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.     

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.     

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10² CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10² CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10³ and 3.6 × 10³ CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.     

Detection of anisakids in fish and seafood products by real-time PCR

Anisakids are a group of widely distributed nematodes which have acquired high social relevance due to their involvement in foodborne infections caused by consumption of raw or undercooked seafood. A TaqMan^®-LNA probe real-time assay targeting the cytochrome oxidase subunit I (COI) was developed allowing the simultaneous detection of the most important anisakids species present in fish and seafood products.The determination of the detection limit in terms of ppm was 1 ppmFor the validation of method developed, twenty fish and cephalopod samples were experimentally contaminated with anisakid. It was checked that in cases in any anisakids species was present, it was detected because the Ct was always less than 35 and did not produce any case false negatives. The main novelty of this work lies in the fact that it can be applied to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. The proposed methodology is rapid, robust, highly sensitive and readily adaptable in routine molecular diagnostic laboratories, and can be employed as molecular screening method in order to assess the food security.     

Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products

In this work, a specific, sensitive, and accurate technique was presented for simultaneous detection of Salmonella spp., Shigella spp., and Staphylococcus aureus in food products, three of the more frequent foodborne pathogens that were usually reported in a variety of food matrices. An internal amplification control (IAC) was added in a multiplex PCR (mPCR) reaction system as an indicator of false negative result that can come from the presence of PCR inhibitors in food products. In the presence of inhibitor, no signal would result for the target genes as well as the IAC which results in a positive signal, thereby, eliminating false negative results. To ensure detection of only the viable cells, the effects of sodium deoxycholate (SD) in combination with propidium monoazide (PMA) treatment in the presence of dead cells and viable cells were investigated. Results showed that PMA treatment alone could not effectively inhibit the detection of 10⁷ CFU/mL of dead Salmonella Typhimurium, Shigella sonnei, and S. aureus from PCR amplification. However, the SD in combination with PMA treatment gave negative results for PCR amplification of dead S. Typhimurium, S. sonnei, and S. aureus in pure culture and food products. When the developed SD-PMA-mPCR assay in combination with IAC was applied to detect the spiked food (milk, ground beef), the LOD of SD-PMA-mPCR assay for S. Typhimurium, S. sonnei, and S. aureus inoculated individually or inoculated simultaneously into milk or ground beef were 10¹ CFU/mL or 10¹ CFU/g after 15 h enrichment. The results suggested that the SD-PMA-mPCR assay in combination with IAC held promise for the detection of foodborne S. Typhimurium, S. sonnei, and S. aureus.     

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.     

Simultaneous detection of Vibrio cholerae,Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio vulnificus in seafood using dual priming oligonucleotide (DPO) system-based multiplex PCR assay

In this study, a rapid and reliable multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus in seafood was developed using the dual priming oligonucleotide (DPO) system. Species-specific DPO primers were designed targeting the mdh, vvhA, colH and toxR genes for the discrimination of V. cholerae, V. vulnificus, V. alginolyticus and V. parahaemolyticus, respectively. Compared to conventional PCR assay, the DPO system-based multiplex PCR assay allowed a wider annealing temperature at 48 °C–68 °C to effectively amplify target genes followed an analytical detection limit of <1.5 × 10² CFU/mL (or g) for each Vibrio species in pure cultures or artificially contaminated food matrix. A total of 396 bacterial strains including 209 targets and 187 other bacterial strains were used to test the specificity of the DPO system-based multiplex PCR assay, and results showed that specific PCR product was only observed in target bacterial strains without nonspecific priming. Practical application of the assay indicated that target Vibrio species in seafood, clinical samples and foodborne outbreaks can be accurately detected. This DPO system-based multiplex PCR assay developed in this study would be a powerful tool for the rapid and reliable detection of the target Vibrio species.     

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.     

Development and evaluation of a real-time PCR assay for detection of lobster,a crustacean shellfish allergen

Crustacean shellfish are a leading cause of food allergy in American adults, and the Food Allergen Labeling and Consumer Protection Act requires that different types of crustacean shellfish be distinguished from each other. In general ELISA assays are not capable of differentiating crustacean type, but PCR assays are. In this work, a real-time PCR assay for lobster, a crustacean shellfish allergen, was developed and evaluated. Food matrices were spiked with lobster meat at 0.1, 1, 10, 100, 1000, 10⁴, and 10⁵ parts per million (ppm). In addition to testing of several different food matrices, method performance was determined using conditions which have historically proven challenging for PCR analyses, specifically food matrices with low DNA content and acidic pH levels, as well as foods that were treated with combined high temperature and pressure. Real-time PCR standard curves were generated from spiked, treated foods and analyzed with respect to linear range and reaction efficiency. In most cases, the assay was linear over 6–8 orders of magnitude; lower limits of detection were 0.1–1 ppm and reaction efficiencies were within the preferred range of 100 ± 10%. A notable exception occurred in the case of heat treatment at acidic pH, which resulted in severe delay or complete loss of amplification signals.     

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL⁻¹ and 100 ng mL⁻¹ of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g⁻¹ (110–120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.     

Development of a PCR protocol to detect ochratoxin A producing moulds in food products

Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 10²–10⁴ cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs.     

Quantification and differentiation of Bacillus cereus group species in spices and herbs by real-time PCR

Spores of Bacillus (B.) cereus group species are frequent contaminants in foodstuffs including spices and herbs. However, the distribution of individual B. cereus group species is unknown as standard cultural methods are insufficient for differentiation. Real-time PCR is an alternative method to detect, differentiate and quantify B. cereus group species in food.In our study we applied a combination of previously described real-time PCR assays to detect and quantify the B. cereus group (excluding B. cytotoxicus) with simultaneous discrimination of B. pseudomycoides and cry1-positive B. thuringiensis as well as differentiation of B. weihenstephanensis from B. cereus group species with non-rhizoid colony morphology. For testing food matrices, which can also include PCR inhibiting substances, an internal amplification control was included. In total, five DNA extraction kits were tested on pure spore suspensions to select the one with the best recovery rate when coupled to real-time PCR. The Qiagen DNeasy Blood & Tissue Kit performed best with a limit of detection (LOD) of approximately 100 cfu/ml for spores of each B. cereus, B. weihenstephanensis, B. thuringiensis and B. pseudomycoides strain. However, applied to allspice, paprika, pepper and oregano artificially contaminated with B. cereus spores the LOD was ≥10⁵ cfu/g. In contrast, using the open-formula cetyltrimethylammonium bromide (CTAB) method LODs of 10² to 10³ cfu/g were achieved for paprika, pepper and oregano. For allspice, the LOD was 10⁶ cfu/g.Our quantitative multiplex real-time PCR coupled to DNA extraction by the CTAB method provides a sensitive culture independent technique with the potential to quantitatively detect and differentiate B. cereus group species in several spices and herbs.     

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.     

Double antibodies sandwich enzyme-linked immunosorbent assay for the detection of Alicyclobacillus acidoterrestris in apple juice concentrate

Two heterologous antibodies against Alicyclobacillus acidoterrestris (A. acidoterrestris) were obtained from Sprague Dawley (SD) Rats and Japan White Rabbits by immunizing them with an A. acidoterrestris strain isolated from apple juice concentrate (AJC). By using these two antibodies, we established a double antibodies sandwich ELISA (DAS-ELISA) method for the rapid detection of A. acidoterrestris in AJC, with the detection limit of 5 × 10³ CFU/mL. There was no cross-reaction with seven common foodborne microorganisms by employing this established essay, and the coefficient of variation (CV) within and among independent runs were less than 5.0% and 10% respectively, indicating of satisfactory specificity and repeatability. This DAS-ELISA assay was successfully applied to detect A. acidoterrestris in AJC samples when coupled with selective incubation for 12 h, and the results were agreed well with those obtained from the routine K medium method. Further, this method shows advantage over indirect ELISA (ID-ELISA) and SPA-ELISA assays previously reported by our group, facilitating the use of this newly developed assay method in food products.     

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice.     

A novel screening approach based on six real-time PCR systems for the detection of crustacean species in food

Analysis of crustacean species plays a role in authenticity issues as well as allergen detection. A real-time PCR-based screening assay was developed for the detection of crustaceans in food. In order to cover most relevant species in one analytical step, PCR systems were newly developed for the detection of shrimps (Penaeidae), lobster (Homarus sp.), Common shrimp (Crangon crangon), river prawns (Macrobrachium sp.) and Chinese mitten crab (Eriocheir sinensis). In addition a published system targeting Northern prawn (Pandalus borealis) was selected. All PCR-systems are based on mitochondrial 16S rRNA gene sequences and were optimized to be run with a standard program at a universal annealing temperature of 60 °C. Validation experiments confirmed a sensitivity of the PCR systems of 0.01–0.1 genome copies or 0.04–2.5 pg DNA, respectively. Specificity was demonstrated with 25,000 copies of pure genomic DNA from about thirty plant and animal species relevant in food and the performance in food matrix was evaluated. Finally primer and probes were pre-spotted steadily on 96-well PCR plates and the practical applicability of the assay was proven with selected food products. The assay enables detection of multiple species of market relevance.     

Evaluation of carbonyl species in fish oil: An improved LC–MS/MS method

Fish oil, a common source of omega-3 fatty acids, can be easily oxidized to generate carbonyl species, thus, the measurement of these compounds in fish oil are significant for food safety. Here, a comprehensive and sensitive method to quantify forty-four carbonyl species including toxic substances such as acrolein, glyoxal, methylglyoxal and trans-4-hydroxy-2-hexenal in fish oil has been developed and validated. The samples were derivatized with 2,4-dinitrophenylhydrazine, cleaned using C18 solid phase extraction, and analyzed by liquid chromatography coupled with tandem mass spectrometry. The calibration curves represented satisfactory linearity (r² > 0.998) and the quality control samples showed favorable precisions within an acceptable range (RSD < 10%). The accuracies ranged from 96 to 109%. The detection and quantification limits ranged from 1.5 to 30 ng/mL and 5–90 ng/mL, respectively. The method was successfully applied to determine carbonyl species in ten fish oil products. The proposed method is suitable for high throughput analysis of carbonyl species in fish oil.     

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.     

Loop-mediated isothermal amplification (LAMP) coupled with bioluminescence for the detection of Listeria monocytogenes at low levels on food contact surfaces

A commercial loop-mediated isothermal amplification (LAMP) based system with bioluminescence, named as 3M™ Molecular Detection Assay (MDA), was validated for the detection of Listeria monocytogenes (3-strain cocktail) at low levels (10⁰, 10¹, 10² CFU/100 cm²) inoculated on stainless steel (SS) and polyethylene (PE) surfaces, with and without (w/o) organic load (OL) of cold-smoked salmon homogenate by comparing with a standard ISO method as reference. The results of this study revealed that a commercial LAMP-based method performed equally effective compared with ISO method at inoculum levels of 10⁰ and 10²/100 cm², showing 100% specificity and sensitivity, respectively. At 10¹ CFU/100 cm², a slight reduction in the effectiveness of LAMP-based method was observed most likely due to the use of single enrichment step in the procedure of LAMP-based method. The LAMP-based method was shown to be rapid and reliable detection technique for L. monocytogenes present at low numbers on food processing surfaces, regardless of organic residues, and can be applicable in seafood industry.