Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.     

A Rapid and Simple DNA Extraction Procedure to Detect Salmonella spp. and Listeria monocytogenes from Fresh Produce Using Real-time PCR

DNA isolation procedures significantly influence the outcome of PCR-based detection of human pathogens. Unlike clinical samples, DNA isolation from food samples, particularly from fresh and fresh-cut produce has remained a formidable task and has hampered the sensitivity and accuracy of molecular methods. We utilized a commercially available filter-based DNA isolation method (FTA) in conjunction with real-time PCR-based detection of Salmonella spp. and Listeria monocytogenes. The protocol uses filter paper discs impregnated with a patented chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. Use of the FTA method in conjunction with real-time PCR for the detection of Salmonella spp. and L. monocytogenes was compared with two commercially available DNA isolation procedures that are commonly used for high throughput real-time PCR pathogen detection systems. Both pathogens were successfully detected from artificially inoculated fresh and fresh-cut produce such as alfalfa sprouts, cilantro, green onion, broccoli, prepacked mixed salad, and spinach at low cell numbers (four to seven colony forming units per 25 g initial inoculum level before enrichment). The FTA protocol had distinct advantages of simplicity, biosafety, and compatibility for high throughput screening. This DNA preparation protocol was rapid, sensitive, required minimal handling, and reduced interference from produce-associated inhibitors of real-time PCR. Mention of brand names does not constitute an endorsement by the U.S. Department of Agriculture above others of a similar nature not mentioned.     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Validation of Real-Time PCR and Enzyme-Linked Fluorescent Assay-Based Methods for Detection of Salmonella spp. in Chicken Feces Samples

A comparative study of enzyme-linked fluorescent assay (ELFA)-based methods and real-time polymerase chain reaction (PCR)-based methods using three and two different sample preparation protocols, respectively, and the standard culture-based method EN ISO 6579:2002/Amd1:2007, for the detection of Salmonella spp. in chicken feces, was performed on 20 artificially and 68 naturally contaminated chicken feces. Selectivity, relative specificity, relative accuracy, relative sensitivity, and relative detection level were determined. According to criteria established in the methods comparison study included in EN ISO 16140:2003 for validation of alternative microbiological methods, the ELFA-based methods (V1 and V2) as well as a real-time PCR method (PCR2) were comparable to the reference method for the detection of Salmonella in chicken feces. They provided results in 48 h and presented a high sensitivity (97% for all of them). The three methods showed a relative specificity of 94%, V1 being the method which presented the highest relative accuracy (96%). While detection level for V2 and reference method was between 3 and 13 CFU/25 g, PCR2 method was able to detect down to 3 CFU/25 g. In conclusion, both the real-time PCR and the ELFA-based assays can be used as rapid and user-friendly screening methods for detection of Salmonella spp. in chicken feces.     

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.     

Survey of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products by commercial real-time PCR kits

Real-time PCR (RTiPCR) assays including enrichment stage were evaluated for the rapid detection of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products using molecular beacon probes available as commercial kits (WARNEX Genevision, Canada & AES Chemunex detection system, France). The accuracy of the assays was evaluated analyzing 1032 naturally contaminated food samples in combination to the conventional cultural methods. Presence/absence testing of the above pathogens was performed in 25 g samples of each product. In case of L. monocytogenes of 39 positive RTiPCR samples, 37 were confirmed by the cultural method (based on McNemar's test the difference between the two methods is insignificant). The highest incidence of L. monocytogenes in food products was found in desserts and the second highest in frozen pastries. None of the samples were cultural positive but negative in the RTiPCR test. One among the 343 investigated samples was positive for Salmonella spp. by RTiPCR and the cultural method. Out of 333 samples analyzed for E. coli O157:H7 no positive sample was detected. RTiPCR-based methods proved to be powerful tools for fast, sensitive and accurate pathogen detection in raw food ingredients and ready-to-eat products.     

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

A 7-plex PCR assay was developed to achieve an effective detection and identification of serotype Enteritidis of Salmonella spp. and shiga toxin-producing type of Escherichia coli O157 in meat products. Six DNA sequences in the invA and sdfI genes of Salmonella Enteritidis as well as rfbE, eae, stx1, and stx2 genes of E. coli O157:H7 were employed to design primers. The multiplex PCR assay could specifically and sensitively detect and identify target pathogens. Applying the assay to meat samples, the multiplex PCR assay was able to simultaneously detect and identify the two pathogens at a sensitivity of three CFU/10 g raw meats after simple 16 h enrichment in buffered peptone water. Further applying in 21 retail meat samples revealed that two samples were positive for non-shiga toxin producing E. coli O157, one sample was positive for Stx2 producing E. coli O157 and five samples were positive for Salmonella enterica Enteritidis. Taken together, the 7-plex PCR assay is a rapid and reliable method for effectively screening single or multiple pathogens occurrences in various meat products, and could also identify the Salmonella enterica Enteritidis from all Salmonella spp. and shiga toxin producing type from all E. coli strains. Considering as a non expensive screening tool, the multiplex PCR assay has a great potential in complement for food stuff analysis by conventional microbiological tests.     

Development of a nucleic acid lateral flow immunoassay for simultaneous detection of <Emphasis Type="Italic">Listeria</Emphasis> spp. and <Emphasis Type="Italic">Listeria</Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples.     

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.     

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

Syto9 and probe‐based multiplex real‐time PCR assays for simultaneous detection of a group of foodborne pathogens (named SYLC group), targeting Salmonella spp. (invA gene), Yersinia enterocolitica (ystA gene), Listeria monocytogenes (hly gene) and Campylobacter spp. (rrna gene), have been developed. The Syto9 assay generates amplicon DNA melting curve with four peaks of 86.5 ± 0.5, 84 ± 0.5, 81.5 ± 0.5 and 90.5 ± 0.5 °C corresponding Salmonella spp., Y. enterocolitica, L. monocytogenes and Campylobacter spp. targets, respectively. The sensitivities of the Syto9 and TaqMan assays in artificially inoculated chicken wing rinses were in a range of 3.2 × 10² to 3.1 × 10⁴ and 9.8 × 10² to 1.9 × 10⁴ colony‐forming units per millilitre, respectively, depending on the pathogen. All tested target strains (n = 100) were correctly detected by the both assays, whereas nontarget strains (n = 100) demonstrated no cross‐reactivity representing 100% specificity. The assays are suitable for application in qualitative and quantitative detection of SYLC group pathogens in food matrices.     

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.     

A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1–10³ cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10²–10³ cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

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.     

A New Validated Real-Time PCR-Based Method for the Specific and Fast Detection of Cronobacter spp. in Infant Formula

In this study, a real-time polymerase chain reaction (PCR)-based method was designed for the fast detection of Cronobacter spp. (a newly proposed genus formerly known as Enterobacter sakazakii) in infant formula. The real-time PCR was positively tested with 70 Cronobacter strains, including members of all the species of this genus, and 88 non-Cronobacter strains. This new PCR-based system was validated against the reference standard ISO/TS 22964: 2006 (ISO International Organization for Standardization 2006) using 70 food matrices including powdered infant formula, follow-up formula, and hydrolyzed cereals for infants. The detection limit of the technique was found to be of 1 cfu in 10 g of food, fulfilling the requirements of the European Commission. The time of analysis, which comprises around 3–6 days using the reference method, is considerably reduced to less than 24 h using the real-time PCR-based system hereby described, allowing food industry a faster release of the stocks for commercialization. Moreover, this method includes an internal amplification control, co-amplified during each PCR run to verify the results.     

Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella

Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng–50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10⁴ CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.     

Development of an Internal Amplification Control Using Multiplex PCR for the Detection of Listeria monocytogenes in Food Products

The bacterial pathogen Listeria monocytogenes is responsible for listeriosis, a food-borne disease, which may result in severe illness and possible death. Large outbreaks of listeriosis have been associated with food products including soft cheeses and ready to eat food products. Polymerase chain reaction (PCR) is a molecular identification method for food-borne pathogens; however, a drawback of this method is that false-positive or false-negative results may occur. To validate the accuracy of the PCR as a powerful molecular tool for pathogen detection, it is important that false-negative results be distinguishable from true-negative PCR results. The aim of this study was to design and include an internal amplification control (IAC) within the PCR to coamplify with L. monocytogenes in order to identify false-negative results of L. monocytogenes from ostrich meat and camembert cheese samples. The IAC had to be incorporated into the PCR without loss of specificity and sensitivity on the detection limit of L. monocytogenes and was developed and tested for use in a multiplex PCR detection system. A region of the pUC19 plasmid was selected as the IAC for this study. The optimal concentration at which pUC19 would coamplify with L. monocytogenes was determined to be 0.001 pg/μL. Following an enrichment procedure, the minimum number of organisms detected in a spiked food sample by the PCR was 8 CFU/mL L. monocytogenes; the same detection limit was attained when the pUC19 IAC was included in the PCR. An optimal pUC19 IAC concentration increased the reliability of the PCR for food diagnostic purposes.     

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.     

Development and application of a new multiplex real‐time PCR assay for simultaneous identification of Brucella melitensis,Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes are important foodborne pathogens in milk and milk products, which are responsible for a variety of diseases that pose serious hazards to public health and food safety. The objective of this study was to develop a novel multiplex RTi‐PCR for the detection of B. melitensis, C. sakazakii and L. monocytogenes and to characterise the potential risk of these pathogens in raw milk and cheese. The raw milk (n = 25) and cheese samples (n = 20) were analysed by multiplex RTi‐PCR assay and detected for quantification of the three pathogens. In this study, B. melitensis, C. sakazakii and L. monocytogenes were simultaneously identified using BMEII0466, mms operon and hly as target genes, respectively. The multiplex RTi‐PCR assay that was developed showed good sensitivity and selectivity for the pathogenic microorganisms (r² = 0.986–0.997). Multiplex RTi‐PCR results showed that most of the samples were contaminated with the pathogens screened.