Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels

tmRNA - a novel high-copy-number RNA diagnostic target - its application for Staphylococcus aureus detection using real-time NASBA

A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL(-1) in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target

Real-time pcr method combined with a matrix lysis procedure for the quantification of listeria monocytogenes in meat products

In this study a real-time PCR method has been developed for the specific quantification of the foodborne pathogen Listeria monocytogenes on meat products through the gene hlyA. The PCR was combined with a matrix lysis that allowed the obtaining of the microorganisms without sample dilution and the elimination the PCR inhibitors from dry-cured ham. The qPCR method calibration curve had an efficiency of 100.4%, limits of detection and quantification were 30.1 ± 6.2 CFU/g which is under the legal limit of L. monocytogenes in ready-to-eat products, and an analytical variability <0.25 log hlyA gene copies/reaction. The analysis was performed simultaneously with the reference method ISO 11290-2. The comparison of the qPCR-matrix lysis results with the reference method showed an excellent correspondence, with a relative accuracy between 95.83–105.20%. Finally, the method was applied to commercial derived meat samples and the pathogen was quantified in one of the commercial samples assayed in 69.1 ± 13.9 CFU/g while the reference method did not quantify it. The optimized qPCR showed higher precision and sensitivity than the reference method at low concentrations of the microorganism in a shorter time. Therefore, qPCR-matrix lysis shows a potential application in the meat industry for L. monocytogenes routine control. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Isolation and Identification of Foodborne Pathogens of Special Interest in Food Safety

[ES] La seguridad alimentaria es una prioridad para la población y en la actualidad cobra mayor importancia por ciertas tendencias alimentarias como el consumo de alimentos crudos y la distribución generalizada de alimentos orgánicos, que pueden ser la causa de enfermedades transmitidas por alimentos. Para garantizar la seguridad alimentaria, la detección de estos microorganismos debe realizarse de manera rápida y eficiente. Par eso, el método de cultivo microbiológico se considera el oficial para la detección de estos patógenos. Sin embargo, adolece de importantes inconvenientes, ya que no solo requiere mucho tiempo, sino que también es laborioso y consume muchos recursos. Además, puede ser limitado con respecto a la detección de bacterias fisiológicamente alteradas y/o estresadas durante el almacenamiento y la conservación. En este trabajo se ha desarrollado un protocolo sencillo y rápido para la detección simultánea de E. coli, L. monocytogenes, S. aureus y S. enterica en alimentos, mediante la combinación de una etapa de co-cultivo en medio líquido y la detección por PCR múltiple. Se ha evaluado la eficiencia de varios medios de enriquecimiento y se seleccionó el agua de peptona tamponada como el medio óptimo para el co-cultivo de las cuatro bacterias diana. También se optimizaron las condiciones de PCR múltiple y se aplicaron tanto a co-cultivos como a muestras de alimentos inoculados artificialmente, lechuga orgánica y carne picada. Después de la optimización, la PCR múltiple desarrollada fue capaz de detectar las cuatro bacterias simultáneamente, hasta con una inoculación inicial de 10^0 UFC/mL. En presencia de ambas matrices alimentarias inoculadas, tras la etapa de co-cultivo, la PCR múltiple pudo detectar simultáneamente las 3 bacterias E. coli, S. enterica y L. monocytogenes, mientras que S. aureus se ha detectado por PCR simplex, a partir del mismo extracto de ADN del co-cultivo. Los resultados obtenidos permiten concluir que el uso de un paso de co-cultivo en Agua peptona tamponada, antes de la detección por PCR simple y múltiple, puede facilitar la detección simultánea de las cuatro bacterias potencialmente presentes en las matrices alimentarias. La presencia o ausencia de la bacteria diana en los alimentos se confirma en unas 30 horas, lo que reduce el tiempo requerido para la detección en comparación con el tiempo mínimo de 7 días por método cultural. Asimismo, permite reducir el número de medios de cultivo y reactivos, para el aislamiento e identificación de bacterias que no son detectadas por PCR y que no están presentes en las matrices alimentarias, lo que supone un importante ahorro económico.[CA] La seguretat alimentària sempre és una prioritat per a la població i en l' actualitat cobra major importància per certes tendències alimentàries, com el consum d' aliments crus i la distribució generalitzada d' aliments orgànics, que poden ser la causa de malalties transmeses per aliments. Per garantir la seguretat alimentària, la detecció d' aquests microorganismes s' ha de realitzar de manera ràpida i eficient. Per a això, el mètode de cultiu microbiològic es considera l' oficial per a la detecció d' aquests patògens. Però, hi ha importants inconvenients, ja que no només requereix més temps, sinó que també és laboriós i consumeix molts recursos. A més, pot ser limitat pel que fa a la detecció de bacteris fisiològicament alterats i/o estressats durant l'emmagatzematge i la conservació. En aquest treball s'ha desenvolupat un protocol senzill i ràpid per a la detecció simultània d' E. coli, L. monocytogenes, S. aureus i S. enterica en aliments, mitjançant la combinació d' una etapa de co-cultiu en medi líquid i la detecció per PCR múltiple. S'ha avaluat l'eficiència de diversos mitjans d'enriquiment i s'ha seleccionat l'aigua de peptona tamponada com el medi òptim per al co-cultiu dels quatre bacteris diana. També es van optimitzar les condicions de PCR múltiple i es van aplicar tant a co-cultius com a mostres d'aliments inoculats artificialment, enciam orgànic i carn picada. Després de l'optimització, la PCR múltiple desenvolupada va ser capaç de detectar els quatre bacteris simultàniament, fins a una inoculació inicial de 10^0 UFC/mL. En presència d' ambdues matrius alimentàries inoculades, després l' etapa de co-cultiu, la PCR múltiple va poder detectar simultàniament els 3 bacteris: E. coli, S. enterica i L. monocytogenes, mentre que S. aureus s' ha detectat per PCR simple, a partir del mateix extracte d' ADN del co-cultiu. Els resultats obtinguts permeten concloure que l' ús d' un pas de co-cultiu en Aigua de peptona tamponada, abans de la detecció per PCR simple i múltiple, pot facilitar la detecció simultània dels quatre bacteris potencialment presents en les matrius alimentàries. La presència o absència del bacteri diana en els aliments es confirma en unes 30 hores, la qual cosa redueix el temps requerit per a la detecció en comparació amb el temps mínim de 7 dies per mètode cultural. Així mateix, permet reduir el nombre de mitjans de cultiu i reactius, per a l' aïllament i identificació de bacteris que no són detectats per PCR i que no estan presents en les matrius alimentàries, la qual cosa suposa un important estalvi econòmic.[EN] Food safety is a priority for the population and is nowadays more important than ever due to certain dietary trends such as the consumption of raw foods and the widespread distribution of organic foods, which may be the cause of foodborne diseases. To ensure food safety, the detection of these microorganisms must be done quickly and efficiently. Although, the microbiological culture method is considered to be the official method for the detection of these food-borne pathogens, it suffers from significant drawbacks, such as time-consuming, laborious and expensive, in addition it may be limited regarding the detection of physiologically altered and/or stressed bacteria, during storage and preservation. In this work has been developed a simple and rapid protocol for the simultaneous detection of E. coli, L. monocytogenes, S. aureus and S. enterica in food, by combining a liquid co-culture step and detection by multiplex PCR. The efficiency of several enrichment media was evaluated and buffered peptone water was chosen as the optimal medium for the co-culture of the four target bacteria. Then, optimized multiplex PCR conditions were applied to both the co-cultures and the samples of artificially inoculated foods, organic lettuce and ground meat. After optimization, the developed multiplex PCR was able to simultaneously detect the four bacteria, up to an initial inoculation of 10^0 CFU/mL. In the presence of the two inoculated food matrices, after a co-culture step, the multiplex PCR could simultaneously detect the 3 bacteria: E. coli, S. enterica and L. monocytogenes, whereas, S. aureus has been detected by simplex PCR, from the same co-culture DNA template. The results obtained allow conclusion that the use of a co-culture step in Buffered Peptone Water, before detection by simplex and multiplex PCR, can facilitate the simultaneous detection of the four bacteria potentially present in the food matrices. The presence or the absence of the target bacteria in food is confirmed in approximately 30 hours, which reduce the time required for the detection compared to the minimum time of 7 days by cultural method. Also, it allows to reduce the number of culture media and reagents, for the isolation and identification of bacteria that are not detected by PCR and which are not initially present in the food matrices, which represents a significant economic savings.Boukharouba, A. (2022). Isolation and Identification of Foodborne Pathogens of Special Interest in Food Safety [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182828TESI

Fast, Affordable and multiplexed foodborne pathogen detection on miniaturized devices

Food poisoning is a global public health concern affecting, not only developing, but also developed countries, where several pathogens are responsible for these foodborne diseases causing high number of hospitalization and death. The objective of this thesis was to develop an improved methodology for the multiplex detection of Salmonella spp., Shiga Toxin-producing E. coli (STEC) and L. monocytogenes, based in DNA detection, and its integration in a miniaturized device. To achieve this goal, the different steps of the analysis, including the sample pre-treatment, DNA amplification and visualization of the results were addressed in order to reduce the time of analysis, reduce the cost, and allow naked-eye detection. The methodology developed in this project allowed for the multiplex detection of the three targets, reducing the time of analysis to only 9 h vs 7 days by traditional approaches with similar sensitivity and specificity to qPCR. Furthermore, the possibility to integrate the amplification step in a miniaturize device opens the door for the development of Point-of-Care systems

Developments in nanoparticles for use in biosensors to assess food safety and quality

The following will provide an overview on how advances in nanoparticle technology have contributed towards developing biosensors to screen for safety and quality markers associated with foods. The novel properties of nanoparticles will be described and how such characteristics have been exploited in sensor design will be provided. All the biosensor formats were initially developed for the health care sector to meet the demand for point-of-care diagnostics. As a consequence, research has been directed towards miniaturization thereby reducing the sample volume to nanolitres. However, the needs of the food sector are very different which may ultimately limit commercial application of nanoparticle based nanosensors. © 2014 Elsevier Ltd

Simultaneous Detection of Four Main Foodborne Pathogens in Ready-to-Eat Food by Using a Simple and Rapid Multiplex PCR (mPCR) Assay

[EN] The increasing consumption of organic or ready-to-eat food may cause serious foodborne outbreaks. Microbiological culture for detection of food-borne pathogens is time-consuming, expensive and laborious. Thus, alternative methods such as PCR are usually employed for outbreaks investigation. In this work we aimed to develop a rapid and simple protocol for the simultaneous detection of E. coli, L. monocytogenes, S. aureus and S. enterica, by the combination of an enrichment step in a single culture broth and a multiplex PCR (mPCR) assay. The effectiveness of several enrichment media was assessed by culture and PCR. Buffered Peptone Water was selected as the optimum one. Then, mPCR conditions were optimized and applied both, to pure co-cultures and artificially inoculated food samples (organic lettuce and minced meat). In culture medium inoculated at 100 CFU/mL, mPCR was able to detect the four microorganisms. When performed on artificially food samples, the mPCR was able to detect E. coli, S. enterica and L. monocytogenes. In conclusion, Buffered Peptone Water broth can effectively support the simultaneous growth of E. coli, S. aureus, L. monocytogenes and S. enterica and could be, thus, used prior to a mPCR detection assay in ready-to-eat food, therefore considerably reducing time, efforts and costs of analyzes.FundingThis research was funded by Ministerio de Ciencia e Innovacion, Spain, Grant number PID2019-105691RB-I00. Miguel Garcia-Ferrus is the recipient of a PEJ2018- 003746A Grant from Ministerio de Economia, Industria y Competitividad, Spain.Boukharouba, A.; González Pellicer, A.; García-Ferrús, M.; Ferrús Pérez, MA.; Botella Grau, MS. (2022). Simultaneous Detection of Four Main Foodborne Pathogens in Ready-to-Eat Food by Using a Simple and Rapid Multiplex PCR (mPCR) Assay. International Journal of Environmental research and Public Health. 19(3):1-18. https://doi.org/10.3390/ijerph1903103111819

Rapid Detection of \u3cem\u3eListeria monocytogenes\u3c/em\u3e

Listeria monocytogenes is a foodbome pathogen that can cause severe illness and even death. It is found in dairy and meat products. The focus is on rapid detection since conventional methods are time consuming (4-5 days). Pre-enrichment steps, as part of those methods, are time consuming. Our objective was to develop a detection system without a pre-enrichment step, giving a final result within 2 to 4 h. In the concept of the need for speed, a detection system with an antibody-based capture technique, followed by polymerase chain reaction (PCR), was developed. Glass beads coated with a Listeria polyclonal antibody were added to the food sample. After a static incubation/capturing step, beads-cell complexes were separated from the food, and boiled to lyse the cells and release the DNA. In a final PCR/electrophoresis step the DNA samples were analyzed. The use of a flow-based capturing system (ImmunoFlow) was also investigated. Using a bead-antibody complex in this ImmunoFlow setup has several advantages, including the possibility of concentrating the microorganisms out of large food samples (with flow through setup), the exclusion of a pre-enrichment step, and the potential for automation. Besides buffer solution (Tris), different kinds of milk, e.g., pasteurized, Ultra High Temperature (UHT), and raw milk, were also investigated. The detection limit in buffer solution was 1 x 106 CFU/ml no matter if the ImmunoFlow system or the static incubation was used. For the different pasteurized milk samples, the detection limit varied between 1 x 107 and 1 x 108 cells/ml in the static procedure. For UHT and raw milk, however, capturing of Listeria monocytogenes cells was not possible in the static or the ImmunoFlow setup. In conclusion, we developed a rapid and specific detection system for Listeria monocytogenes at high concentration in pasteurized milk using a static capturing procedure. The total test time for this detection system is less than 4 h, which is much faster than the present detection systems (which are using an enrichment step prior to testing). Implementing a real-time PCR system after capture would further reduce this detection time