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Lactococcus garvieae, Yersinia ruckeri and Flavobacterium psychrophilum are three of the most important pathogens in worldwide rainbow trout aquaculture (Oncorrhynchus mykiss Walbaum). In this work, a multiplex quantitative PCR (qPCR) method for the simultaneous detection of the three pathogens was developed and tested in a commercial trout farm. Fifty-four spleen and brain samples from a trout farm were analyzed using both multiplex qPCR and classic microbiological methods. When comparing qPCR results and classic plate diagnostic techniques no negative deviations were observed, which resulted in a 100% relative sensitivity of the multiplex qPCR method developed. In addition, efficiency of the qPCR method ranged between 97.5% and 108.8%. The specificity of the combination of primers and probes was successfully tested in 57 target and non-target bacterial strains Hence, the multiplex qPCR method developed in the present study could be used as a reliable diagnostic tool for the detection of L. garvieae, Y. ruckeri and F. psychrophilum in rainbow trout farms, enabling faster diagnostics (only a few hours) and contributing to a quick response and management of bacterial disease outbreaks.

Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively This work was supported by a partnership agreement project between the Confederación Hidrográfica del Guadalquivir and INL for the development of a system for early detection of zebra mussels through analysis of environmental DNA, and by project Nanotechnology Based Functional Solutions (NORTE-01-0145-FEDER-000019), supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. S. A. acknowledges the Portuguese funding institution FCT – Fundação para Ciência e Tecnologia for Ph.D. scholarship SFRH/BD/140396/2018 SI

Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.

Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens (Salmonella spp. and Escherichia coli O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex “same-day” detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen’s kappa of 0.92, with a Limit of Detection95 below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 104 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.

The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/&micro, L of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.

Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.

The safety of using nano- and microparticles is a developing concern. In this study, we conducted risk assessments of chitosan microparticles (CMs) using in vitro human epithelial cell lines and in vivo animal model, Caenorhabditis elegans. After engineering of various CMs, we screened four CMs based on antimicrobial activity, which is a potential usage for disease treatment caused by multidrug resistant bacteria, and evaluated for risk assessments. CMs, with strong antimicrobial activity, and inorganic nanoparticles (SiO2, TiO2, and ZnO) did not cause toxicity in human cells measured by cell membrane integrity, mitochondria activity, and reactive oxygen species concentration. However, when applied to C. elegans, only CMs generated with low molecular weight chitosan and tripolyphosphate at 0.1% did not affect the lifespan, while the other CMs and inorganic nanoparticles shortened the lifespan, suggesting that they may cause subtle toxicity. These results suggest that C. elegans could be a sensitive animal model to measure low level of toxicity of nano- and microparticles. Taken together, although CMs do not cause toxicity at working concentrations of antimicrobial activity in human epithelial cells, they may cause toxicity at high concentration, suggesting that nano- and microparicles should be thoroughly investigated before they are applied.

In the present study a multiplex real-time PCR method was developed for early detection of diseased fish infected by Aeromonas salmonicida, Vibrio anguillarum, and/or Tenacibaculum maritimum. The method consisted of the detection of three species-specific genes after DNA extraction with a commercial kit. Three types of samples were tested, and the results were compared with those of traditional diagnosis. The method obtained a limit of detection of 104 cfu/mL (2 x 102 cfu/tube). Additionally, 27 samples from fish showing signs of disease were correctly diagnosed by the developed methodology, demonstrating its suitability for implementation in aquaculture.

Campylobacteriosis is one of the most important reported zoonosis worldwide. Besides poultry other sources of infection have been described. In the current study, the incidence of Campylobacter spp. was assessed over a five-month period in mussel samples harvested from one of the most important producing areas (Galicia, NW Spain) in Europe. Out of 91 samples screened, 8% were positive and identified as C. lari by MALDI-TOF and whole genome sequencing. All were detected during the colder months (February and March). The antimicrobial resistance and virulence genes analysis indicated that all were multi-resistant to at least 4 antimicrobials. They were negative for the presence of 5 virulence-related genes. This is the first report of this zoonotic pathogen in mussels from one of the most important shellfish producing regions in Europe. The genomes of these 7 C. lari isolates were released to the genome public database at NCBI.

European Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect

Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.

Dairy products have been implicated in foodborne infections caused by different bacterial pathogens. Among them, Listeria monocytogenes is of particular concern due to its ubiquity, resistance to sanitation processes and high mortality rates resulting from infection. These issues make the development of novel methods for the rapid detection of this bacterium of high interest. The evaluation of a novel multiplex real-time Recombinase Polymerase Amplification method including an internal amplification control is reported in the present work. The method performance was compared to that of the European reference method (ISO 11290-1) for the detection of the species in samples from 40 commercial products, including 14 UHT milk samples, 16 hard cheese samples, 6 infant dairy preparation samples and 4 fresh cheese samples. A limit of detection below 10 cfu/25 g or mL sample was achieved, and values higher than 90% were obtained for relative sensitivity, specificity, accuracy, positive and negative predictive values and the index (kappa) of concordance. Analysis was achieved within one working day, compared to the six days required using the ISO method. Moreover, slight modification of the ISO 11290-1 method to include secondary enrichment in half Fraser broth resulted in the confirmation of all positive samples.

Spain is the third mussel producer worldwide, and the main supplier to the European market, coming 98% of this production from Galicia. Pathogenic species from the genus Vibrio pose a considerable public health threat as the causative agents of both sporadic and epidemic human infections. Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus are the most significant pathogens within this genus. The aims of this study were to investigate the prevalence of human pathogenic Vibrio species in Galician mussels from different geographical areas, and determine their pathogenic profiles by qPCR as rapid diagnostic method. Out of the 101 samples, 72% of the V. parahaemolyticus positive samples, carried at least one virulence factor (tdh and/or trh), being trh was the most prevalent (37%). The highest number of positive samples was found during October and September. During the period of study neither Vibrio vulnificus nor V. cholerae were detected. After enrichment, positive samples were tested for antibiotic resistance genes (ermB and tetQ) by qPCR. No resistance was detected against tetracycline. On the contrary, 52% of the samples were positive for ermB, and out of these, 78% also had, either one or both, V. parahaemolyticus virulence factors.

Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ∼10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.

Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.

The current study was conducted in order to reduce the overall time needed in a previously validated qPCR method, and extend the procedure for the simultaneous detection of an additional pathogen (Shigella spp.).The original method was modified by extending the primary enrichment and eliminating the secondary enrichment. Additionally, a rapid commercial DNA extraction kit was evaluated against our reference protocol taking into consideration DNA concentration obtained, purity of the DNA extracts evaluating two absorbance ratios (260/280 and 260/230), and Cq and final fluorescence after qPCR analysis. Comparable results were obtained with both DNA extraction methods, but the commercial kit performed worse with low bacterial concentrations. A total of 84 spiked and blind samples were analyzed with the rapid protocol without finding significant differences with respect to the original method regarding qPCR efficiency (90–103%), and all the method’s parameters that were previously analyzed (above 91%). Additi...

Listeria monocytogenes is still one of the most significant food-borne pathogens. The reason for this importance relies on its capacity to survive under adverse conditions, such as low pH and/or low temperature, and more important is its high lethality, which can reach 30 % of infected individuals. At present, there are several meth- ods available for its detection and enumeration, and a great effort has been focused on the development and evalua- tion of detection protocols. However, most enumeration approaches, based on culture techniques, take several days to complete, and most alternative protocols do not reach the legal limit required for this bacterium (100 cfu/g). The cur- rent study presents the complete development and evalua- tion of a Most-Probable-Number method combined with a real-time PCR confirmation step, which may be accom - plished in 28 h. The method was evaluated in five differ - ent food types at different inoculation ranges and was also compared against the ISO 11290-2: 1998 which is the inter- nationally accepted reference method. No discrepancies were observed with respect to the expected results of either inoculated or natural samples, thus proving the suitability of the method for its direct application in the food industry for the reliable control of this pathogen. Our MPN-qPCR proved to be faster than other molecular and traditional methods, as well as to have high sensitivity, specificity and accuracy.

Real-Time PCR is a very powerful tool with multiple applications in food microbiology, being pathogen detection the most important for food safety. In early January 2013 an increase in the incidence of Listeria monocytogenes was observed in a mussel-processing facility in the north-west of Spain. Then, a previously in-house validated qPCR method was applied to detect the contamination origin in the processing line. By the end of the same month a total of 62 samples from different spots in the processing plant were analyzed, obtaining 25 positive results by qPCR. All the isolates, identified as L. monocytogenes, presented the same biochemical profile and belonged to the same molecular typing group (Group 3). After the identification and elimination of the contamination source, 25 additional samples were analyzed over the following nine months, without any positive sample. Results obtained showed that the qPCR method is a suitable technique to identify the exact source of contamination that could appear in food processing plants, saving time respect to traditional culture methods.

The aim of the work, as part of the FP7 Beads project, was to isolate potentially probiotic bacteria targeted to shellfish, which could facilitate depuration of commercial molluscs. A total of 365 bacteria were isolated from shellfish digestive gland and screened for activity against different pathogens. One bacterial isolate obtained from Mytilus galloprovincialis , designated as 3M21 and identified as Enterococcus hirae , presented antibacterial activity against Listeria monocytogenes , Listeria innocua , Enterococcus faecalis , and antiviral activity against Hepatitis A ATCC VR-1402 and murine Norovirus (MNV-1), culturable in vitro . This strain was sensitive to chloramphenicol, fusidic acid, ampicillin, vancomycin and gentamicin. The active substance produced by 3M21 was partially characterized and shown to be proteic, with a molecular weight of 6.95 kDa determined by Tris–Tricine electrophoresis, high thermostability (30 min at 100 °C), resistant to a wide pH range (2–9) and to treatment with certain surfactants (Tween 20, 80 and EDTA at 2 mM). After its partial purification, the bacteriocin was encapsulated in 3% of alginate and an alternative using BaCl 2 instead of CaCl 2 in the hardening bath significantly improved its encapsulation. Although the use of probiotic bacteria is gaining in popularity in the aquaculture industry, its application in shellfish depuration has seldom been studied. The isolation, characterization and microencapsulation of a bacterium with antipathogen activity already acclimatized to seafood environment, as the isolate 3M21, could increase the efficiency of shellfish depuration process.

The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.

Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.

Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100% as well as the positive and negative predictive values.

In recent years the incidence of vibriosis has greatly increased, raising the concern among consumers about the innocuity of certain food products. Previous studies demonstrated various advantages of molecular methods, including qPCR, for the screening of food-borne pathogens. The new method developed in the present study allows fast and reliable detection of the main human pathogenic Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus). Specificity of the combination of primers and probes was successfully tested against several bacterial species and strains (44 different strains). Evaluation of the qPCR efficiency reported a value of 94% with the simultaneous amplification of the internal amplification control. The evaluation of the quality of the method was based on six parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values as well as Kappa index of concordance. Each of the values obtained were higher than 96%. Additionally a very low limit of detection was determined for the developed method (less than 10 cfu/25 g). All the parameters of the method analyzed were obtained from the analysis of a wide variety of foodstuffs, water samples and reference material from proficiency tests, and compared against the culture reference method.

Microbiological analysis of food is an integrated part of microbial safety management in the food chain. Monitoring and controlling foodborne pathogens are traditionally carried out by conventional microbiological methods based on culture-dependent approaches in control laboratories and private companies. However, polymerase chain reaction (PCR) has revolutionized microbiological analysis allowing detection of pathogenic microorganisms in food, without the necessity of classical isolation and identification. However, at present, PCR and quantitative polymerase chain reaction (qPCR) are essential analytical tools for researchers working in the field of foodborne pathogens. This manuscript reviews recently described qPCR methods applied for foodborne bacteria detection, serving as economical, safe, and reliable alternatives for application in the food industry and control laboratories. Multiplex qPCR, which allows the simultaneous detection of more than one pathogen in one single reaction, saving considerable effort, time, and money, is emphasized in the article.

Shiga toxin-producing Escherichia coli are a known cause of serious illnesses, and a major public health concern. The coastal environment is the receptacle of agricultural and urban wastewater effluents, thus STEC can be present. In the current study their occurrence and prevalence in mussels (Mytilus galloprovincialis) cultured in the southern Rias of Galician was assessed. This region is interesting due to its economic importance as Galicia produces the 98% of the mussels cultured in Spain, being this country the third world producer. From June 2012 until September 2013, 241 mussel samples were collected, being 112 positive for eae and 17 also positive for stx1,2. An increase was observed in the winter months for both genes, which seemed correlated with a generalized decrease in water salinity. Serotype O157:H7 was not detected, but one isolate resulted resistant to six antibiotics, denoting the presence of multidrug resistant bacteria in this area. Practical applications A critical step for adequate risk assessments is the evaluation of potential threats. In this sense, very few microbiological parameters are analyzed in bivalve molluscs, being many human pathogens not assayed. Shiga toxin-producing Escherichia coli was detected for the first time in mussels harvested in Galicia, a region of critical importance worldwide for the mussel industry. This study highlights that additional studies to better characterize the microbial populations of this important region are still needed, and furthermore, that measures to control microbial pathogens, such as depuration, must not be taken lightly.

In recent years a new genetic target for Vibrio cholerae detection has been reported, but its application was limited to clinical samples. This target, lolB, has never been applied to either food or environmental samples. In the present study the development, as well as the evaluation and pre-validation, of a real-time PCR method based on lolB, is described. The method included a newly designed hydrolysis probe to enhance its specificity. After comparison against other molecular and traditional methods, similar results were obtained regarding relative sensitivity, relative specificity, relative accuracy, positive and negative predictive values and index kappa of concordance (all higher than 91%), as well as a very low limit of detection (2 cfu/25 g). Additionally, after the analysis of more than 160 different food and environmental samples, its applicability in the food industry was completely demonstrated.

15 páginas, 2 figuras, 2 tablas, The microbiological quality of 18 commercially available in Spain ready-to-eat fish products containing Engraulidae was evaluated through application of the corresponding ISO procedures for total mesophilic aerobic microbial counts, detection and enumeration of enterobacteria, and detection of Staphylococcus spp. All isolates were identified to the species level using two different biochemical methods: the API® test and the Biolog® identification system. The most commonly occurring contaminants found were Enterobacteriaceae—such as Citrobacter freundii and other Citrobacter species, Enterobacter cloacae, Cronobacter sakazakii, Hafnia alvei, Pantoea, Proteus ssp., and Escherichia coli. The presence of such opportunistic pathogens and contaminant microflora was confirmed in 61% of the foods sampled., This study was supported by Project Grant No. 09TAL002CT from the Xunta de Galicia, Spain.