Development of a rapid and sensitive quantum dot-based immunochromatographic strip by double labeling PCR products for detection of Staphylococcus aureus in food

Staphylococcus aureus was listed as the 2nd common food-borne pathogens, resulting in diseases such as pneumonia, pseudomembranous colitis, pericarditis and even sepsis. A rapid, simple and sensitive detection method is required to monitor food in cases of contamination by S. aureus. Hence, a novel immunochromatographic test strip (ICTS), based on the two-component reaction (i.e. digoxigenin and anti-digoxigenin, quantum dots-conjugated streptavidin and biotin) in test line with QDs as indicator, was developed for the rapid and sensitive detection of S. aureus. Genomic DNA from bacteria lysis by boiling was extracted easily and rapidly with silica-coated magnetic nanoparticles, and a species-specific gene was amplified by PCR using digoxigenin/biotin-labeled primers. The fluorescence of captured QD labels on the test line and control line served as signals was observed by UV light. The sensitivity and specificity of the ICTS were estimated using bacteria spiked food samples (artificially mixed with cell counts (10⁸ CFU/mL for each bacterium) of Escherichia coli, Listeria monocytogenes and Lactobacillus plantarum). The limit of detection for S. aureus was 3 × 10⁰ CFU/mL and 3 × 10¹ CFU/g in spiked milk powder and meat samples, respectively, which was not affected by those non-S. aureus strains. Our results showed that QDs-based ICTS was promising for rapid and sensitive detection of S. aureus within 2 h. Hence, this protocol might be useful for screening and monitoring the contamination of S. aureus in food products, and helpful for promoting the prevention and control of communicable disease caused by food-borne pathogen.     

Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples

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 <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of T_t is needed for quantification purposes.     

Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 10² CFU/mL, as low as 7.5 × 10⁰ CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 10¹ CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 10³ CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.     

Development of a novel target-enriched multiplex PCR (Tem-PCR) assay for simultaneous detection of five foodborne pathogens

In the present study, a novel target-enriched multiplex PCR (Tem-PCR) assay was developed for simultaneous detection of Salmonella spp., Listeria (L.) monocytogenes, Staphylococcus (S.) aureus, Escherichia (E.) coli O157:H7 and Shigella spp.. DNA primers including universal primer and composite primer pairs were used in this Tem-PCR assay. Of which, the universal primer was linked to the 5'-end of each specific primer designed targeting Salmonella invA gene, Staphylococcus aureus femA gene, Shigella ipaH gene, Listeria monocytogenes hly gene and Escherichia coli O157:H7 rfbE gene, respectively, generating the composite primers. During PCR amplification, the composite primer pairs were employed to enrich target genes of different pathogens in initial cycles and the universal primer was employed to enrich the amplicons produced with the composite primers priming in later PCR cycles. Significantly, the Tem-PCR assay overcomes the amplification disparity resulting from primers competition observed in conventional multiplex PCR assay. With the evaluation of the Tem-PCR assay for the detection of these five foodborne pathogens, the assay showed high specificity to the target bacteria with an analytical detection limit of <2.0 × 10² CFU/mL for each, and practical detection capability, suggesting a novel multiplex PCR method for detecting foodborne pathogens.     

Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples

Cronobacter spp. (Enterobacter sakazakii) is an opportunistic pathogen and is linked with life-threatening infections in neonates. The organism has been isolated from a wide variety of foods and environments. In this study, a Taqman real-time PCR assay incorporating an internal amplification control (IAC) was developed and evaluated for specific detection of Cronobacter spp. in foods. Previously reported macromolecular synthesis (MMS) operon sequence was selected for specificity, and 67 bacterial strains, including four strains of Cronobacter spp., were evaluated. All Cronobacter strains were successfully identified, however no cross-reactivity was observed with non-Cronobacter strains. Detection limit of the assay in pure culture and formula infant without enrichment was 1.2 × 10³ CFU/ml (1.2 × 10¹ CFU/assay). After 24 h enrichment in broth, as few as 10⁰ CFU/ml or g of Cronobacter could be detected in artificially contaminated food samples (infant formula, sterilized milk and chicken meat). The detection limit of the real-time PCR assay however remained unaffected in the presence of 10⁸ CFU/ml Salmonella typhimurium in another analysis. A total of 92 food samples were analyzed for the presence of Cronobacter, out of which two pork samples were found as positive by real-time PCR, whereas only one was detected by the ISO standard method. The adjusted real-time PCR assay can be adopted to rapidly detect Cronobacter spp. in food samples with high specificity and sensitivity, and can prevent false negative results by using the IAC.     

Evaluation of a novel antimicrobial solution and its potential for control Escherichia coli O157:H7, non-O157:H7 shiga toxin-producing E. coli,Salmonella spp., and Listeria monocytogenes on beef

The goal of this study was to evaluate the efficacy of a novel antimicrobial solution made with chitosan, lauric arginate ester, and organic acids on Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and non-O157 shiga toxin-producing E. coli cocktails and to test its potential to be used as a marinade for raw beef. Fresh beef top round steaks were surface-inoculated with the pathogen cocktails at approximately 2.5 or 4.5 Log CFU/cm², marinated with the antimicrobial solution (AMS), and then stored at 4 °C for 6, 24, and 48 h. Three commercially available marinades were used for comparison. Results revealed that AMS had the most antimicrobial effect regardless of the type or inoculation level of pathogens (P < 0.05). After 6 h, the AMS marination reduced all pathogens to levels below the limit of detection (<1 Log CFU/cm²), resulting in a 3.5 Log CFU/cm² reduction. When AMS was diluted with autoclaved distilled water by 5 times (AMS 1:5) or 10 times (AMS 1:10), its antimicrobial efficacy was impacted by marination time, the inoculated pathogens, and the inoculation levels. This study demonstrates that the developed antimicrobial solution has a great potential to be used during marination by consumers to ensure better food safety.     

Development of a quantitative real-time PCR assay for viable Salmonella spp. without enrichment

Propidium monoazide (PMA) combined with molecular quantitative real-time PCR (qPCR) has been widely used for only detection of viable bacteria. However, recent studies indicated PMA did not fully inhibit the detection of dead Salmonella. In this study, we developed a more effective PMA Taqman-based qPCR than previous studies to quantify viable Salmonella spp. in raw shrimp. This method has high specificity by using 60 strains belonging to 23 species. The optimization of the PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10⁶ CFU/mL dead cells, while only 10³–10⁴ CFU/mL dead cells could be inhibited in previous reports. This assay could detect viable Salmonella spp. at as low as 36 CFU/mL in pure culture and 100 CFU/g in raw shrimp. By comparing with PMA-qPCR, qPCR and plate counting for quantifying Salmonella in samples, this PMA-qPCR was obviously superior to qPCR and had good agreement with plate counting. In conclusion, this effective method can be used as an available tool to quantify viable Salmonella spp. in raw shrimp.     

In-field detection of multiple pathogenic bacteria in food products using a portable fluorescent biosensing system

Foodborne diseases caused by pathogenic bacteria have increasingly become a major public health issue worldwide. Rapid, simple, and accurate detection methods are urgently needed for in-field screening of bacterial pathogens. In our previous work, rapid detection methodologies have been established based on fluorescent nanobiosensors for simultaneous separation and detection of multiple foodborne pathogenic bacteria. In this research, a portable fluorescent biosensing system was designed and built and further assessed for in-field detection of three main types of bacterial pathogens that have been associated with the outbreaks of foodborne illness. Using the developed fluorescent nanobiosensor coupled with nanobead-based immunomagnetic separation, we conducted blind tests with the portable device to simultaneously detect E. coli O157:H7, L. monocytogenes, and S. Typhimurium in different food products in three cities selected from three big agricultural provinces in China. Specificity tests showed low interference of this multiplex biosensor from non-targets in food samples. The detection could be done from sampling to results within 60 min. Limits of detections of this method for E. coli O157:H7, L. monocytogenes, and S. Typhimurium were determined to be 10², 10³, and 10³ CFU/mL in lettuce, shrimp, and ground beef, respectively. Recovery tests were also investigated and this method was evaluated to be accurate comparing with the gold standard culturing method. Therefore, it is feasible for this portable fluorescence biosensing system to be used in rapid and in-field screening of multiple foodborne pathogenic bacteria in foods, such as vegetables, livestock meat, and sea food. And together with fluorescent nanobiosensors, it provides a promising alternative tool to traditional culturing method, or even conventional ELISA and PCR based methods.     

A multiplex polymerase chain reaction coupled with high-performance liquid chromatography assay for simultaneous detection of six foodborne pathogens

A multiplex polymerase chain reaction (PCR) coupled with high-performance liquid chromatography (HPLC) assay was developed to simultaneously detect Salmonella, Campylobacter jejuni, Listeria monocytogenes, Yersinia enterocolitica, Streptococcus hemolyticus and Staphylococcus aureus from foods. Six pairs of specific PCR primers were designed according to Salmonella invA, C. jejuni cdtA, L. monocytogenes pfrA, S. aureus femA, Y. enterocolitica 16S rRNA and St. hemolyticus cfb. Following the development of multiplex PCR, the PCR products were subjected to HPLC analysis. Unique HPLC peak profile for each PCR product represented corresponding bacterial strain, suggesting a better alternative to conventional PCR gel electrophoresis with ethidium bromide (EB) staining. The specificity analysis of the multiplex PCR-HPLC with 121 bacterial strains and 4 yeast strains showed that the method was highly specific for the target pathogens. One thousand and three hundred ninety-four blind samples were used to evaluate the practical diagnostic capability of the method, and results showed that eighty-eight food samples contaminated with single or multiple pathogens were detected by the method, which accorded with the testing results via conventional method. All data demonstrated that the multiplex PCR-HPLC is an efficient diagnostic method for rapid identification of the six foodborne pathogens. The simplicity and high sensitivity of the method may lead to improved management of food safety and foodborne diseases.     

Rapid and standardized methods for detection of foodborne pathogens and mycotoxins on fresh produce

Due to the increase in consumption of fresh produce regarding to the health demand in the last decades, a considerable portion of foodborne outbreaks has been trackbacked to contaminated fresh produce, which have appeared as highly possible vehicles for foodborne outbreaks nowadays. Delays in detection of pathogens and mycotoxins on fresh produce hindered the trace-back investigations in finding the source and revealed the urgent need of rapid and reliable methods. In the frame of this review, we summarized available fast, reliable and standardized methods (conventional, molecular, rapid and recently developed methods) used for detection of the most common foodborne pathogens and mycotoxins which are the most likely causative agents of outbreaks caused by contaminated fresh produce.     

Review of Salmonella detection and identification methods: Aspects of rapid emergency response and food safety

Salmonella has been recognized as a major and important foodborne pathogen for humans and animals over more than a century, causing human foodborne illness as well as high medical and economical cost. Accordingly, the effort to develop efficient and reliable Salmonella detection methods continues. This paper reviews and describes the development and application of commercially available Salmonella detection methods. These are categorized into several groups based on the principle applied: conventional culture methods, immunology-based assays, nucleic acid-based assays, miniaturized biochemical assays, and biosensors. Conventional culture methods serve as the basis in food testing laboratories despite rather laborious and time-consuming protocols. Considerable progress in rapid methods using emerging technologies yield faster answers and higher throughput of samples. This paper also shows and analyzes Salmonella test results and summarizes the features and limitations of the studies involving Salmonella detection methods developed for emergency response, mainly by food emergency response laboratories participating during recent fiscal years. The emergency response laboratories utilize Salmonella detection methods possessing properties that include simplicity, versatility, high sensitivity, good specificity, and cost efficiency. Collaboration of the food emergency response laboratories in the development of these technologies is important essentially to compare for the purpose of continually improving Salmonella detection methods.     

Investigation into the antibacterial behavior of suspensions of magnesium oxide nanoparticles in combination with nisin and heat against Escherichia coli and Staphylococcus aureus in milk

The antibacterial activities of magnesium oxide nanoparticles (MgO NP) alone or in combination with nisin against Escherichia coli and Staphylococcus aureus were investigated. Synergistic antibacterial effects existed and at lower levels of nisin when compared to when nisin was used alone. Also the antibacterial activities of MgO NP in combination with other antimicrobials (nisin and heat) against E. coli and S. aureus were investigated in milk. A synergistic effect of MgO NP in combination with nisin and heat was observed as well. Scanning electron microscopy was used to characterize the morphological changes of E. coli after antimicrobial treatments. It was revealed that MgO NP treatments in combination with nisin distort and damage the cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells. This is the first report describing the antibacterial activity of MgO NPs and nisin in milk. It leads the way to development of treatment combinations which could result in a decrease in pasteurisation temperatures and the level of MgO NP required for pasteurising milk and maintaining pathogen control.     

Sextuplex PCR combined with immunomagnetic separation and PMA treatment for rapid detection and specific identification of viable Salmonella spp., Salmonella enterica serovars Paratyphi B,Salmonella Typhimurium,and Salmonella Enteritidis in raw meat

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 10¹ CFU/mL in pure culture and 10² CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.     

Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars

Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 10⁶ CFU/ml in pure culture or 1 × 10⁶ CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 10² CFU/ml (4.3 × 10² CFU/ml for S. Typhimurium, 3.7 × 10² CFU/ml for S. Paratyphi B, 7.2 × 10² CFU/ml for S. Typhi) in pure culture and 10³ CFU/g (4.3 × 10³ CFU/g for S. Typhimurium, 3.7 × 10³ CFU/g for S. Paratyphi B, 7.2 × 10³ CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce.     

Temporal patterns in the occurrence of Salmonella in raw meat and poultry products and their relationship to human illnesses in the United States

The prevalence and level of microbial pathogens on various commodities often exhibit seasonal patterns. As a consequence, the incidence of foodborne illness tends to follow these trends. Of the various product classes, the occurrence of microbial contamination can be high on raw meat and poultry products, with Salmonella potentially occurring in all meat and poultry product classes. Since 1999, the Food Safety and Inspection Service in the United States has collected samples of meat and poultry products and analyzed them for the presence of Salmonella. This study uses a common modeling approach to estimate the seasonal change in the proportion of test-positive samples for seven classes of raw meat and poultry products. The results generally support the hypothesis of a seasonal increase of Salmonella during the summer months. The proportions of test-positive samples decrease rapidly in the late fall for all product classes except chicken and ground turkey, which remain somewhat elevated through late winter. A comparison of the pathogens' seasonal pattern in meat and poultry with human cases reveals that the seasonal increase in human cases precedes the seasonal increase in meat and poultry by between one and three months. These results suggest that while contaminated meat and poultry products may be responsible for a substantial number of human cases, they are not necessarily the primary driver of the seasonal pattern in human salmonellosis.     

Rapid and simultaneous detection of Salmonella,Shigella, and Staphylococcus aureus in fresh pork using a multiplex real-time PCR assay based on immunomagnetic separation

The incidence of foodborne infections caused by Salmonella sp., Shigella sp. and Staphylococcus (S.) aureus in fresh pork is increasing each year, which poses a great potential threat to public health. In this study, a rapid and simultaneous detection for these three pathogens from fresh pork samples was developed by combining immunomagnetic separation (IMS) with multiplex real-time PCR (RT-PCR). Magnetic beads coated with specific antibodies were used to capture and purify the pathogens from 250 mL matrix prepared by both spiked and commercial samples, followed by DNA extraction. Then, multiplex RT-PCR was applied with three sets of specific primers and probes. The limit of detections were evaluated in 67 spiked pork samples and were 2.0 CFU/g for Salmonella, 6.8 CFU/g for Shigella, and 9.6 CFU/g for S. aureus. The sensitivity, specificity, and accuracy of IMS-multiplex RT-PCR method were 99.2%, 100%, and 99.5%, respectively. One hundred fifty-one samples were tested using the IMS-multiplex RT-PCR and culture methods, and a comparison of the results showed that the former was a potentially reliable method for rapid and effective detection of Salmonella sp., Shigella sp., and S. aureus in fresh pork.     

Survival of E. coli O157:H7 Staphylococcus aureus,Shigella flexneri and Salmonella spp. in fermenting `Borde', a traditional Ethiopian beverage

The survival and inhibition of food-borne pathogens during the fermentation of different foods and beverages is documented. This prompted the study to evaluate survival of Staphylococcus aureus, Shigella flexneri, Salmonella spp. and Escherichia coli O157:H7 during the fermentation of `Borde', a traditional Ethiopian fermented low-alcohol beverages. The pH of mixtures of `Borde' ingredients at the start of fermentation was between 4.28 and 4.31. At ambient temperatures all test strains grew well in the control and reached counts as high as log 6.6 cfu/ml for E. coli O157:H7 and >log 7 cfu/ml for S. aureus, S. flexneri, and Salmonella spp. When co-inoculated with lactic acid bacteria (LAB), counts of E. coli O157:H7, S. aureus and S. flexneri were >log 2 cfu/ml and >log 1.5 cfu/ml at 12 h and at 16 h, respectively. At 24 h, these counts were <log 1.3 cfu/ml. The count of Salmonella spp., however, was <log 2 at 16 h and they were eliminated at 24 h. LAB-fermented `Borde' had final pH of 3.85 and titratable acidity of 0.41%. At refrigeration temperature, there was no marked growth of test strains in the control. In the presence of LAB, counts of S. aureus were reduced by 2 log units whereas the counts of the other test strains were reduced only by <1 log unit. LAB increased by 1 log unit between 12 and 16 h. The survival of test strains at levels of >2 log cfu/ml between 12 and 16 h indicated health hazard as fermentation of `Borde' is completed within 12 h and consumed within 4 h thereafter.     

Detection of Salmonella spp. in spices and herbs

The detection of microbial contaminations in spices and herbs is a challenging task due to their strong antimicrobial effects, which potentially increase the risk for false-negative results. Therefore, the present study mainly focuses on the detection of Salmonella spiked to cinnamon and oregano. Both condiments completely inhibited the proliferation of Salmonella at a 1:10 (w/w) dilution. Consequently, the supplementation of the buffered peptone water with K₂SO₃ as well as the application of higher initial dilutions was investigated. While no detrimental effect of K₂SO₃ was observed during the growth of 14 different Salmonella isolates, it even improved their detection in condiments. An effect, which was also determined with increased dilution ratios. For detection, a quantitative approach via enumeration of the colony-forming units (CFUs), and qualitative approaches via the culture-based detection according to ISO 6579 and via the nucleic acid-based detection with the 3M Molecular Detection System (MDS) were performed. Subsequently, the limit of detection (LOD) was determined, which was <5 CFU 25 g⁻¹ for both qualitative approaches. Furthermore, the persistence of Salmonella DNA spiked to parsley was determined with the MDS. Despite of the modifications, the LOD for Salmonella spiked to oregano was significantly lower prior than after enrichment, pointing to the requirement for further improvements. Last but not least, a ring trial was performed, which emphasized the importance for a reliable detection.