Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.     

Commercially Available Rapid Methods for Detection of Selected Food-borne Pathogens

Generally, the enumeration and isolation of food-borne pathogens is performed using culture-dependent methods. These methods are sensitive, inexpensive, and provide both qualitative and quantitative assessment of the microorganisms present in a sample, but these are time-consuming. For this reason, researchers are developing new techniques that allow detection of food pathogens in shorter period of time. This review identifies commercially available methods for rapid detection and quantification of Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and Shiga toxin-producing Escherichia coli in food samples. Three categories are discussed: immunologically based methods, nucleic acid-based assays, and biosensors. This review describes the basic mechanism and capabilities of each method, discusses the difficulties of choosing the most convenient method, and provides an overview of the future challenges for the technology for rapid detection of microorganisms.     

Polymerase Chain Reaction for the Rapid Detection and Serovar Identification of Salmonella in Food and Feeding Stuff

The polymerase chain reaction (PCR) is one of the most important rapid methods for the sensitive and specific detection of pathogenic and spoilage microorganisms. The method is increasingly applied in surveillance and monitoring programs to detect pathogens, especially for ensuring the safety and quality of food. The food-borne pathogen Salmonella together with Campylobacter is the most predominant bacterial pathogen in Europe, causing approximately 160,000 confirmed human cases per year. During the last two decades, the importance of Salmonella for food safety triggered the development of dozens of PCR-based detection methods. They promise significant advantages compared to the traditional culture-based methods with respect to speed, easiness, reliability, sensitivity, cost effectiveness, and automation. However, many of them are not applicable because of lacking validation procedures. Meanwhile, PCR has internationally been standardized and guidelines as well as standards for the validation of alternative methods, such as PCR, were established. This review will give an overview of the historical development of PCR-based methods for the detection of Salmonella including validation aspects and summarizes the state-of-the-art for the detection of Salmonella in food and feeding stuff by real-time PCR. Furthermore, current multiplex PCR-based serovar-specific identification methods will be reviewed.     

Rapid detection of Salmonella from vegetable rinse-water using real-time PCR

A PCR-based method for the detection of Salmonella spp. from fresh vegetable rinse-water was developed. The method is a modification of an existing Association of Official Analytical Chemist (AOAC)-approved protocol and is compatible for high throughput analysis. The protocol is sensitive enough to detect contamination in mixed-salad (made up of approximately 80% leaf lettuce, 10% red cabbage and 10% carrots by weight) that was washed with water, which was artificially contaminated with Salmonella spp. at the estimated level of 1–10 cells ml⁻¹. The modified protocol, which includes a confirmatory melt–curve analysis of PCR products, requires 8–10 h. The method should help implementation of the HACCAP program for fresh produce.     

A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk

Intoxications and infections caused by food-borne pathogens represent an increasing public health problem, and diagnostic tests in multiplex format are needed for the rapid identification of food contaminations caused by more than one microbial species. We have developed a multiple PCR-based platform for the simultaneous detection of the widespread milk-associated pathogens Salmonella spp., Listeria monocytogenes and Escherichia coli O157. The assay combines an enrichment step in a medium properly formulated for the simultaneous growth of target pathogens, a DNA isolation method, and a multiplex Real-Time PCR detection system based either on dual-labelled probes (mRT-PCR), or on melting curve analysis (mHRM). The second, producing a distinct peak for each amplification product, allows the qualitative assessment of pathogen presence. Moreover, the internal amplification control (IAC) included in the reaction, ensuring the reliability of results, complies with quality management programmes. Inclusivity and exclusivity were 100% each, with a detection limit of 1 CFU for each pathogen in a total of five 25 ml-aliquots of raw milk, and a duration of two working days.The assay represents an alternative approach for the qualitative detection of the cited bacterial species, suitable for a relatively inexpensive screening of several milk samples, reducing the turnaround time and the workload.     

Detection of Listeria monocytogenes in Fresh Produce Using Molecular Beacon—Real‐time PCR Technology

ABSTRACT: The capability of an assay to detect Listeria monocytogenes from artificially inoculated fresh‐cut produce such as cantaloupe and mixed salad was demonstrated. An oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (Molecular Beacon, MB) was used in a real‐time polymerase chain reaction (PCR) assay. As few as 4 to 7 colony‐forming units (CFU) of L. monocytogenes per 25 g of artificially contaminated produce could be detected. A comparison of 2 commercially available kits using MB‐PCR (iQ‐Check, Bio‐Rad Laboratories) and conventional PCR (BAX, Dupont Qualicon) was performed on artificially inoculated produce. The time required to detect L. monocytogenes (from produce to PCR) was considerably shorter for the iQ‐Check protocol (approximately 26 h) compared with the BAX‐PCR (approximately 52 h). The iQ‐Check protocol was also used to confirm the identity of the L. monocytogenes isolates obtained during a microbiological screen of conventional and organic leaf lettuce and alfalfa sprout samples from local supermarkets. The iQ check protocol was successful in differentiating L. monocytogenes isolates from other Listeria spp. such as L. welshimeri, L. innocua, and L. ivanovii. This is the 1st report of the application of the MB probe being used for real‐time detection of L. monocytogenes in whole and fresh‐cut produce.     

Simultaneous detection of Listeria monocytogenes and Salmonella by multiplex PCR in cooked ham

A multiplex PCR (m-PCR) assay with an internal amplification control (IAC) was developed for the simultaneous detection of Salmonella spp. and Listeria monocytogenes through invA and prfA genes, respectively. To ensure the detection of the pathogens in cooked ham, samples were enriched in both buffered peptone-water and Half Fraser broth. Subsequently, equal volumes of enrichment broths were mixed and DNA purification was performed prior to m-PCR reaction, saving considerable time and effort. The m-PCR also proved to be very useful as a simple and ready-to-go method for simultaneous confirmation of presumptive L. monocytogenes and Salmonella spp. colonies directly from agar plates without any DNA extraction steps.     

Outbreaks and factors influencing microbiological contamination of fresh produce

Fresh fruits and vegetables are nutritionally well‐recognised as healthy components in diets. The microbiological foodborne outbreaks associated with the consumption of fresh produce have been increasing. Salmonella spp., Escherichia coli O157:H7, Staphylococcus aureus, Campylobacter spp. and Listeria monocytogenes are the most common pathogens that contaminate fresh produce. This review discusses recent foodborne outbreaks linked to fresh produce, factors that affect microbiological contamination and measures that could be adopted to reduce the foodborne illnesses. © 2016 Society of Chemical Industry     

Evaluation of the Microbial Safety of Child Food of Animal Origin in Greece

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.     

Effectiveness of edible coatings to inhibit browning and inactivate foodborne pathogens on fresh-cut apples

Multiple formulations of edible coating solutions were developed and evaluated for their ability to maintain the quality and minimize populations of foodborne bacteria on fresh-cut apples. Fresh-cut apples were artificially inoculated with Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes, and dip-treated with coating solutions. Changes in populations of pathogens and molds and yeasts (M&Y) were evaluated after solution treatments and during storage at 4°C for 35 days. Changes in color, texture firmness, and weight loss during storage were also determined. Among the formulations, the solution containing 1% of three organic acids (acetic, lactic, and levulinic acids), 2% N-acetyl-l-cysteine or L-cysteine with 0.5% chitosan demonstrated the best effectiveness for the simultaneous reduction of pathogenic and spoilage microorganisms, control of browning, and maintenance of the quality of fresh-cut apples. The treated apple slices had nondetectable pathogens and M&Y (<1 log CFU) and the least quality changes for 35-day storage. The developed solutions can be used at processing plants, retail stores, or homes to protect fresh-cut apples or other fresh-cut fruits and vegetables from microbiological and quality deterioration.     

Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

The present work is focused on the development of a TaqMan multiplex real-time PCR method for the detection of Salmonella, Shigella and L. monocytogenes in seafood, meat and ready-to-eat products. The aim of this study is to detect the three pathogens in one single test including an enrichment medium for the simultaneous growth of the bacteria of interest and an Internal Amplification Control (IAC) to monitor PCR inhibitors. For this purpose, three genes were selected, invA for Salmonella, ipaH for Shigella and hlyA for L. monocytogenes. Also, no. 17 broth without dextrose and further modified by adding Tween 80 was used for the enrichment step. Specificity of the method was checked against a panel of 24 non-target bacterial strains. RT-PCR efficiency obtained for the simultaneous amplification of all three pathogens was 102.5% for Salmonella, 108.9% for Shigella and 106.4% for L. monocytogenes. The limit of detection (LOD) was evaluated in seafood, meat and ready-to-eat products, being established within 3 and 22 cfu in 25 g of sample for the three bacteria analyzed. Seventy-eight samples were analyzed with multiplex RT-PCR including spiked and natural samples collected from different laboratories. Even though several RT-PCR methods have been developed for the detection of Salmonella, Shigella and L. monocytogenes, as far as we know this is the first method developed for the simultaneous detection of these three pathogens, coupling RT-PCR with an enrichment in the same broth and being tested in a wide range of different processed food samples with a low LOD. The application of this method can significantly reduce costs and time of analysis in laboratories, what would be reflected in a faster response in those risk situations when they are detected.     

Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk—Sheep are not small cows

Mastitis, inflammation of the mammary gland, is an important cause of disease, mortality, and production losses in dairy and meat sheep. Mastitis is commonly caused by intramammary infection with bacteria, which can be detected by bacterial culture or PCR. PathoProof (Thermo Fisher Scientific Ltd., Vantaa, Finland) is a commercially available real-time PCR system for the detection of bovine mastitis pathogens. Sheep differ from cattle in the bacterial species or bacterial strains that cause mastitis, as well as in the composition of their milk. The aim of this study was to evaluate whether the PathoProof system was suitable for detection of mastitis pathogens in sheep milk. Milk samples were collected aseptically from 219 udder halves of 113 clinically healthy ewes in a single flock. Aliquots were used for bacteriological culture and real-time PCR-based detection of bacteria. For species identified by culture, the diagnosis was confirmed by species-specific conventional PCR or by sequencing of a housekeeping gene. The majority of samples were negative by culture (74.4% of 219 samples) and real-time PCR (82.3% of 192 samples). Agreement was observed for 138 of 192 samples. Thirty-four samples were positive by culture only, mostly due to presence of species that are not covered by primers in the PCR system (e.g., Mannheimia spp.). Two samples were positive for Streptococcus uberis by culture but not by PCR directly from the milk samples. This was not due to inability of the PCR primers to amplify ovine Streptococcus uberis, as diluted DNA extracts from the same samples and DNA extracts from the bacterial isolates were positive by real-time PCR. For samples containing Staphylococcus spp., 11 samples were positive by culture and PCR, 9 by culture only, and 20 by PCR only. Samples that were negative by either method had lower bacterial load than samples that were positive for both methods, whereas no clear relation with species identity was observed. This study provides proof of principle that real-time PCR can be used for detection of mastitis pathogens in ovine milk. Routine use in sheep may require inclusion of primer sets for sheep-specific mastitis pathogens.     

A New Real-Time PCR Assay for the Specific Detection of Salmonella spp. Targeting the bipA Gene

A straightforward real-time polymerase chain reaction (PCR)-based assay was designed and evaluated for the detection of Salmonella spp. in food and water samples. This new assay is based on the specific detection of the bipA gene of Salmonella, which encodes a protein of the guanosine triphosphate (GTP)-binding elongation family that displays global modulating properties, by regulating a wide variety of downstream processes. The new method correctly identified all 48 Salmonella strains used in the inclusivity test, and did not detect all 30 non-Salmonella species tested. The method was evaluated by analyzing 120 diverse food and water samples enriched in buffered peptone water. The bipA-based real-time PCR assay showed 100% efficiency, sensitivity, and specificity compared to the invA-based method previously published, which was developed as a part of a European project for the standardization of PCR methods in food microbiology. The assay includes an independent internal amplification control (IAC) in each reaction to control false negative results.     

Efficacies of Garlic and L. sakei in Wine‐Based Marinades for Controlling Listeria monocytogenes and Salmonella spp. in Chouriço de Vinho,a Dry Sausage Made from Wine‐Marinated Pork

Chouriço de vinho is made from roughly minced (10 to 30 mm) pork and fat, seasoned with a marinade made from wine, salt, garlic, and other facultative seasonings used according to the recipe of each producer. The batter is maintained at 4 to 7 ºC for 24 to 48 h. It is then stuffed into natural thin pork gut, cold smoked and matured at a low temperature for 1 to 4 wk. The effect of garlic used in wine‐based marinade and a starter culture of indigenous Lactobacillus sakei on the behavior of Listeria monocytogenes and Salmonella spp. in the processing of chouriço was investigated. The garlic (as powder and fresh juice) was found to contribute (P < 0.05) to the control of both pathogens in broth. Garlic dose, as tested within the usual limits used for seasoning, did not impact the reduction of pathogens. Garlic‐wine‐based marinade and a starter culture of indigenous L. sakei contribute to controlling L. monocytogenes and Salmonella spp. in the processing of chouriço. Their presence was responsible for the loss of viability of L. monocytogenes and Salmonella spp. following 5 d of drying, even sooner than situations where no garlic was used. The results of the present work show that the use of a wine‐based marinade with garlic has an important role in ensuring the safety of the product.     

Detection of Bacillus spores using PCR and FTA filters

Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.     

Study of Arcobacter spp. contamination in fresh lettuces detected by different cultural and molecular methods

Arcobacters are considered potential emerging food and waterborne pathogens. However, there is no data on the presence of Arcobacter spp. in fresh vegetables. Therefore the objective of this research was to study the presence of Arcobacter spp. in fresh lettuces.Fifty fresh lettuces purchased from different local shops in Valencia (Spain) were analyzed. The assay was performed simultaneously by cultural and molecular methods. Isolates were identified by real-time, multiplex PCR and restriction fragment length polymorphism analysis of PCR-amplified DNA fragment (PCR-RFLP). Finally, all the isolates were genotyped using the randomly amplified polymorphic DNA (RAPD-PCR) method.Arcobacter sp. was detected in 10 of the 50 samples (20%) by real-time PCR, being A. butzleri the unique detected species by mPCR. The detection levels obtained by conventional PCR (7 samples/50, 14%) were slightly lower. These seven samples were found to be positive also by culture isolation. All 19 obtained isolates were identified as A. butzleri by multiplex PCR and PCR-RFLP. Great genetic heterogeneity among the isolates was observed by RAPD-PCR profiling.To our knowledge, this is the first study in which Arcobacter spp. is detected in fresh vegetables such as lettuces. Although these foods are generally considered safe, given the large quantities consumed and the fact that further cooking is absent, lettuce could be a source of Arcobacters of public health concern.     

Microbiological Quality and Safety of Fresh Fruits and Vegetables at Retail Levels in Korea

The aim of this study was to evaluate the microbiological quality and safety of fresh produce at retail level in Korea in order to periodically update information and establish available risks associated with consumption of fresh fruits and vegetables. The samples from different markets located in 3 provinces of South Korea were collected. The protocol in the Korean Food Standards Codex was applied and generic Escherichia coli, coliforms, aerobic mesophilic bacteria (AMB), and yeast and mold (YM) in 360 packaged and unpackaged fresh fruits and vegetables were analyzed. Presence of pathogens was examined using real‐time polymerase chain reaction (q‐PCR) after enrichment of samples. For all, the microbial counts ranged from 1.7 to 10.6 log cfu/g for AMB, 2.2 to 7.9 log cfu/g for coliforms, and 5.5 to 7.9 log cfu/g for YM. Three lettuce samples were contaminated by E. coli with a bacterial load ranging from 2 to 4 log cfu/g. Salmonella spp. were not detected in any fresh produce. Listeria monocytogenes, E. coli O157:H7, and Staphylococcus aureus were found in 1 (0.6%), 3 (0.8%), and 5 (1.4%) fresh produce samples, respectively. Bacillus cereus (50.3%) and Clostridium perfringens (13.3%) had the highest prevalence. These results indicate the need for employing strict control measures and developing preventive strategies to improve the quality and safety of fresh produce in Korea.     

Salmonella real-time PCR-Nachweis

Salmonella belongs to the most important bacterial pathogens worldwide causing disease in humans and animals mainly by the oral uptake of contaminated food. Consequently, detection methodologies for Salmonella from food items are meaningful for routine laboratories. Here, we describe two different real-time PCR based methods for the detection of Salmonella in food. The procedure begins with a cultural pre-enrichment in buffered peptone water for 18–24 hours at 37 °C followed by a selective enrichment step in Rappaport-Vassiliadis medium for at least six hours at 42 °C. Next, the microbial DNA is extracted and finally Salmonella-DNA is specifically detected by the real-time PCR. Both methods differ in the Salmonella target gene sequence. One assay amplifies a 285-bp-DNA-Fragment of the invA-gene, and the other a 95-bp-DNA-Fragment of the ttrC/ttrA-gene. An internal amplification control indicates the correct carrying out of the PCR. The duration of both detection methods is between 24 and 28 hours.     

Multiplex PCR for the simultaneous detection of Salmonella spp. and Salmonella Enteritidis in food

Multiplex PCR assay (mPCR) for the detection of Salmonella spp. and S. Enteritidis was developed in this study using artificially contaminated chicken carcasses. The assay showed 100% specificity to detect approximately 1 CFU of Salmonella in 10 g of chicken skin after non‐selective enrichment. The mPCR was evaluated in Minas cheese, fresh pork sausage and chicken carcasses commercially available. Salmonella spp. was detected in nine of sixty‐six chicken carcasses, five of fifty‐two cheese samples, and five of fifty‐two sausage samples. The serovar Enteritidis was detected in two samples of contaminated sausage. The mPCR results were confirmed by conventional culture and biochemical identification of the isolates. Serotyping confirmed the presence of S. Enteritidis in sausage samples and showed contamination by serovars Schwarzengrund and Montevideo in chicken carcasses.