of PCR positive samples. The molecular system can be successfully applied to the test of suspect colonies.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from L. Jesús Garcia-Gil writing in Real-Time PCR in Food Science: Current Technology and Applications:

Campylobacter is a microaerophilic, spiral shaped, Gram-negative bacterium comprising 16 species. Although many of these species are thermotolerant, i.e. able to grow at 42 degrees C, C. jejuni, C. coli, C lari, and C. upsaliensis are the most prevalent foodborne pathogens. The need for a fast detection of these bacteria in foodstuff has fostered the development of rapid method, most of them based on PCR techniques. Nevertheless, the use of the appropriate targets has limited the development of reliable methods. This difficulty arises, in part, from the fact that target genes used commonly, either virulence genes or ribosomal, contain high variability, even among strains. This has serious implications, for instance, as false negative results. As a consequence, the number of available PCR protocols to detect thermotolerant Campylobacters is very limited. The use of strongly conserved, housekeeping genes as PCR targets has resulted in a good approach to the ideal real-time PCR based method. The difficulty in such a task is actually reflected in the scarce officially certified tools commercially available.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from David Rodríguez-Lázaro, Nigel Cook and Marta Hernández writing in Real-Time PCR in Food Science: Current Technology and Applications:

A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labeling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programs of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications