BACTERIOLOGY

INTRODUCTION
Nucleic acids present in food are of no nutritional value but are characteristic for the various biological components in complex products. Analysis of specific nucleic acids in food allows the determination of the presence or absence of certain constituents in complex products or the identification of specific characteristics of single food components. As DNA is a rather stable molecule, processed food is generally analyzed using DNA-based method, but DNA is also the preferred analyte for seeds, raw material and ingredients. DNA-based detection systems for genetically modified food and food-borne pathogens have been developed recently. Furthermore, the detection of plant and animal species as well as allergens and certain ingredients or contaminants in the final food products has been shown to be feasible with DNA-based methods. Because of its high sensitivity, its specificity and rapidity the polymerase chain reaction (PCR) is the method of choice for this purpose

BACKGROUND
The principle Technological advances have increased the speed of diagnostic testing for many diseases. However, for bacterial food poisoning, stool culture, which can take up to 1 week, is still the only method routinely used for diagnosis in most UK microbiology laboratories,methdologies emerging for the rapid diagnosis of food poisoning are immunoassays, which detect antigens or antibodies from pathogens, and polymerase chain reaction (PCR), a commonly used technique to amplify and detect pathogenic DNA/RNA. Both techniques may significantly reduce the detection time for pathogens in faecal or food samples, compared with traditional culture methods.
This systematic review focused on the use of rapid tests for six bacterial food-borne pathogens: Salmonella, Campylobacter, Escherichia coli O157, Clostridium perfringens, Staphylococcus aureus and Bacillus cereus. Diagnostic accuracy was assessed, and an economic model was subsequently developed, assessing costs and cost-effectiveness of PCR and immunoassays, compared with culture.

ABSTRACT
Public awareness of microorganisms transmitted by food, which pose a severe threat to human health, has increased dramatically in recent years. Some of the agents, which have been responsible for numerous cases and outbreaks of food-borne illness and also many deaths, are Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7 and other Shiga toxin-producing E. coli, Yersinia enterocolitica, Clostridium perfringens, Giardia, Cryptosporidium, Cyclospora, hepatitis A virus, and noroviruses. Thus, there is a need for rapid, sensitive, and reliable methods for detection of food-borne pathogens and for investigating cases and outbreaks of illness caused by these agents. Advancements in technology have resulted in the development of various types of rapid methods for detection, identification, and enumeration of food-borne pathogens. In addition to providing results in a shorter length of time than conventional culture-based methods, rapid assays are often more sensitive, specific, and more accurate than classical methods. Rapid methods include miniaturized biochemical kits and antibody- and nucleic acid-based assays. The PCR is a nucleic acid amplification technique, which has become a very useful tool for rapid detection, identification, and typing of pathogenic microorganisms both in the clinical setting and in food and environmental microbiology laboratories. The PCR is a rapid technique, with both high sensitivity and specificity. In recent years, the PCR has advanced from end-point detection to detection of product while the reaction is occurring. This is referred to as ¿real-time¿ PCR, and the technique can be used to quantify target DNA. Furthermore, there is currently much interest in the development of rapid diagnostic systems that have the capability to link sample processing, preparation, and simultaneous detection of multiple pathogens and new threats, including potential bio-threat agents. Use of rapid methods, particularly techniques of PCR are important for safety of food supply





PCR IN FOOD ANALYSIS INCLUDING ADVANCEMENTS
PCR is an in vitro method to generate copies of a defined DNA sequence. In principle, a specific DNA fragment, flanked by two oligonucleotides serving as primers for the reaction, is amplified by a thermostable polymerase. The reaction consists of three functional steps per cycle of amplification, each determined by a different temperature to allow melting of the double-stranded DNA, annealing of the primers, and extension of the primers by the polymerase. Typically, 25-40 cycles of this temperature profile are run to produce a detectable quantity of copies of the template DNA fragment. Primers are short single-stranded DNA molecules, usually 18 to 35 bases in length, designed to bind selectively to the complementary sequences of the target DNA segment. During primer annealing one primer has to bind in forward and the other one in a defined distance in reverse orientation to the separated DNA strands. Therefore, designing PCR primers is a critical step in PCR analysis, because the PCR primers need to have the required specificity. In other words, DNA sequences characteristic for the organism or constituent to be identified have to be available





PCR ANALYSIS INCLUDES FOLLOWING STEPS

Isolation of DNA from the food, amplification of the target sequences by PCR, separation of the amplification products by agarose gel electrophoresis and estimation of their fragment size by comparison with a DNA molecular mass marker after staining with ethidium bromide (figure below) and finally a verification of the PCR results by specific cleavage of the amplification products by restriction endonuclease or the more time-consuming, but also more specific, transfer of separated amplification products onto membranes followed by hybridisation with a DNA probe specific for the target sequence. Alternatively amplification products may be verified by direct sequencing or a second nested or semi-nested PCR. A very convenient approach is to perform PCR amplification and verification in one single run by using a target-specific fluorescent-labelled oligonucleotide probe in a real-time PCR system. Real-time PCR requires more expensive laboratory equipment, but allows the gel free product detection without the


need to open the PCR tubes after amplification again. This approach is therefore less time-consuming and labour-intensive. It implies a lower risk of contamination and there is no need to use mutagenic staining dyes such as ethidium bromide. With real-time PCR also highly accurate quantitative results can be obtained.
Real-time PCR systems rely upon detection and quantitation of a signal generated from a fluorescent reporter that increases in direct proportion to the amount of PCR product generated (shown in figure). Today two different real-time systems are in use, the capillary cyclers and the block cyclers. A capillary cycler makes use of performing PCR in a small reaction volume in a glass capillary exposed to a temperature regulated air stream, which results in very rapid thermal cycling protocols. A PCR with 35 cycles is completed within about 25 minutes, but due to the thermocycler available only 32 single PCR reactions can be run at the same time. The block cyclers use in general the 96 microtiter-plate format. Thus, 96 single PCR reactions can be run simultaneously in about 1-1.5 hours for a PCR with 35 cycles. These real-time systems can employ either a target-specific oligonucleotide probe with a reporter dye and quencher dye attached such as Hybridisation probe that binds to all double-stranded DNA. The target-specific probes anneal specifically to the single-stranded DNA within the region flanked by the two PCR primers. Due to the proximity of the quencher to the reporter, the quencher suppresses the fluorescence emitted by the reporter by Förster Resonance Energy Transfer (FRET) through space. During PCR the reporter is separated from the quencher, which results in an increase of the reporter signal. The increase in reporter fluorescence is proportional to the amount of the specific PCR product. The cycle number required until the fluorescence level crosses a threshold of detection (C T = threshold-cycle) is used to calculate quantitative data . Green possesses a selective affinity to double-stranded DNA, but does not react with single-stranded DNA or with RNA.. However, amplification products are characterized by melting point analysis. Non-specific products should therefore be distinguished from the desired PCR product by a difference in the melting points.





SAMPLING AND SAMPLE PREPARATION
The sampling plan determines how representative the result of the analytical procedure is, whereas quality and quantity of the analytes may vary depending on sample preparation . Thus, sampling and sample preparation are crucial steps in the process of identification and quantification of a certain food component. The limit of detection of an analytical method as a whole is determined, not by the most sensitive part of the procedure, but by the least, which is in most cases sampling and, therefore, the outcome of analytical detection procedures is as good as the quality of sampling. Adequate sampling plans are dependent on the type of material to be analyzed (raw materials, pure ingredients or finished processed food). The higher the degree of heterogeneity, the more critical will be the choice of the appropriate sampling plan, because the sample has to be representative for the material to be analyzed. Both, the number of samples to be taken and the appropriate sample size will be defined by the degree of heterogeneity of the material to be analyzed and by the threshold limit up to which the result should be reliable. Furthermore, appropriate sample size is, at least in part, determined by the sensitivity of the analytical method. Sample representativity must also be maintained during subsequent reduction of the field sample to laboratory and test samples. For routine DNA analysis, 1 to 5 g is generally considered a maximum realistic test portion size , but due to the high sensitivity of the PCR even between 100 mg and 350 mg of vegetal material is sufficient for DNA extraction.
Quality and yield of the isolated DNA are two critical factors in DNA preparations for PCR analysis. With the exception of grains, fruits and other raw materials, the products to be analyzed such as food samples, contain material that has undergone varying levels of processing, including physical, chemical and enzymatic treatment that influences the quality and amount of the DNA. Mechanical treatment results in fragmentation of DNA, heat treatment in DNA degradation, low pH in increased chemical hydrolysis and depurination of DNA and enzymatic treatment may result in an enzymatic hydrolysis or modification of the DNA. Moreover processing may lead to a complete degradation or removal of the DNA. Failures in extracting detectable DNA levels have so far been reported for soybean sauce, margarine, sugar, refined oils and distilled ethanol produced from genetically modified potatoes .
Quality and quantity of the DNA obtained for PCR varies not only according to the material under examination and the degree of processing the material has been subjected to, but also according to the DNA extraction method applied. Because of the existence of a great number of very different products which have to be analyzed, a unique DNA isolation procedure for all the different matrices does not exist . The yield and purity of the extracted DNA could be improved by adapting the extraction procedure to the matrix of the DNA source. Fat, polysaccharides, polyphenols and other secondary compounds are reported to pose a major problem in PCR analysis, since these compounds can irreversibly interact with proteins and nucleic acids and may therefore act as PCR inhibitors. The exclusion of PCR inhibitors is a very crucial point in PCR analysis. In principle, two different DNA isolation protocols with plenty of variations are used for DNA extraction from complex food matrices: the CTAB method and commercially available DNA-binding silica columns. The classical protocol for DNA isolation is based on an incubation of the samples in the presence of a detergent such as cetyltrimethylammonium bromide (CTAB) or sodium dodecylsulfate (SDS) and a treatment with organic solvents such as chloroform and phenol, respectively, followed by precipitation of DNA with isopropanol or ethanol. The second protocol is based on commercially available DNA-binding resins as ready to use kits. In general, the classical detergent-based methods have been reported to result in higher DNA yields but poorer DNA quality compared to the DNA-binding resins and the extraction is more time-consuming . However, the DNA yields received with the recently commercialized DNA purification kits for food samples are comparable to those obtained with the classical detergent-based methods.
CONTROL REACTION
Once the primers are designed and the conditions for a robust assay are optimized, PCR is a very sensitive, rapid, and relatively easy to handle assay for the safety and quality assurance of agricultural commodities, food and animal feed. The total detection time, that is the period of time from when the samples were taken until the PCR products were visualized, is approximately 6-8 hours. However, to exclude false positive and/or false negative results in the PCR analysis several controls have to be included into the methodology. To exclude false negative results, the absence of inhibitors has to be shown by a control PCR using an internal standard or by spiking the DNA preparation with the target sequence. It is also possible to check DNA quality by a separate PCR using a target sequence always present in the product to be analyzed. False negative results might also be due to the inactivation of PCR reagents. To assure the quality of the PCR reagents, a positive control is used, that is the quality of the PCR reagents is tested by performing a PCR close to the limit of detection with pure target DNA. To avoid misinterpretation due to false positive results because of a contamination of reagents or the laboratory itself, a negative control is applied, that is performing the complete PCR assay from extraction to amplification without a DNA source.
GMO ANALYSIS
The introduction of genetically modified organisms (GMO) and food derived thereof has generated a demand for appropriate analytical methods to assure compliance with legislation. Therefore, there is a need to monitor and verify the presence and amount of GMOs in agricultural crops and in products derived thereof. In addition, it is important to know whether the GMO detected is authorized. Thus, reliable detection and quantitative analytical methods are urgently needed. So far, GMO detection and quantification relies either on DNA detection using PCR, or on protein detection using immunological assays such as the Enzyme-Linked ImmunoSorbent Assay (ELISA). Prerequisites for the development of immunological detection methods are the availability of high affinity antibodies directed against the protein to be detected and an active expression of the newly introduced gene in the tissue to be analyzed. It should be noted, however, that genetic modifications do not necessarily result in the production of a new protein and even if, protein expression levels are not always sufficient for detection purposes. In addition, the new protein may be produced only in certain parts of the organism or its level varies in different tissues or during different phases of the physiological development. Since immunoassays require antigens with a native structure, any conformational change in the epitope structure of the protein to be detected renders the assay ineffective. Because food processing frequently leads to such conformational changes, protein detection using immunoassays is limited to grains, raw materials as well as fresh and unprocessed food. A further drawback of the immunoassays is their inability to identify the source of the protein binding to the antibody. Therefore, such protein-based detection methods will not be able to distinguish between two GM varieties expressing the same protein, even if different genetic constructs were used for transformation. Beyond simple detection, it is necessary to determine whether the product under investigation contains GM material above or below a specified threshold concentration. Within the European Union for example, Regulation (EC) No 1829/2003 on genetically modified food and feed amended September 22 nd, 2003 fixes a threshold of 0.9% on an ingredient level for adventitious or technically unavoidable contamination of GM material in a non-GM background. Therefore, labelling becomes mandatory, if GM material derived from one organism is present in a product in a proportion above 0.9% on the ingredient level, but the regulation does not specify whether this is mass per mass, genome per genome or any other unit. The quantitative nature of immunoassays is well established and quantitative results from protein assays are expressed on mass equivalents, however, precise calculation of GM material is only possible by normalising the transgenic to an internal housekeeping protein. In addition, quantification using protein-based methods is very difficult because expression levels of proteins may vary in different tissues as well as with age and environmental conditions. Another factor affecting determination of the concentration of GM material by immunoassays is the above mentioned inability to identify the source of the protein binding to the antibody. As the concentration of a specific transgenic protein may vary in crop varieties, it is, for example, impossible to determine the total concentration of GM material in a product containing those varieties in unknown proportions with a single immunoassay targeting the transgenic protein, even if the product consists of only one ingredient, such as maize flour. Thus, immunoassays will not be developed for analyzing processed food; however, they are used successfully to test seeds, raw materials and less processed materials consisting of only one organism .
The first official method for the detection of a food product derived from genetic engineering was PCR-based and published in 1993 in the Collection of Official Methods under Article 35 of the German Federal Foodstuffs Act (now Article 64 of the German Act on Food, Commodities and Animal Feed) . Since this time several official PCR-based methods for different genetically modified organisms used for food production have been published . DNA sequences of the genes for the new traits, of marker or selection genes and of the promoter or terminator could act as a target in PCR. The first PCR-based methods for GMO detection were screening methods or construct-specific approaches. Since most of the transgenic crops approved in the early years of GMO commercialization contained the 35S promoter of Cauliflower Mosaic Virus and/or the NOS terminator of Agrobacterium, these genetic elements were used as target sequences for a general screening . This had the advantage of getting a hint on the presence of transgenic sequences from a wide number of different GMOs. The absence of an amplification product points to the absence of transgenic sequences, whereas the presence of an amplification product points to the presence of a GMO or a contamination by Cauliflower Mosaic Virus or Agrobacterium, respectively. Since it is not possible to identify the GMO itself by using the screening approach, a unique DNA-sequence for the GMO to be identified has to be used as a target sequence in a follow-up PCR. As a unique DNA sequence, a region of the newly introduced DNA spanning the boundary of at least two adjacent genetic elements was used (construct-specific approach) . However, for an unequivocal identification of a GMO by PCR, primer selection has to be based on target sequences that are characteristic for the individual transgenic organism, such as the cross-border region between integration site and the newly introduced genetic element of a specific GMO or specific sequence alterations due to truncated gene versions or altered codon usage (event-specific approach) . Therefore, a prerequisite for designing specific primers for the identification of GMOs by PCR is the availability of detailed information on their molecular make-up. Unfortunately, information about the molecular make-up of non-authorized GMOs is in general not available and therefore, it is almost impossible to detect the presence of non-authorized GMOs. In addition, there might be differences on the genotype level with respect to the engineered trait between the cultivated, commercialized crop lines and the originally approved GMO line, because of using the latter in crossing/backcrossing programmes. In general, the exact genetic composition of the commercialized line is not known. Furthermore, it is impossible with the already available GMO detection assays to distinguish between two independent but related GMOs and one single GMO in which both traits have been combined by breeding (gene stacking).
Within the European Union it becomes necessary to assess compliance with the 0.9% threshold regulation by the determination of the amount of each of the GMOs present in the individual ingredients from which it has been prepared if a product has been shown to contain one or more authorized GMOs. The aim of these quantitative approaches is not an absolute but a relative quantification, hence, not the absolute amount of material derived from genetic engineering will be determined in a product but the percentage of GM material with respect to a certain food ingredient, such as the percentage of transgenic soybeans in the whole soybean fraction of a certain ingredient. For relative GM material concentrations in food mixtures, the quantification of a GMO-specific gene has to be normalized to a species-specific reference gene. In practice, accurate relative quantification might be achieved by one relative quantification (determination of ΔCT using multiplex PCR) or a combination of two absolute quantification reactions; one for the GMO-specific gene and a second for the species-specific reference gene. Taking into consideration the respective number of copies per genome and with the assumption that the GMO material has been submitted to the same treatment as the non-GMO material, the measurement can be expressed as a genome percentage. This ratio can be considered to be equal to the cell ratio, which is generally equal to the mass ratio. Calculation of the relative amount of GM material is obviously dependent on the number of inserted target sequences and as mentioned above, the approved and the cultivated lines may show differences on the molecular level in respect to these target sequences. Currently, real-time PCR can be considered the most powerful tool for the detection and quantification of GMOs in a wide variety of agricultural and food products. The accuracy of the method is certainly dependent on the type of GMO, food matrix, and processing involved, and the analysis of samples containing low concentrations of GMOs may show a great error.

DETECTION OF FOOD-BORNE PATHOGENS
Food-borne illness, because of the contamination of food with pathogenic microorganisms is still one of the major problems of the food industry, especially in countries with poor hygienic conditions. Therefore, the rapid detection of pathogens such as Salmonella sp., Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, pathogenic Escherichia coli, Bacillus cereus, Shigella sp. or Staphylococcus aureus and other microbial contaminants in food is critical for ensuring the safety of consumers. Traditional methods to detect food-borne bacteria often rely on time-consuming growth in culture media (up to 4 days), followed by isolation, biochemical identification, and sometimes serology. Recent advances in technology make detection and identification faster and more convenient, but some questions about their sensitivity and specificity still remain. Molecular biological methods that use antibodies and nucleic acids to detect specific food-borne bacterial pathogens were scarcely known a decade and a half ago. Few scientists could have predicted that these tools of basic research would come to dominate the field of food diagnostics. Today, a large number of cleverly designed assay formats using these technologies are available commercially for the detection in food of practically all major established pathogens and toxins, as well as of many emerging pathogens. These tests range from very simple antibody-bound latex agglutination assays to very sophisticated DNA amplification methods. ELISA´s have been the methods of choice to speed up the analysis until now, but PCR-based detection systems are about to dominate the field . Within limited workload, PCR-based methods may provide confirmed results within about 30 hours without the necessity to routinely isolate pure cultures.
Although PCR-based assays are in general more specific, sensitive, and faster than conventional microbiological methods, the complexities of food matrices continue to offer unique challenges that may preclude the direct application of these molecular biological methods. Consequently, a short cultural enrichment followed by physical separation of the organisms from the culture medium is still required for food samples prior to analysis with these assays. The cultural enrichment prior to DNA extraction and PCR analysis results in a dilution of PCR inhibitors and an increased number of target cells and therefore in a higher sensitivity. The major advantage of the cultural enrichment however, is the fact that only viable cells are detected. On the other hand, the cultural enrichment prevents the determination of the initial number of bacterial cells present in the food sample. This is not really a drawback, because in general world-wide legislation needs only the verification of the absence of any viable cell in a defined amount of food. In addition, a few systems based on the isolated mRNA have been established . Since mRNAs are short living molecules, a signal in the PCR system could be generated only by viable cells. These systems have the advantage of omitting the cultural enrichment. However, they are not considered to be suitable for routine analysis of food samples, because of the high likelihood of false positive (DNA contamination of the RNA preparation) and false negative (instability of the mRNA) results.
PCR systems for the detection of food-borne pathogens target in general genes involved in pathogenicity of these bacteria. Thus, a positive PCR result only indicates that bacteria with those gene sequences are present and that they have the potential to be toxigenic, but not, that the gene is actually expressed and that the toxin is made. Therefore, rapid methods are generally used as screening techniques, with negative results accepted as is, but positive results requiring confirmation by the appropriate official method, which, in many instances, is cultural. Although confirmation may extend analysis by several days, this may not be an imposing limitation, as negative results are most often encountered in food analysis. The greater detection sensitivity of PCR-based methods may also affect existing microbiological specifications for food; this undoubtedly will have repercussions on the regulatory agencies, food manufacturers, and also consumers.

SPECIES IDENTIFICATION
Interspecies meat adulteration or preparation of meat products by mixing meats is a common procedure in most countries. This is of major concern for many consumers, particularly in relation with ambiguous or improper labelling, adulteration with cheaper meats, or religious specifications such as “kosher” food for Jews and “halal” foods for Muslim prohibitions. Therefore, there is a need for rapid and reliable methods for species identification in such food commodities. Protein-based species identification is specific and sensitive, but cross-reactions of closely related species cannot be ruled out. Furthermore, due to protein denaturation species identification by protein-based methods is known to be especially difficult when meat products have been thermally processed. These problems can be solved with DNA-based methods. Early methods based on hybridization of specific probes were time-consuming and often not sufficiently sensitive. Today PCR-based methods have become very important and are widely used. In principle, two different approaches are applied in the area of species identification: the use of species-specific primers or the use of primers binding to conserved regions of a certain gene present in all species to be identified and subsequent identification of the species by restriction fragment length polymorphism (RFLP) . To achieve a high sensitivity in the PCR assays, genes present in a high copy number within the genome are used as targets. These genes include rDNA (5S, 12S, 18S) (13,15,67) genes or mitochondrial DNA (cytochrome C) genes (14,26,68). It was reported that the presence of 1% of an animal species in a mixed meat product could be easily verified . In some cases detection limit was even better. Therefore, these assays can be useful for the accurate identification of animal species present in meat products, avoiding mislabelling or fraudulent species substitution in meat mixtures.
DETECTION OF FOOD CONSTITUENTS (ingredients or contaminants)
PCR-based methods are also increasingly used for the detection of ingredients and contaminants in food products such as soy in meat products , wheat in non-wheat products or allergens in diverse food . The approach applied is essentially the same as for species identification in the meat area. DNA is isolated directly from the food products followed by direct identification of the ingredient or contaminant by a species-specific PCR.
Currently the method of choice for allergen detection in food is the ELISA. ELISA assays are sensitive and specific test, able to yield fully quantitative results. In some instances sensitivity and specificity of the currently available immunological assay may not be satisfactory and a PCR-based assay might be preferred. Even if the employment of a DNA analysis in allergen detection is seen as an attractive alternative to immunological methods it is discussed controversially. PCR-based assays do not detect the allergen or any specific protein itself. Therefore, the result cannot be tied to actual allergenic exposure. Moreover food processing can affect proteins (allergens) and DNA rather differentially and, protein and DNA could be separated during certain processing steps yielding erroneous results regarding the presence of allergens in the product. Despite these limitations, DNA-based methods offer many advantages over protein-based methodologies, preliminary that the target DNA is efficiently extracted under harsh denaturing conditions and is less affected than the extraction of proteins from food matrices . Another advantage of analysing DNA is its stability against geographical and seasonal variations, which may vary protein composition.
CONCLUSION
DNA-based methods are increasingly used for the detection of foreign food constituents, such as microbial pathogens, or the presence of genetically modified crop material. Furthermore, the detection of plant and animal species as well as allergens, certain ingredients or contaminants in the final food products has been shown to be feasible with DNA-based methods. The methods are in general fast, very specific and provide a sensitive tool for the detection of specific food constituents. The choice of the analytical method applied is however, mainly dependent on the food concerned (availability of specific PCR primers) and on the history of processing involved during food production (degradation or even removal of DNA). However, it has to be considered that PCR-based assays detect only the presence of DNA from a living entity. However, this is not always the compound of concern. PCR-based assays do not detect for example the allergen or mycotoxin itself. Therefore, the PCR-based result cannot be tied to actual allergenic exposure or an actual contamination of the sample with mycotoxins. Nevertheless, PCR-based detection methods provide an excellent alternative to more traditional methodologies in the quality and safety assurance of food.

REFERENCES http://www.worldfoodscience.org/cms/?pid=1003869
http://ars.usda.gov/research/publications/publications.htm?seq_no_115=208078&pf=1