Section I. Real-time PCR Basics

1. Introduction to the Real-time PCR

David Rodríguez-Lázaro and Marta Hernández

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.

2. Current Challenges in Real-time PCR Diagnostics in Food Science

David Rodríguez-Lázaro, Nigel Cook and Marta Hernández

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.

3. Sample Preparation for Real-time PCR in Food Science

Tomáš Kuchta

Sample preparation including DNA isolation is described as the first procedural step preceding the analysis of food by real-time PCR. Principles of, applications of and prerequisites for direct DNA isolation from food matrix are presented, providing information on chaotropic solid phase extraction, solubilization with cetyltrimethylammonium bromide followed by liquid-liquid extraction, and on immunomagnetic separation. Importance of and procedures for testing the amplifiability of the isolated DNA, determination of the recovery and recovery rate of DNA from food as well as procedures improving the efficiency of DNA isolation from "difficult" food matrices are given. Various techniques for DNA quantitation, including UV spectrometry, fluorimetry and determination of amplifiable DNA by PCR, are described and their use and usefulness are discussed. The effectivity of individual approaches at the analysis of various food matrices is discussed. For the application field of rapid detection of pathogenic bacteria in food, information on cultivation enrichment, immunoseparation, centrifugation and microfiltration is provided. The applicability of different sample preparation methods at the detection of various pathogenic bacteria in several food product types is discussed. Attention is paid also to DNA isolation from enriched samples, including description of rapid techniques for partial DNA separation, bacterial cell lysis and removal of PCR inhibitors.

4. Internal Amplification Controls in Real-time Polymerase Chain Reaction-Based Methods for Pathogen Detection

Nigel Cook, Gabriel A de Ridder, Martin D'Agostino and Maureen B Taylor

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.

Section II. Food Microbiology

5. Standardization of Real-time PCR Methods in Food Microbiology

Kornelia Berghof-Jäger

International law requires that only food suitable for consumption may reach the market. To meet this demand, thorough microbiological testing must be performed on raw materials, the manufacturing process and finished products. Real-time PCR methods are particularly well-suited for this testing as they are fast, precise and very specific. Multiple methods, including real-time PCR, exist for testing the same analyte. These are favored according to regional preferences and regulatory requirements. However, global trade could be simplified if there was an international consensus on a set of analytical standards. The International Organization for Standardization (ISO) and the European Organization for Standardization (CEN) are platforms for generating standards through open, balanced and consensus-driven processes. To avoid duplication of work and structures, an agreement on technical co-operation between ISO and CEN (Vienna Agreement) was approved in 1991. This allows for focused expertise to be used efficiently to benefit international standardization. Currently, a few general Standards exist which describe the basic requirements of PCR methods. General standardization documents focusing on performance characteristics as well as basic requirements and definitions of real-time PCR are in development. Standards for specific detection of the food-borne pathogens Clostridium botulinum, Yersinia, STEC, Vibrio and viruses are also in progress. In parallel, standardization of real-time PCR-based methods for the detection of genetically modified organisms (GMO) and allergens in food are ongoing.

6. Real-time PCR Detection of Foodborne Pathogenic Salmonella spp.

Burkhard Malorny, Dietrich Mäde and Charlotta Löfström

Infections by Salmonella enterica are a significant public health concern worldwide. Salmonellae form a complex group of bacteria consisting of two species, six subspecies and more than 2500 serovars (serotypes). Mainly through ingestion of contaminated food or feed, they cause self-limiting gastrointestinal disease in a wide range of mammalian hosts. Within the last decade, numerous real-time PCR assays have been developed for rapid detection of salmonellae in potentially contaminated food or feed. Some of them were extensively validated and are useful for diagnostic laboratories. Furthermore, effective sample preparation prior to the analytical real-time PCR assay avoids inhibitory substances disturbing the PCR and contributes to a high sensitivity. We discuss appropriate sample preparation methods including enrichment procedures for various food items and analytical real-time PCR assays for the detection of Salmonella and give a state-of-the-art summary what targets are used and how valid the assays are to apply as diagnostic tool. Furthermore, recommendations for selection of an appropriate real-time PCR method are presented.

7. Real-time PCR Methods for the Detection of Listeria monocytogenes in Foods

David Rodríguez-Lázaro and Marta Hernández

Listeria monocytogenes is a bacterial pathogen that causes serious localized and generalized infections in humans. Traditional detection methods of this pathogen involves two enrichment steps and a final isolation in two specific culture media, and a final confirmation using biochemical and/or molecular techniques, and therefore more than 5 days are needed for a final confirmation. An alternative to accelerate results in diagnostics of this pathogen in food is the application of real-time PCR-based methods. They can provide highly selective results with a considerable reduction of the time for analysis. This new methodology has been implemented satisfactory in a wide spectrum of food categories including those more relevant to the transmission of this pathogen to humans.

8. Real-time PCR Detection of Foodborne Pathogenic Escherichia coli

Patricia Elízaquível, Gloria Sánchez and Rosa Aznar

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 chapter the different approaches proposed to circumvent the difficulties to detect pathogenic E. coli in food and the most frequently targeted genes are presented.

9. Detection of Pathogenic Thermotolerant Campylobacter species by Real-time PCR

L. Jesús Garcia-Gil

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°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.

10. Detection of Pathogenic Yersinia enterocolitica by Real-time PCR in Foods

Dietrich Mäde

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.

11. Real-time PCR Detection of Foodborne Pathogenic Vibrio

Luciana Croci and Elisabetta Suffredini

Bacteria of the genus Vibrio are naturally occurring in aquatic environments, the majority of them being non-pathogenic to humans, whereas some species have been associated with human disease. In particular V. cholerae, V. parahaemolyticus and V. vulnificus are known to be important human pathogens. The detection of pathogenic vibrios mostly relies on conventional cultural methods that often require further confirmation. Recently molecular methods (conventional PCR and Real Time PCR) to test food and environmental samples for the presence of different potentially pathogenic Vibrio spp have been developed. An overview of the Real Time PCR methods proposed by different authors is reported. For each of the three vibrios (V. cholerae, V. parahaemolyticus and V. vulnificus) the primers and probes targeting, in single or multiplex format, species-specific genes or their pathogenic factors are summarised. Real Time PCR is efficient, rapid and easy to perform, resulting very useful in the qualitative and quantitative detection of pathogenic vibrios in seafood. On the other hand, PCR may amplify dead cells providing false positive results or may be influenced by the presence of the inhibitors providing false negative results. The proposals of overcoming these problems through a treatment of cultures with ethidium bromide monoazide (EMA) to detect only viable cells and through the addition of an IAC (internal control) for inhibition control are described.

12. Real-time PCR Detection of Foodborne Pathogenic Staphylococcus aureus

B. Stessl and M. Wagner

The real incidence of Staphylococcus aureus the ethological cause of staphylococcal food poisoning (SFP) is unknown due to underreported cases and the misinterpretation of symptoms (vomiting, diarrhoea) also caused by other agents as B. cereus and Norovirus. Conventional microbiological methods recommended by EU regulation 2073 are time-consuming and often lead to "false negative" results as viable S. aureus cells are not detectable but still a higher amount of heat-stable enterotoxins was produced. In the last years several multiplex PCR detection methods targeting staphylococcal enterotoxin coding genes have been described as complementation to direct enterotoxin detection by Enzyme Linked Immunosorbent Assay (ELISA). To gain a better insight in the enterotoxin gene status of samples quantitative real-time PCR assays have been developed. The lack of qPCR in distinguishing live and dead bacteria could be neglected as an unexpected S. aureus food poisoning outbreak causes more economic loss as a restrained food lot.

13. Real-time PCR Detection of Foodborne Pathogenic Clostridia

Kathie Grant and Corinne Amar

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 chapter focuses on the practical application of real-time PCR detection assays for these two clostridial foodborne pathogens.

14. Real-Time PCR and other Molecular Detection Methods for Foodborne Pathogenic Viruses

Doris Helen D'Souza, Marta Hernández, Nigel Cook and David Rodríguez-Lázaro

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.

15. Real-Time PCR Detection of Foodborne and Waterborne Parasites

George D. Di Giovanni, Gregory D. Sturbaum, and Huw V. Smith

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 chapter 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.

Section III. Food Quality

16. Real-time PCR Analysis of Genetically Modified Organisms

Arne Holst-Jensen

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 chapter 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 chapter will provide several examples.

17. Real-time PCR Analysis of Food Allergens and Gluten

Carmen Diaz-Amigo and Bert Popping

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 chapter discusses suitable systems for detection of DNA of ingredients and foods containing allergenic proteins, potential pitfalls and multiplex capabilities of such systems.

18. Real-time PCR Methods for Identification of Animal or Plant Species

Barbara Brežná and Ľubica Piknová

Real-time PCR authentication of the biological species in food is justified by creating fair market environment for producers by discouraging fraud. Authenticity also needs to be guaranteed, when local food-manufacturing traditions are protected by regulations, such as Protected Geographical Status (PGS) framework of the European Union. Furthermore, the consumers have a right to correct information about the food content, so that they are able to make informed choices regarding possible food allergies, special diets based on nutritional quality or religious and cultural taboos. Last but not least, endangered biological species need to be monitored along the food chain to fight poaching and illicit trade. The real-time PCR assays for authentication of species are summarized in tables and the considerations regarding the assay design are discussed. The choice of suitable DNA marker is crucial for the specificity of the real-time PCR assay. The assay should be empirically validated on a panel of samples, which are desired to score as positive, for instance cultivars of the species, as well as broad range of those desired to score as negative. Detection of almond and distinction between bread wheat and durum wheat illustrate the problematic situations regarding assay specificity. The sensitivity of the qualitative assay can be enhanced by choice multi-copy gene as a marker, however single-copy genes are considered more suitable for quantitative assays due to copy number stability. Amplification control should be included in real-time PCR assays to add reliability to the results. The optimal approaches to determine the limit of detection and to ensure correct quantification are still debated.