Detection of Salmonella enteritidis and Salmonella typhimurium in foods using a rapid,multiplex real-time recombinase polymerase amplification assay

Salmonella has been recognized as a major foodborne pathogen for humans and animals. In this study, a multiplex real-time recombinase polymerase amplification (RPA) was developed for simultaneous detection of Salmonella enterica serovars, Salmonella enteritidis and Salmonella typhimurium, from chicken, eggs, lettuce, and papaya. The reaction was performed for 20 min at 35°C, and the detection limit of the assay was 10² CFU/ml for pure culture. In food application, the limit of detection (LOD) of S. enteritidis and S. typhimurium using multiplex real-time RPA without enrichment procedure was 10² CFU/25 g, respectively. After enrichment, the LOD of S. enteritidis and S. typhimurium was 10 CFU/25 g. Moreover, the result for Salmonella spp. was not significantly different from those obtained using a culture-based method. Additionally, the assay has a lower cross-reactivity with other pathogenic microorganisms and a good stability performance. Thus, the developed multiplex RPA assay could be used as a rapid tool for the detection of S. enteritidis and S. typhimurium in food.     

大肠杆菌O157:H7量子点免疫层析试纸条的研制

研制一种大肠杆菌O157:H7量子点免疫层析试纸。利用自制水溶性量子点静电偶联大肠杆菌O157:H7单克隆抗体,将大肠杆菌O157:H7单克隆抗体和羊抗兔二抗划线于硝酸纤维素膜分别作为检测线和质控线,制备双抗体夹心法检测大肠杆菌O157:H7的量子点免疫层析试纸。该试纸条能在5min内完成检测,检测限制为1×104 CFU/mL,对常见的8种食源菌无交叉反应。基于量子点的大肠杆菌O157:H7免疫层析试纸操作简便,灵敏度和特异性较好,可用于食品快速检测。     

Development of a novel dual priming oligonucleotide system-based PCR assay for specific detection of Salmonella from food samples

In this study, a novel dual priming oligonucleotide (DPO) system-based polymerase chain reaction (PCR; DPO system-based PCR) assay, which detected the fimY gene of Salmonella, was developed for the fast food testing. The DPO system-based PCR assay allowed a wide range of annealing temperatures at 48–68°C to efficiently amplify fimY gene with an analytical detection limit of 1.2 × 10² CFU/ml for Salmonella in pure cultures and artificially contaminated food matrix. Significantly, the presence of a bubble-like polydeoxyinosine (polyI) linker in the DPO system brought an unparalleled high specificity in the identification of target bacteria, and consequently, the false positives and mismatches of PCR process can be eliminated in priming. Applying the DPO system-based PCR assay to 285 collected food samples revealed that 29 samples were positive in this assay, in accordance with the results of conventional culture-based method, indicating a potential diagnostic capability. The high specificity of the DPO system-based PCR indicates its great potential to be a quick, reliable and practical method for the detection of Salmonella in foods.     

Microfluidic rapid quantification of Salmonella enterica serovar Typhimurium collected from chicken meat using immunomagnetic separation after formaldehyde treatment

Foodborne pathogens such as Salmonella enterica serovar Typhimurium, which cause self-limiting gastroenteritis, are important in food safety and public health. Detection of Salmonella in complex food matrices is required for rapid and effective monitoring of contaminated food products. In this study, we collected Salmonella Typhimurium in chicken using immunomagnetic separation after formaldehyde treatment. Collected Salmonella Typhimurium cells were then quantified microfluidic device or fluorescence microscopy. The recovery ratio obtained with immunomagnetic separation after formaldehyde treatment was 77 (±5)% for the microscope count and 63 (±9)% for the microfluidic count. According to the fluorescence microscope images, the food matrix was effectively removed from the collected cell suspension. The counts of Salmonella Typhimurium cells obtained by the microfluidic device and fluorescence microscope were closely correlated (R² = 0.993) with a range of 3.3 (±0.5) × 10³ to 1.2 (±0.5) × 10⁶ cells g⁻¹. Our method provides quantitative detection of Salmonella Typhimurium within 3–4 h and could be used to efficiently evaluate microbial risk of quality control of foodstuffs and prevention of foodborne disease.     

Rapid detection of Salmonella spp. using magnetic resonance

The globalization of the world's food trade calls for rapid and accurate detection of foodborne pathogens to ensure safety of foods for human consumption, to prevent outbreaks and management of foodborne infectious diseases. Currently, commercial detection methods for pathogenic microbials require multiple days for sample‐to‐answer results. In this study, we demonstrated a highly sensitive and rapid detection of a microbial pathogen using Molecular Mirroring (M²) technology and Lab‐in‐the‐Box system based on nuclear magnetic resonance that works rapidly and efficiently for the detection of Salmonella. This technology detected Salmonella at 1 cfu/reaction in water. In tuna, the M² technology detected 1 cfu/g with 5 hr of enrichment and analysis with a T₂ signal of 342 ms. In addition to sensitive detection and minimal enrichment, this methodology detected pathogens from inhibitory mediums. Therefore, this technology can be widely applied to other fields such as environmental monitoring, public health and safety, national security, and medical diagnosis.

Practical applications

The combination of molecular biology and nuclear magnetic resonance technology represents a novel, rapid, sensitive, and highly specific methodology for the detection of Salmonella spp. in tuna compared to standard conventional methods. Practical applications of the M² technology have been tested with human samples, animal samples, and food samples to detect microbial pathogens before and after food processing, thus is ideal to protect public health and to ensure food safety. Furthermore, this biosensor analytical technology can be applied to almost any medium or target of interest in the field of food safety, clinical diagnostics, and biosurveillance.     

Dynamic predictive model for the growth of Salmonella spp. in liquid whole egg

Abstract: A dynamic model for the growth of Salmonella spp. in liquid whole egg (LWE) (approximately pH 7.8) under continuously varying temperature was developed. The model was validated using 2 (5 to 15 °C; 600 h and 10 to 40 °C; 52 h) sinusoidal, continuously varying temperature profiles. LWE adjusted to pH 7.8 was inoculated with approximately 2.5–3.0 log CFU/mL of Salmonella spp., and the growth data at several isothermal conditions (5, 7, 10, 15, 20, 25, 30, 35, 37, 39, 41, 43, 45, and 47 °C) was collected. A primary model (Baranyi model) was fitted for each temperature growth data and corresponding maximum growth rates were estimated. Pseudo‐R² values were greater than 0.97 for primary models. Modified Ratkowsky model was used to fit the secondary model. The pseudo‐R² and root mean square error were 0.99 and 0.06 log CFU/mL, respectively, for the secondary model. A dynamic model for the prediction of Salmonella spp. growth under varying temperature conditions was developed using 4th‐order Runge–Kutta method. The developed dynamic model was validated for 2 sinusoidal temperature profiles, 5 to 15 °C (for 600 h) and 10 to 40 °C (for 52 h) with corresponding root mean squared error values of 0.28 and 0.23 log CFU/mL, respectively, between predicted and observed Salmonella spp. populations. The developed dynamic model can be used to predict the growth of Salmonella spp. in LWE under varying temperature conditions. Practical Application: Liquid egg and egg products are widely used in food processing and in restaurant operations. These products can be contaminated with Salmonella spp. during breaking and other unit operations during processing. The raw, liquid egg products are stored under refrigeration prior to pasteurization. However, process deviations can occur such as refrigeration failure, leading to temperature fluctuations above the required temperatures as specified in the critical limits within hazard analysis and critical control point plans for the operations. The processors are required to evaluate the potential growth of Salmonella spp. in such products before the product can be used, or further processed. Dynamic predictive models are excellent tools for regulators as well as the processing plant personnel to evaluate the microbiological safety of the product under such conditions.     

Molecular Enrichment for Qualitative Molecular Pathogen Detection in Food

Reliable detection of low levels of pathogenic microbial contaminants is one of the most important prerequisites to guarantee safe food production. In this work, the use of molecular enrichment (ME) subsequent to the sample preparation method Matrix-Lysis was evaluated solely for qualitative molecular pathogen detection from complex matrices. It could be successfully shown that the LOD of an all-molecular pathogen detection approach for food was effectively lowered by a factor of ten from 1.60?×?10¹ CFU/ml to 1.60?×?10⁰ CFU/ml due to ME.     

Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10³ to 10⁸ CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10³ CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF.     

高荧光CdTe量子点荧光探针测定Cu~(2+)

以亚碲酸钠为碲源,巯基丙酸(MPA)和柠檬酸三钠为稳定剂在水相中一步合成了具有良好荧光性质的Cd Te量子点,并利用Cd Te量子点与Cu2+混合后会发生荧光猝灭的现象,建立Cu2+测定方法,并对反应条件如缓冲体系的浓度和p H值、反应时间、量子点浓度以及试剂添加顺序进行优化。优化结果为:在p H 6.6、浓度为0.01 mol/L的磷酸一氢钠—磷酸氢二钠缓冲液中,量子点浓度5×109mol/L,以量子点—缓冲液—金属离子的添加顺序反应30 min,可得到最佳反应效果。研究显示,最佳试验条件下,Cu2+浓度为0~5×107mol/L范围内,Cd Te量子点的荧光强度猝灭程度与Cu2+浓度之间呈良好的线性关系,线性相关系数R2=0.988 4,检出限为1×108mol/L。     

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.     

Impedimetric Determinaton of Total, Mesophilic and Psychrotrophic Zounts in Raw Milk

A rapid impedimetric determination for total, mesophilic and psychrotrophic counts in raw milk showed correlations (between impedance detection times and standard plate counts) of -0.96, -0.95, and 0.96, respectively. Mesophiles were most often seen as the predominant population, the impedimetric method allowed for these samples containing above 1.0 × 10⁵ CFU/ml to be screened out within 4 hr. Psychrotrophic levels of 1 × 10⁵ CFU/ml and above were screened within 21 hr, while total concentration of samples containing above 1 × 10⁵ CFU/ml were screened within 16 hr.     

Evaluation of a Polymerase Chain Reaction–Based System for Detecting Salmonella Species from Pork Carcass Sponge Samples

Pork carcass sponge samples (n = 230) collected from 10 Taiwanese slaughter plants were screened for Salmonella using 2 methods: BAX® for Screening/Salmonella, a polymerase chain reaction‐based detection system and a culture method using SM‐ID agar as the selective plating medium. The BAX method identified 14 samples as positive for Salmonella. The 8 samples identified as Salmonella‐positive by the SM‐ID method were also BAX‐positive. Inoculation studies showed BAX detected Salmonella in samples having initial 1.4 × 10¹ cfu/mL Salmonella inoculum prior to the enrichment process in the presence of 3.0 × 10⁶ cfu/mL of non‐Salmonella florae. BAX provides a rapid screening alternative to the culture method for Salmonella detection.     

Magnetoelastic biosensor for the detection of Salmonella typhimurium in food products

In this article, a magnetoelastic sensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium in food products is described. The remote query nature of magnetoelastic sensors enables the detection of bacterial species in sealed and opaque containers. Bacterial binding to the antibody on the sensor surfaces changed the resonance parameters, and these changes were quantified by the shift in the sensor’s resonance frequency. Response of the sensors to increasing concentrations (5 × 10¹–5 × 10⁸ cfu/ml) of S. typhimurium in three different food products (water, fat-free milk and apple juice) was studied and similar responses were observed. These results were also further ascertained by Scanning Electron Microscopy (SEM) studies. A detection limit of 5 × 10³ cfu/ml, with a sensitivity of 139 Hz/decade was obtained for the sensors tested in water samples, as compared to 129 Hz/decade in apple juice and 127 Hz/decade in fat free milk. A 2 × 0.4 × 0.015 mm sensor was employed in all the investigations. The dissociation constant K _d and the binding valencies for S. typhimurium spiked in water samples was 435 cfu/ml and 2.33 respectively; as compared to 309 cfu/ml and 2.38 for apple juice; and 1389 cfu/ml and 1.85 for fat free milk samples. Bacterial binding was specific and a divalent binding was observed.     

High-throughput detection of spore contamination in food packages and food powders using tiered approach of ATP bioluminescence and real-time PCR

A rapid and quantitative method for detection of Bacillus spores in food/non-alcoholic beverage packages and food powders has been developed using filtration-based ATP bioluminescence and real-time PCR, targeting the sporulation gene (spo0A). In combination with heat activation, the presence and amount of viable bacterial spores (i.e., Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus thuringiensis) was determined within 20 min through ATP signal amplifications. The detection limits of heat activation-ATP bioluminescence assay for B. amyloliquefaciens and B. licheniformis spores on food packages were 1.4 × 10² and 1.0 × 10³ CFU/cm², respectively. In contaminated food powders, B. thuringiensis spores could be detected by the ATP assay within the range of 7.9 × 10⁰ to 3.2 × 10⁴ CFU/mg powder while the PCR detection limit was 614 CFU/mg. Linear relationships between luminescent signal (RLU/mg) and plate count (CFU/mg) were found. The same sample after heat activation-ATP assay could be directly used for real-time PCR as a streamlined detection to confirm the identity of Bacillus spores in food packages and food powders even though some bacterial DNA loss was observed. This tiered approach, filtration-based one-tube ATP luminescence method as a rapid, viable screening and using real-time PCR as confirmation, could serve as a high-throughput tool for the detection of Bacillus spores in the food and beverage industry.     

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.     

Rapid detection of Salmonella in milk by a nuclear magnetic resonance biosensor based on the streptavidin-biotin system and O-carboxymethyl chitosan target gadolinium probe

Rapid and sensitive detection of foodborne pathogens is of great importance for food safety. Here, a set of nuclear magnetic resonance (NMR) biosensors based on a O-carboxymethyl chitosan target gadolinium (Gd) probe was developed to quickly detect Salmonella in milk by combining NMR technology and bioimmunotechnology with membrane filtration technology. First, O-carboxymethyl chitosan (O-CMC) was biotinylated to prepare biotinylated O-carboxymethyl chitosan (biotin–O-CMC) through amide reaction, and biotinylated magnetic complexes (biotin–O-CMC–Gd) were obtained by using O-CMC, which has strong chelating adsorption on Gd. The target probe was obtained by combining biotin–O-CMC–Gd with the biotinylated antibody (biotin–antibody) via streptavidin (SA) by introducing the SA–biotin system. Then, Salmonella was captured by the target probe through antigen–antibody interaction. Finally, NMR was used to measure the longitudinal relaxation time (T₁) of the filtrate collected by membrane filtration. This NMR biosensor with good specificity and high efficiency can detect Salmonella with the sensitivity of 1.8 × 10³ cfu/mL within 2 h; in addition, it can realize the detection of complex samples because of its strong anti-interference capability and may open up a new method for rapid detection of Salmonella, which has a great application potential.     

Piezoelectric Biosensor for Detection of Salmonella typhimurium

A flow injection analysis (FIA) system was developed based on a piezoelectric biosensor for detection of Salmonella typhimurium. The anti-Salmonella spp antibody was immobilized onto the gold electrode coated quartz crystal surface through a polyethylenimine-glutaraldehyde (PEG) technique and dithiobis-succinimidyl propionate (DSP) coupling. The PEG technique proved more successful for FIA applications than the DSP coupling. The biosensor had responses of 23–7 Hz in 25 min when the PEG immobilization technique was employed, with R²0.94 for Salmonella typhimurium concentrations of 5.3.10⁵ to 1.2.10⁹ CFU/mL.     

Effect of carvacrol and thymol on Salmonella spp. biofilms on polypropylene

The present study evaluated the effects of carvacrol and thymol against Salmonella spp. biofilm on polypropylene. The efficacy of the compounds was assessed by quantifying Salmonella spp. cells during and after biofilm formation on polypropylene and performing scanning electron microscopy. During biofilm formation, carvacrol and thymol, at subinhibitory concentrations, reduced bacterial counts about 1–2 log, while established Salmonella spp. biofilms were reduced about 1–5 log by carvacrol and thymol, at MIC or 2× MIC. The greatest reduction in carvacrol‐treated biofilms, about 5 log, was observed with 156 and 312 μg mL^?1 (MIC and 2× MIC) in established Salmonella Typhimurium ATCC 14028 biofilms. Thymol showed the greatest reduction, about 4 log, at 624 μg mL^?1 (2× MIC) against mature Salmonella Enteritidis biofilm. Carvacrol and thymol reduced the number of Salmonella spp. cells on polypropylene, suggesting their potential for the control of Salmonella spp. biofilms.     

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.