Rapid Detection Of Foodborne Pathogens Research Articles

Recent Advances in Peptide Nucleic Acids for Rapid Detection of Foodborne Pathogens

Peptide nucleic acids (PNAs) are DNA/RNA analogs in which sugar-phosphate backbone is replaced by N-2-aminoethylglycine repeating units. Since PNA contains a neutral skeleton, there is no electrostatic repulsion, resulting in significant stability of its hybrid structure with complementary oligonucleotides. At present, PNA has taken the place of DNA probe in many studies. There are several disadvantages of cellular uptake of PNA, so modifications in PNA backbone or covalent coupling with cell-penetrating peptides are necessary to improve its delivery inside the cells. In recent years, PNA has been extensively used in the rapid detection of microorganisms, such as fluorescence in situ hybridization, PCR amplification, biosensor, and gene chip. The structure and characteristics of PNA probe, the hybridization method, and the design principle of PNA probes are introduced in this review, and the application progress of PNA probes in the rapid detection of foodborne pathogens is summarized. On this basis, the advantages and disadvantages of PNA probes are analyzed, and the future development trend is prospected.

Application of Biosensors for Detection of Pathogenic Food Bacteria: A Review.

The use of biosensors is considered a novel approach for the rapid detection of foodborne pathogens in food products. Biosensors, which can convert biological, chemical, or biochemical signals into measurable electrical signals, are systems containing a biological detection material combined with a chemical or physical transducer. The objective of this review was to present the effectiveness of various forms of sensing technologies for the detection of foodborne pathogens in food products, as well as the criteria for industrial use of this technology. In this article, the principle components and requirements for an ideal biosensor, types, and their applications in the food industry are summarized. This review also focuses in detail on the application of the most widely used biosensor types in food safety.

Rapid Classification of Single Bacterium Based on Backscattering Microscopic Spectrum—A Pilot Study

Rapid detection of foodborne pathogens is one of the most effective ways to solve food safety problems. To achieve rapid and noninvasive detection and classification of foodborne pathogens, we modified a fiber confocal backscattering micro-spectral system to suit an extremely small biological sample, that is, a bacterium. This system offers single-bacterium level, label-free, convenient, and environmentally friendly characterization. Three categories of common foodborne pathogens (Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus) were measured. The scattering spectrum ranging from 450 to 900 nm was selected, and by the model of principal component analysis (PCA) and error back propagation algorithm of back propagation neural network (BPNN), the backscattering microscopic spectra of three categories of pathogens were dimensionality reduced, identified and classified. The results showed that the identification accuracy of three categories of pathogens was above 90%, under neutral, acidic and alkaline culturing conditions，respectively. The preliminary results demonstrated the feasibility of using confocal backscattering microscopic spectra combined with PCA and BPNN algorithm to identify and classify single bacterium in a rapid, noninvasive, and label-free manner.

Single-cell classification of foodborne pathogens using hyperspectral microscope imaging coupled with deep learning frameworks

A high-throughput hyperspectral microscope imaging (HMI) technology with hybrid deep learning (DL) framework defined as “Fusion-Net” was proposed for rapid classification of foodborne bacteria at single-cell level. HMI technology is useful in single-cell characterization, providing spatial, spectral and combined spatial-spectral profiles with high resolution. However, direct analysis of these high-dimensional HMI data is challenging. In this work, HMI data were decomposed into three parts as morphological features, intensity images, and spectral profiles. Multiple advanced DL frameworks including long-short term memory (LSTM) network, deep residual network (ResNet), and one-dimensional convolutional neural network (1D-CNN) were utilized, achieving classification accuracies of 92.2 %, 93.8 %, and 96.2 %, respectively. Taking advantage of fusion strategy, individual DL framework was stacked to form “Fusion-Net” that processed these features simultaneously with improved classification accuracy of up to 98.4 %. Our study demonstrated the ability of DL frameworks to assist HMI technology in single-cell classification as a diagnostic tool for rapid detection of foodborne pathogens.

An in situ-Synthesized Gene Chip for the Detection of Food-Borne Pathogens on Fresh-Cut Cantaloupe and Lettuce.

Fresh foods are vulnerable to foodborne pathogens which cause foodborne illness and endanger people’s life and safety. The rapid detection of foodborne pathogens is crucial for food safety surveillance. An in situ-synthesized gene chip for the detection of foodborne pathogens on fresh-cut fruits and vegetables was developed. The target genes were identified and screened by comparing the specific sequences of Salmonella Typhimurium, Vibrio parahemolyticus, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O157:H7 from the National Center for Biotechnology Information database. Tiling array probes were designed to target selected genes in an optimized hybridization system. A total of 141 specific probes were selected from 3,227 hybridization probes, comprising 26 L. monocytogenes, 24 S. aureus, 25 E. coli O157:H7, 20 Salmonella Typhimurium, and 46 V. parahemolyticus probes that are unique to this study. The optimized assay had strong amplification signals and high accuracy. The detection limit for the five target pathogens on fresh-cut cantaloupe and lettuce was approximately 3 log cfu/g without culturing and with a detection time of 24 h. The detection technology established in this study can rapidly detect and monitor the foodborne pathogens on fresh-cut fruits and vegetables throughout the logistical distribution chain, i.e., processing, cleaning, fresh-cutting, packaging, storage, transport, and sale, and represents a valuable technology that support the safety of fresh agricultural products.

Electrical DNA Biosensor Using Aluminium Interdigitated Electrode for Salmonella Detection

Nowadays there are many alternative methods that have been discovered and developed for the rapid detection of foodborne pathogens that can cause food poisoning. Unfortunately, majority of them still requires improvement in sensitivity and selectivity issues to be of any practical use daily. In this research, biosensors was prepared from 5 µm gap Aluminium interdigitated electrode (Al IDE) to detect Salmonella enterica typhi (S. typhi). The IDE sensors in the biosensor field is extremely interest in these days due to the high number of finger electrodes as comb structure which can gain high sensitivity through electrical measurements. S. typhi is a serious food borne pathogen, makes typhoid disease which causes many deaths annually in worldwide. Functionalization steps of the Al IDE to create biosensor was based on silanization by APTES, immobilization with carboxylic functionalized S. typhi ssDNA probes and blocking agent with tween-20 were the major functionalization steps. The functionalized steps were electrically characterized using current voltage measurements. The selectivity measurement was performed with specific target was identified electrically using complementary, non-complementary and single base mismatch ssDNA target.

Rapid and sensitive detection of Salmonella Typhimurium using nickel nanowire bridge for electrochemical impedance amplification

Rapid detection of foodborne pathogens is crucial to prevent the outbreaks of foodborne illnesses. In this study, a sensitive electrochemical aptasensor was developed using aptamer coated gold interdigitated microelectrode for target capture and impedance measurement, and antibody modified nickel nanowires (NiNWs) for target separation and impedance amplification. First, the interdigitated microelectrode was modified with the biotinylated aptamers against Salmonella typhimurium through electrostatic absorption of streptavidin onto the microelectrode and streptavidin-biotin binding. Then, the target Salmonella cells were magnetically separated and concentrated using the NiNWs modified with the anti-Salmonella typhimurium antibodies to form the bacteria-NiNW complexes, and incubated on the microelectrode to form the aptamer-bacteria-NiNW complexes. After an external arc magnetic field was developed and applied to control the NiNWs to form conductive NiNW bridges across the microelectrode, the enhanced impedance change of the microelectrode was measured and used to determine the amount of target bacteria. This electrochemical aptasensor was able to quantitatively detect Salmonella ranging from 102 to 106 CFU/mL in 2 h with the detection limit of 80 CFU/mL. The mean recovery for the spiked chicken samples was 103.2%.

Rapid and sensitive detection of Salmonella with reduced graphene oxide-carbon nanotube based electrochemical aptasensor

Rapid detection of foodborne pathogens is crucial as ingestion of contaminated food products may endanger human health. Thus, the objective of this study was to develop a biosensor using reduced graphene oxide-carbon nanotubes (rGO-CNT) nanocomposite via the hydrothermal method for accurate and rapid label-free electrochemical detection of pathogenic bacteria such as Salmonella enterica. The rGO-CNT nanocomposite was characterized using Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction and transmission electron microscopy. The nanocomposite was dropped cast on the glassy carbon electrode and further modified with amino-modified DNA aptamer. The resultant ssDNA/rGO-CNT/GCE aptasensor was then used to detect bacteria by using differential pulse voltammetry (DPV) technique. Synergistic effects of aptasensor was evident through the combination of enhanced electrical properties and facile chemical functionality of both rGO and CNT for the stable interface. Under optimal experimental conditions, the aptasensor could detect S. Typhimurium in a wide linear dynamic range from 101 until 108 cfu mL−1 with a 101 cfu mL−1 of the limit of detection. This aptasensor also showed good sensitivity, selectivity and specificity for the detection of microorganisms. Furthermore, we have successfully applied the aptasensor for S. Typhimurium detection in real food samples.

Effect of the Number of ME Elements on Capture Rate of a Small Number of Pathogens in the Prototype of Phage Filter with a Comparison of Rocking Method

Foodborne illness is a frequent public health problem because there is potential for food to be contaminated by pathogens at any point from farms to the dinner tables. Each year, the cost of foodborne illness in the U.S. is enormous. The direct and indirect costs associated with foodborne illness are more than $15.6 billion per year. For rapid detection of foodborne pathogens, Magnetoelastic (ME) biosensor is one of the methods to detect pathogens on food surface and liquids. As the multistate outbreak caused by Romaine lettuce contamination in 2018, current microbiological testing methods, required by Food and Drug Administration (FDA)’s Bacteriological Analytical Manual (BAM), shows shortages. According to the requirement, a new method, named 3D bio-molecular filter, was developed to capture, concentrate, and isolate a small number of pathogens in large volume of liquid streams. The filter consists of ME filter elements, a pair of permanent magnetic plates, and testing glass tube. ME filter elements are magnetically held and arrayed in a testing glass tube. The target pathogen in a large volume of liquid will be capture by ME filter elements when a large volume of liquid passes through. A prototype of a compact edition was developed in this paper to support this method. The device consists of high magnetic plates, which provide uniform high magnetic force to hold ME filter elements, and a square glass tube to pass through the liquid with target pathogen. In this work, E2 phage is immobilized on ME filter elements to capture, concentrate, and isolate Salmonella Typhimurium in processing water. Two different methods to capture Salmonella, rocking method and 3D biomolecular filter method, are compared. The capture rate and effects of the number of ME filter elements on Salmonella capture efficiency will be discussed.

Effects of Different Broth Enrichment upon Phage Magnetoelastic Biosensor for Fast Detecting Low <i>Salmonella</i> Counts on Problematic Produce

According to the FDA Bacteriological Analytical Manual (BAM) for Salmonella identification in produce, two pre-enrichment steps with 48 hours of incubation are the golden procedures. Lactose broth is recommended for the first pre-enrichment step medium for leafy greens, and the universal pre-enrichment (UP) broth is for tomatoes. However, the suggested broths were evaluated to have the maximum performance using the culture-dependent methods, and may not be applied to other methods, such as biosensor detection platform. A wireless bacteriophage magnetoelastic (ME) biosensor has been recently developed for real-time or rapid detection of food-borne pathogens in various foods. This affinity-based biosensor utilizes a phage oligonucleotide as the probe to capture target bacteria. In this study, the efficiencies of different pre-enrichment media for early detection of low Salmonella on spinach leaves and tomatoes use ME biosensors to shorten detection time. Four broths of modified peptone water, Lennox broth (LB), lactose broth, and UP broth were selected in this study. Various pre-enrichment times for ME biosensor detection were investigated. After spiking 4 cfu/g Salmonella on the tomatoes surfaces, the phage biosensor was able to detect Salmonella within 5 hours of pre-enrichment comparing to 24 hours in the FDA procedures. For Salmonella spiked spinach leaves, the same medium showed Salmonella positive within 7 hours. This study demonstrated that LB broth is the best medium to shorten pre-enrichment time to pass Salmonella number detection thresholds for ME biosensor detection in spinach and tomatoes when comparing to FDA procedures.

Rapid detection of Salmonella in milk by biofunctionalised magnetic nanoparticle cluster sensor based on nuclear magnetic resonance

Controlling foodborne pathogens is important to ensure food safety and prevent foodborne diseases. In this study, a Fe3O4 nanoparticle cluster (Fe3O4 NPC) biosensor based on nuclear magnetic resonance was developed for Salmonella detection. The capture antibody of Salmonella was used, and the biotinylated antibody specifically recognised Salmonella at different sites. Fe3O4 NPC modified with streptavidin were used to capture biotinylated antibody by biotin and determine streptavidin interaction. Finally, Fe3O4 NPC immobilised on 96-well microplates were eluted, thereby reducing the transverse relaxation time (T2) of neighbouring water molecules. The method detected Salmonella at 105 cfu mL−1, which was also the limit of detection in spiked milk samples, and showed high selectivity over other non-target bacteria. The proposed biosensor could be a potential tool for sensitive and rapid detection of foodborne pathogens in food, environmental, and agricultural samples.

Rapid detection of Salmonella Typhimurium using magnetic nanoparticle immunoseparation, nanocluster signal amplification and smartphone image analysis

Rapid detection of foodborne pathogens is of great significance for ensuring food safety. In this study, a portable biosensor was developed for rapid and sensitive detection of Salmonella Typhimurium using magnetic nanoparticle immunoseparation, nanocluster signal amplification and smartphone image analysis. The magnetic nanoparticles were conjugated with the monoclonal antibodies by biotin-streptavidin system for specifically capturing the target to form the magnetic bacteria. The polyclonal antibodies and glucose oxidase (GOx) were incubated with calcium chloride to synthesize the immune GOx-nanoclusters (GNCs) for reacting with the magnetic bacteria to form the nanocluster bacteria. The GNCs on the nanocluster bacteria were used to catalyze the glucose into hydrogen peroxide, which was measured using the peroxide test strip. The image of the strip was collected and analyzed using the Hue-Saturation-Lightness color space based smartphone APP for the determination of the target bacteria. This proposed biosensor was able to detect Salmonella Typhimurium with the linear range of 101–105 CFU/mL and the low detection limit of 1.6 × 101 CFU/mL. The mean recovery of Salmonella Typhimurium in the spiked chicken samples was 99.8%. This biosensor has the potential to provide a simple, portable and low-cost method for rapid and in-field detection of Salmonella Typhimurium in foods.

Surface-Enhanced Raman Scattering (SERS) With Silver Nano Substrates Synthesized by Microwave for Rapid Detection of Foodborne Pathogens.

Rapid and sensitive methods have been developed to detect foodborne pathogens, a development that is important for food safety. The aim of this study is to explore Surface-enhanced Raman scattering (SERS) with silver nano substrates to detect and identify the following three foodborne pathogens: Escherichia coli O157: H7, Staphylococcus aureus and Salmonella. All the cells were resuspended with 10 mL silver colloidal nanoparticles, making a concentration of 107 CFU/mL, and were then exposed to 785 nm laser excitation. In this study, the results showed that all the bacteria can be sensitively and reproducibly detected directly by SERS. The distinctive differences can be observed in the SERS spectral data of the three food-borne pathogens, and the silver colloidal nanoparticles can be used as highly sensitive SERS-active substrates. In addition, the assay time required only a few minutes, which indicated that SERS coupled with the silver colloidal nanoparticles is a promising method for the detection and characterization of food-borne pathogens. At the same time, principle component analysis (PCA) and hierarchical cluster analysis (HCA) made the different bacterial strains clearly differentiated based on the barcode spectral data reduction. Therefore, the SERS methods hold great promise for the detection and identification of food-borne pathogens and even for applications in food safety.

An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR

BackgroundStaphylococcus aureus is an important human pathogen causing a variety of life-threatening diseases. Rapid and accurate detection of Staphylococcus aureus is a necessity for prevention of outbreaks caused by this pathogen. PCR is a useful tool for rapid detection of foodborne pathogens, however, its inability to differentiate DNA from dead cells and live cells in amplification severely limits its application in pathogen detection. The aim of this study was to develop an improved assay was developed by incorporating the sample treatments with a surfactant and propidium monoazide (PMA) in qPCR for detection of viable S. aureus cells.ResultsThe cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. Triton x-100 was coupled with PMA for sample treatments for detection of viable S. aureus cells in artificially contaminated milk. The qPCR results indicated that the assay reached high an amplification efficiency of 98.44% and the live S. aureus cells were accurately detected from the triton-treated spiked milk samples by the PMA-qPCR assay.ConclusionsThe qPCR assay combined with treatments of PMA and surfactants offers a sensitive and accurate means for detection of viable S. aureus cells. Cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. The information on sample treatment with surfactants to improve the dead cell DNA removal efficiency in qPCR by increasing PMA’s permeability to dead cells can be used for other pathogens, especially for Gram-positive bacteria.

Rapid Identification of Mixed Enteropathogenic Bacteria by Means of Au Nanoparticles@Bacteria Using Portable Raman Spectrometer.

Rapid detection of food-borne pathogens is the most critical and urgent issue among all the current food safety problems. As enhanced substrate, nanoparticles are widely used in surface enhanced Raman scattering (SERS) because of unique optical and physicochemical properties. In this study, Au nanoparticles with monodisperse and good reproducibility were synthesized by using sodium citrate reduction method. Applying Au nanoparticles sol as enhanced substrate, a portable Raman spectrometer had been applied for rapid detection of single and mixture pathogenic bacterial contamination by SERS. The results indicated that Escherichia coli, Salmonella typhimirium, Shigella flexner and Staphylococcus aureus showed specific Raman phenotypes at 600∼1700 cm-1. Generally, different bacteria could be easily and instantly recognized by its Raman phenotypes. The PC-LDA classification model was set up by combined bacterial Raman phenotypes with the multivariate statistical analysis. With the short-time inoculation, four enteropathogenic bacteria could be rapidly, precisely, sensitively and specifically identified. Furthermore, the model also had a good ability to predict the mixed contamination. This research provides the possibility of rapid detection in the food and biomedical fields.

Whole-bacterium SELEX of DNA aptamers for rapid detection of E.coli O157:H7 using a QCM sensor

The rapid detection of foodborne pathogens is critical to ensure food safety. The objective of this study is to select aptamers specifically bound to Escherichia coli O157:H7 using the whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and apply the selected aptamer to a QCM (quartz crystal microbalance) sensor for rapid and sensitive detection of target bacteria. A total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of counter selection against a mixture of Staphylococcus aureus, Listeria monocytogenes, and Salmonella Typhimurium, were performed. The aptamer pool from the last round was cloned and sequenced. One sequence S1 that appeared 16 times was characterized and a dissociation constant (Kd) of 10.30nM was obtained. Subsequently, a QCM aptasensor was developed for the rapid detection of E. coli O157:H7. The limit of detection (LOD) and the detection time of the aptasensor was determined to be 1.46×103 CFU/ml and 50min, respectively. This study demonstrated that the ssDNA aptamer selected by the whole-bacterium SELEX possessed higher sensitivity than previous work and the potential use of the constructed QCM aptasensor in rapid screening of foodborne pathogens.

Rapid Pathogen Detection by Surface Swab Sampling and Wireless Biosensing

This paper presents a method that combines surface swab sampling and wireless biosensing for rapid detection of foodborne pathogens on solid surfaces. The method can be applied to any surfaces, including the surfaces of food processing tables and fresh produce, for food safety improvement. Surface swab sampling is an inexpensive and easy-to-use method of collecting pathogens. A desired area or the whole area of a target surface (if needed) can be swabbed to collect pathogens rapidly. In this study, five commercially available swabs were compared. We first investigated the water absorption capacities of the swabs and then analyzed the efficiencies of capture and release of a model pathogen, Salmonella Typhimurium. Plate counting was employed to quantify the initially inoculated cells and collected cells by surface swabbing, and the efficiency of capture and release was calculated.

A sensitive biosensor using double-layer capillary based immunomagnetic separation and invertase-nanocluster based signal amplification for rapid detection of foodborne pathogen

Combining double-layer capillary based high gradient immunomagnetic separation, invertase-nanocluster based signal amplification and glucose meter based signal detection, a novel biosensor was developed for sensitive and rapid detection of E. coli O157:H7 in this study. The streptavidin modified magnetic nanobeads (MNBs) were conjugated with the biotinylated polyclonal antibodies against E. coli O157:H7 to form the immune MNBs, which were captured by the high gradient magnetic field in the double-layer capillary to specifically separate and efficiently concentrate the target bacteria. Calcium chloride was used with the monoclonal antibodies against E. coli O157:H7 and the invertase to form the immune invertase-nanoclusters (INCs), which were used to react with the target bacteria to form the MNB-bacteria-INC complexes in the capillary. The sucrose was then injected into the capillary and catalyzed by the invertase on the complexes into the glucose, which was detected using the glucose meter to obtain the concentration of the glucose for final determination of the E. coli O157:H7 cells in the sample. A linear relationship between the readout of the glucose meter and the concentration of the E. coli O157:H7 cells (from 102 to 107 CFU/mL) was found and the lower detection limit of this biosensor was 79 CFU/mL. This biosensor might be extended for the detection of other foodborne pathogens by changing the antibodies and has shown the potential for the detection of foodborne pathogens in a large volume of sample to further increase the sensitivity.

Rapid Pathogen detection By Surface Swab Sampling and Wireless Biosensing

This paper presents a method that combines surface swab sampling and wireless biosensing for rapid detection of foodborne pathogens on solid surfaces. The method can be applied to any surfaces, including the surface of food processing tables and fresh produce, for improving food safety. Surface swab sampling is an inexpensive and easy-to-use method of collecting pathogens. A desired area or the whole area of a target surface (if needed) can be swabbed to collect pathogens rapidly. In this study, we first investigated the efficiency of capture and release of a model pathogen, Salmonella Typhimurium, in swab sampling. A Salmonella-contaminated surface (2 × 2 cm2 flat polyethylene slab) was prepared by using a spot-inoculation method. Five drops of a Salmonella solution (20 ul/drop at concentrations from 5X108 cfu/ml to 5X103 cfu/ml) were inoculated on the polyethylene surface and allowed to dry in a biosafety cabinet for 2 hours. Swab sampling was then conducted at a controlled temperature and humidity to capture the pathogen. Finally, the swabs used were dipped in a TBS solution for releasing the captured Salmonella cells. Various swab tip materials were tested, including cotton, rayon, and nylon. The effects of swab force and sonication/vortexing on the capture and release efficiency were also investigated. The released Salmonella cells were finally detected and quantified instantaneously by using a wireless phage-based magnetoelastic biosensor. By comparing the quantities of the initially inoculated cells and detected cells, the efficiency of capture and release was calculated.

Direct bacterial loop-mediated isothermal amplification detection on the pathogenic features of the nosocomial pathogen - Methicillin resistant Staphylococcus aureus strains with respiratory origins.

Loop-mediated isothermal amplification based detection assays using bacterial culture or colony for direct detection of methicillin resistant Staphylococcus aureus(MRSA) had been developed and evaluated, followed by its extensive application on a large scale of clinical MRSA isolated from respiratory origins, including nasal swabs and sputums. Six primers, including outer primers, inner primers and loop primers, were specifically designed for recognizing eight distinct sequences on four targets: 16SrRNA, femA, mecA and orfX. Twenty-seven reference strains were used to develop, evaluate and optimize this assay. Then, a total of 532 clinical MRSA isolates were employed for each detected targets. And the results were determined through both visual observation of the color change by naked eye and electrophoresis. The specific of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 40min. The limit of detections (LOD) of bacteria culture LAMP assays were determined to be 104CFU/ml for 16S rRNA, femA, as well as orfX and 105CFU/ml for mecA, respectively. The established novel assays on MRSA detection may provide new strategies for rapid detection of foodborne pathogens.