Contaminated Food Samples Research Articles

Molecular and metabolic strategies for postharvest detection of heat-resistant fungus Neosartorya fischeri and its discrimination from Aspergillus fumigatus

Abstract Heat Resistant Fungi (HRF) and toxigenic fungi are considered as a serious problem both in the agricultural field and for human health, due to ascospores and mycotoxins production, which can contaminate fruit and, as a consequence, adversely affect the agricultural food chain. One strategy to identify these fungi is the use of modern molecular method, which include the analysis of DNA target regions for differentiation of the fungal species. However, previously developed methods included only the identification of pure strains but not the detection of Neosartorya fischeri in artificially contaminated food samples, such as fruit or juices. Therefore, the aim of presented study was to develop a detection method of Neosartorya fischeri in contaminated strawberry fruit and juice. The other strategy is the use of phenotypic assays to determine the metabolic profile of the fungi in order to facilitate a qualitative and quantitative detection of microorganisms based on specific substrates utilization and mycotoxins production. Accordingly, the study included an evaluation of the differences in phenotype profile between N. fischeri and the phylogenetically close fungus Aspergillus fumigatus, as a strategy of their differentiation and identification. PCR detection assay was developed that revealed the presence of N. fischeri DNA in all tested contaminated samples of fruit and juice. Therefore, it can be concluded that this rapid molecular method is an important tool for the evaluation of the postharvest quality of agricultural raw materials. Moreover, the results suggest that specific metabolic and mycotoxin patterns may be used as N. fischeri detection markers and strategy in discrimination of this fungus from A. fumigatus. The results indicated that N. fischeri and A. fumigatus had a different time period of carbon sources utilization, and particularly N. fischeri presented a more efficient carbon metabolism. Mycotoxins, verruculogen and fumitremorgin C, were detected after 4 days incubation of N. fischeri. Although metabolic assays are not such fast as molecular detection approach, they allow to deeper insight into the pathways activated by heat-resistant and toxigenic fungi. Therefore, both molecular and metabolic strategies of heat-resistant fungus detection and identification are complementary and can be used to measure postharvest quality of fruit and their products.

Simultaneous detection of seven foodborne Enterobacteriaceae pathogens using multiplex PCR

Members of the family Enterobacteriaceae aremajor pathogens associated with gastrointestinal disorderscaused by the consumption of contaminated foods. This study developed a multiplex PCR targetingspecific genes for simultaneous detection and differentiationof seven major Enterobacteriaceae members, namely,Escherichia coli (uidA), Proteussp. (atpD), Salmonella sp. (invA), Enterobacter sp. (16S rRNA), Klebsiella sp. (gyrA), Klebsiella pneumoniae (rpoB) and K. oxytoca (pehX) from contaminated food samples. Four main types of food samples comprise fresh, frozen, processed frozen and processed preserved samples were examined by using simultaneous amplification of those seven-targeted genes from obtained reference strains. The multiplex PCR assay showed detection sensitivity reached 5-10 cfu for each pathogen after 2h of sample incubation on nutrient broth. Generally, the 7plex-PCR assay revealed that the most common pathogens found in 192 different food samples with high frequency was Salmonella which distributed within most of all types of food having the maximum occurrence frequency reached 6.77% in hotdog and sausage samples followed by 5.21% in fresh chicken and beef burger. The occurrence frequency % of E. coli was found to be high in chicken/beef burger samples (6.25%), and then slightly decreased to 5.21%, 4.68% and 4.17% in frozen meat, hotdog/sausage and fresh fish samples, respectively. The processed preserved (chicken/beef luncheon and pastrami) and frozen chicken possessed the lowest presence of foodborne pathogens. This study recommends the use of 7plex-PCR detection system for the rapid and accurate detection of some Enterobacteriaceae pathogens is inexpensively and thus could be used for the regular monitoring of food quality.

Rapid Detection of Food-Borne Escherichia coli O157:H7 with Visual Inspection by Crossing Priming Amplification (CPA)

Escherichia coli O157:H7 is an important food-borne pathogen and has ability to contaminate food, such as water, meat products, and milk. Moreover, E. coli O157:H7 is the main toxin-producing serotype which can produce Shiga toxin type 1 and type 2 and cause intestinal disease. The strong pathogenicity and lethality of Escherichia coli O157:H7 pose a serious threat to human. This study aims to develop a rapid and visual detection assay of E. coli for rfbE, stx1, and stx2 genes by crossing priming amplification (CPA). The limit of detection of CPA assay for rfbE, stx1, and stx2 genes was 3.20 fg/μl, 320 fg/μl, and 320 fg/μl in genomic DNA, while that of in artificially contaminated food samples was 103 cfu/ml, 105 cfu/ml, and 105 cfu/ml, respectively, which was distinctly higher than that of PCR methods. And the specificity of CPA assay was tested by 22 different bacterial strains and except for E. coli O157:H7 ATCC43895 and other E. coli O157:H7 isolated from eggs, milk, and beef, all strains showed negative results. The visible detection assay was conducted by the addition of calcein in the reaction solutions. The CPA assay showed a successful detection of E. coli O157:H7 (Shiga toxin–producing and non-Shiga toxin–producing) within 60 min under 63 °C with high sensitivity and specificity. These results indicated that the CPA assays with calcein can provide an advanced method to achieve the rapid and visual detection of food-borne E. coli O157:H7.

H test for exclusion: A guide to an intervention approach to lessen the risk of aflatoxin‐contaminated foods in sorely contaminated regions

AbstractThis research was done on the assumption that the risk for hepatocellular carcinoma (HCC) development that attributed to the consumption of highly contaminated food (HCF) with aflatoxin, may have a major contribution to the total of expected risk. If this hypothesis is true, it is easy to apply an effective and low‐cost intervention approach by excluding HCF and then subjecting it to treatments before use. To examine this hypothesized, H test for exclusion (Hx) model was developed as a computer program, in order to estimates the daily intake, the margin of exposure, and HCC cases per 105 individuals for selected groups of contaminated food samples. To show how this model works, the model was demonstrated in a real example. The obtained results showed that the actual application of excluding HCF would result in a reduction of the expected cancer cases by 58%. Further, this model illustrates comparisons between permitted and nonpermitted samples with regards to international legislation. The model is particularly suitable for regions where contaminated crops and foods are prevalent. It is also convenient for addressing chronic exposure of commonly consumed food commodities to lower contamination levels.Practical applicationsFor aflatoxin in contaminated crops, the Hx program can be used in order to estimates the daily intake, the margin of exposure (MOE), and the expected hepatocellular carcinoma (HCC) cases per 105. Furthermore, it provides a smooth rapprochement between non‐permitted and permitted samples in accordance with international legislation. In fact, the actual application of the model in regions where contaminated crops and foods are predominant can lead to efficient treatments with limited economic losses by excluding HCF.

Application of digital PCR and next generation sequencing in the etiology investigation of a foodborne disease outbreak caused by Vibrio parahaemolyticus

Globally, V. parahaemolyticus infection is a leading cause of bacterial diarrheal diseases. Pathogenic V. parahaemolyticus strains that produce hemolysins are responsible for these diseases. The composition of pathogenic and non-pathogenic V. parahaemolyticus and the change of the bacterial composition before and after traditional selective enrichment in a single sample associated with disease outbreak remain unclear. We investigated an outbreak by using next generation sequencing and digital PCR to address those questions. NGS showed that the V. parahaemolyticus caused the outbreak belonged to s single clone. In contrast, among the seven non-pathogenic V. parahaemolyticus isolated from the suspected food sample, 4 serotypes and 6 PFGE patterns were identified. And nearly 70,000 SNPs were identified among the non-pathogenic strains. This result confirmed that the outbreak was caused by V. parahaemolyticus. Furthermore, NGS results clearly showed the diversity of non-pathogenic V. parahaemolyticus in a single contaminated food sample. The ratios of non-pathogenic and pathogenic V. parahaemolyticus were 31.41 and 620.11 in the original and enriched food samples respectively showed by digital PCR. Meta-genomic data indicated the top 3 species were Weissella cibaria, Weissella confusa, and Enterobacter cloacae in the original food sample, and Vibrio sp Ex25, Vibrio sp 712i, and V. parahaemolyticus in the enriched sample. Therefore, the combing of NGS and digital PCR results showed that traditional Vibrio selective enrichment media could facilitate the growth of Vibrios, however, it provided no advantages to pathogenic V. parahaemolyticus. Hence, our results indicated that the traditional culture methods alone may lead to wrong conclusions and so improvements in culture methods are needed.

Development and application of a visual loop-mediated isothermal amplification combined with lateral flow dipstick (LAMP-LFD) method for rapid detection of Salmonella strains in food samples

Salmonella strains are major foodborne pathogens for the animals and humans, presenting a significant threat to food safety and public health worldwide. The rapid and accurate diagnosis of Salmonella infection is required for effective control and management of disease epidemics. In this study, we developed a rapid and efficient assay that combines loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD) for detection of the Salmonella hilA gene. Compared with PCR and real-time PCR methods, the LAMP-LFD assay has the same specificity and higher sensitivity, and required only 40 min (10 min for LFD detection) at 65 °C. All 52 strains of Salmonella yielded positive results using the LAMP-LFD assay and showed no cross reaction with 37 tested non-Salmonella strains. The detection limit of the LAMP-LFD assay was 13.5 fg/μl (genomic DNA) and 6.7 CFU/mL (cell), which was 1000-fold more sensitive than conventional PCR and 100-fold more sensitive than real-time PCR. Additionally, the LAMP-LFD method could detect Salmonella in artificially contaminated food samples (milk, pork, beef and chicken) when present as low as 144 CFU/mL or CFU/g and without the use of an enrichment step. Nevertheless, the sensitivity was increased to 1.44 CFU/mL or CFU/g after culturing at 37 °C for 6 h. Fifty food samples (chicken meat) were used to test the practicality of the LAMP-LFD assay. After an enrichment step at 37 °C for 6 h, the results showed 100% accuracy compared to the standard culture-based method (ISO 6579:2002) in which 17 out of 50 food samples gave positive results. Overall, the results demonstrated that the developed LAMP-LFD method targeting the hilA gene is rapid, specific, sensitive and allows ease of operation for Salmonella detection, suggesting the potential for this assay to be used as an alternative to traditional testing methods and could applied in low-resource settings.

Development of a smartphone-based biomimetic sensor for aflatoxin B1 detection using molecularly imprinted polymer membranes

A novel smartphone-based optical biomimetic sensor based on free-standing molecularly imprinted polymer (MIP) membranes was developed for rapid and sensitive point-of-care detection of aflatoxin B1. The developed MIP membranes were capable of selective recognition of the target analyte and, at the same time, of generation of a fluorimetric sensor response, which could be registered using the camera of a smartphone and analysed using image analysis. The developed system provides a possibility of synchronous detection of aflatoxin B1 in 96 channels. UV irradiation of aflatoxin B1, selectively bound by the MIP membranes from the analysed samples, initiated fluorescence of aflatoxin B1 with intensity directly proportional to its concentration. The composition of the MIP membranes used as a recognition element was optimised taking into account data of computational modelling. Two functional monomers (2-acrylamido-2-methyl-1-propansulfonic acid and acrylamide) were identified as optimal for the formation of aflatoxin B1-selective binding sites in the structure of the MIP membranes. Working characteristics of the smartphone-based sensor system were also estimated. The influence of pH and of buffer and NaCl concentrations on the smartphone-based sensor responses were studied. High selectivity of the developed sensor system towards aflatoxin B1 was confirmed in experiments with the close structural analogue of the target analyte - aflatoxin G2, and ochratoxin A. The detection limit for aflatoxin B1 using the smartphone-based sensor systems was found to be 20 ng mL−1 for the sensor based on MIP membranes synthesised with acrylamide as a functional monomer. The storage stability of the recognition elements of the developed sensors was estimated as one year when stored at 22 °C. The possibility to detect the aflatoxin B1 in contaminated food samples was shown. The MIP-membrane-based sensor system provided a convenient point-of-care approach in food safety testing.

Aptamer as capture agent in enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of Aflatoxin B1

Aflatoxin B1 (AFB1), is one of the most toxic mycotoxins found to contaminate various food commodities like cereals, dried fruits, tree nuts, spices and crude vegetable oils. In spite of considerable progress in analytical techniques, there is still a need to develop rapid and highly sensitive detection platforms for AFB1. In this study, AFB1 specific aptamer was used as a capture molecule to develop an enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of AFB1. Under optimized conditions, the assay had a linear detection range from 1 μg to 1 pg with a limit of detection (LOD) of 1 pg/mL in buffer. Conventional ELISA with AFB1 hapten as the capture agent (LOD = 10 pg/mL) was also carried out to compare the results with the present method. Recovery studies in food samples like dried red chillies, groundnut and pepper using both the methods was found to be in the range of 88.49–106.4% at 10 ng/mL and 87.4% to 95.8% at 5 ng/mL for ELASA and 76.56–127.68% at 10 ng/mL and 82–101.2% at 5 ng/mL for ELISA. Higher detection (10 fold) and better recovery using ELASA suggest that the method could offer an early, ultrasensitive, high-throughput, qualitative and semi-quantitative detection of AFB1 in contaminated food samples.

A new single-tube platform of melting temperature curve analysis based on multiplex real-time PCR using EvaGreen for simultaneous screening detection of Shiga toxin-producing Escherichia coli, Salmonella spp. and Listeria monocytogenes in food

Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. and Listeria monocytogenes are continuously reported as causative agents of great concern regarding food safety and widespread contamination in many food varieties. Therefore, their simultaneous detection may be advantageous in terms of cost, time and labor savings and simplicity. This study developed a new, simple platform of multiplex real-time polymerase chain reaction (mRT-PCR) for specific, sensitive and rapid detection of STEC, Salmonella spp. and L. monocytogenes in food. The single-tube mRT-PCR format was developed by combining an 18 h enrichment step in simultaneous enrichment broth, boiling based on DNA extraction assay and a mRT-PCR detection system based on melting curve analysis using a fluorescent dye (EvaGreen) for detection of the presence or absence of the three target bacterial pathogens in food samples. Three specific peaks were clearly detected with average melting temperatures of 84.52 ± 0.90 °C, 87.51 ± 0.54 °C and 79.32 °C ± 0.48 °C for STEC, Salmonella spp. and L. monocytogenes, respectively. The sensitivity and specificity of these newly developed mRT-PCR platforms were further investigated using artificially and naturally contaminated food samples. The relative sensitivity, relative specificity and relative accuracy were all 100%, with a detection limit of 1 cfu for each target pathogen in 25 g of food sample. The developed platform of EvaGreen-based single-tube mRT-PCR for detection of the three target pathogenic bacteria in food samples provided results of absence or presence within 20 h. The newly developed mRT-PCR platform in this study offers a promising approach for simple, rapid, sensitive, specific and accurate detection of the three target bacterial pathogens in food.

A highly selective enrichment broth combined with real-time PCR for detection of Staphylococcus aureus in food samples

A new selective enrichment broth (OSA) was developed for detection of Staphylococcus aureus combined with real-time PCR. Acriflavine, nalidixic acid, potassium tellurite and sodium chloride were added as selective reagents and mannitol, sodium pyruvate, and yeast extract as promoters. The growth of S. aureus (10, 100, 1000 CFU/mL inoculation level) in OSA was superior to that in TSB with 7.5% NaCl (TNaCl). OSA showed a good inhibition ability against 8 strains including 4 Gram-positive and 4 Gram-negative. Further, OSA gave a good recovery of cold-, acid-, and heat-stressed cells comparable to TSBYE but superior to TNaCl. A real-time PCR targeting the nuc gene was developed and the standard curve was established from 9.1 × 107 to 9.1 × 102 cfu/mL with a high regression coefficient of determination (R2 = 0.999) and a high amplification efficiency of 96.362%. Using artificially contaminated food samples (kimbap, milk, and spam) with a level of ∼10 CFU/25 g or mL, OSA better supported the growth of S. aureus and all samples were readily detected after 12 h enrichment by real-time PCR. In summary, OSA was demonstrated as a promising selective enrichment broth for detection of S. aureus combined with real-time PCR with a total assay time of 15 h.

Glucosylation of T-2 and HT-2 toxins using biotransformation and chemical synthesis: Preparation, stereochemistry, and stability

Plant-derived phase II metabolites of T-2 toxin (T2) and HT-2 toxin (HT2) were first described in 2011 and further characterized in the following years. Since then, some efforts have been made to understand their biosynthesis, occurrence, toxicity, toxicokinetics, and finally relevance for consumers. Thus, the probably most important question is whether and how these metabolites contribute to toxicity upon hydrolysis either during food processing or the gastrointestinal passage. To answer this question, firstly, knowledge on the correct stereochemistry of T2 and HT2 glucosides is important as this affects hydrolysis and chemical behavior. So far, contradictory results have been published concerning the number and anomericity of occurring glucosides. For this reason, we set up different strategies for the synthesis of mg-amounts of T2, HT2, and T2 triol glucosides in both α and ß configuration. All synthesized glucosides were fully characterized by NMR spectroscopy as well as mass spectrometry and used as references for the analysis of naturally contaminated food samples to validate or invalidate their natural occurrence. Generally, 3-O-glucosylation was observed with two anomers of HT2 glucoside being present in contaminated oats. In contrast, only one anomer of T2 glucoside was found. The second aspect of this study addresses the stability of the glucosides during thermal food processing. Oat flour was artificially contaminated with T2 and HT2 glucosides individually and extruded at varying initial moisture content and temperature. All four glucosides appear to be more stable during food extrusion than the parent compounds with the glucosidic bond not being hydrolyzed.

Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples.

Rapid detection of Listeria and other microbial pathogens in food is an essential part of quality control and it is critical for ensuring the safety of consumers. Culture-based methods for detecting foodborne pathogens are time-consuming, laborious and cannot detect viable but non-culturable microorganism, whereas viability PCR methodology provides quick results; it is able to detect viable but non-culturable cells, and allows for easier handling of large amount of samples. Although the most critical point to use viability PCR technique is achieving the complete exclusion of dead cell amplification signals, many improvements are being introduced to overcome this. In the present work, the yield of dead cell DNA neutralization was enhanced by incorporating two new sample treatment strategies: tube change combined with a double light treatment. This procedure was successfully tested using artificially contaminated food samples, showing improved neutralization of dead cell DNA.

Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB

The aim of the study was to identify and evaluate specific biomarkers to differentiate within Bacillus cereus group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of B. cereus and B. thuringiensis along with 12 strains representing 2 bacterial groups – B. mycoides, B. pseudomycoides, B. weihenstephanensis (B. cereus group); B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes (non-Bacillus sp.) were identified by applying valid biomarkers (groEL and gyrB). In addition, the presence of B. cereus group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (GroEL) and topoisomerase enzyme protein (gyrB). Direct analysis of samples revealed the specificity towards identification and characterization of the B. cereus group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers groEL and gyrB with a high specificity of 98% and 96% respectively to analyze the total B. cereus group. Further, we also reported the detection limit of groEL and gyrB in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of B. cereus group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.

Improved sample treatment protocol for accurate detection of live Salmonella spp. in food samples by viability PCR.

Culture-based detection is still considered as the standard way for detection of Salmonella in foods, although molecular methods, such as viability PCR (vPCR), have been introduced to overcome some disadvantages of traditional culture methods. Despite the success of the vPCR methodology, the problem of false-positive results is a major drawback, especially when applied to environmental samples, hindering the interpretation of the results. To improve the efficiency of vPCR, many approaches have been introduced by several authors during the last years. In the present work, the combination of PEMAX dye, double tube change, and double photo-activation step was established as a strategy to improve vPCR protocol. By combining these approaches, we developed an improved sample treatment protocol able to neutralize DNA signals of up to 5.0×107 dead cells/sample from both pure culture and artificially contaminated food samples. Our results indicate that vPCR can work reliable and has a potential for high throughput detection of live Salmonella cells in food samples, minimizing false-positive signals.

High-resolution melt curve PCR assay for specific detection of E. coli O157:H7 in beef

Among the disease-causing Shiga toxin-producing Escherichia coli (STEC), E. coli O157:H7 is estimated to cause one-third of the total STEC illnesses and the most cases of hemolytic uremic syndrome (HUS) in the U.S. The uidA gene which is present in the majority of E. coli strains, codes for the synthesis of β-d-glucuronidase (GUD). In E. coli O157:H7, the uidA gene has a single point mutation at the +93 position that leads to an alteration in the amino acid sequence encoding the GUD enzyme. The aim of this study was to distinguish E. coli O157:H7 from other E. coli using a high resolution melt curve (HRM) real-time PCR assay. Based on the uidA mutation in E. coli O157:H7, a reliable PCR assay targeting the uidA gene was developed to differentiate E. coli O157:H7 from other E. coli serotypes and the closely related Shigella spp. The specificity of the assay was validated using a set of 128 pure bacterial DNA samples. Following enrichment and immunomagnetic separation (IMS), the assay was further applied to spiked ground beef and beef trim. Isolates of E. coli O157:H7 formed distinctive melt peaks that were easily distinguishable from those of other E. coli serogroups and Shigella isolates in the PCR plot. Therefore, this assay was able to clearly discriminate E. coli O157:H7 strains from other E. coli and Shigella. With a 5–8 h enrichment time, followed by IMS, 10 CFU of E. coli O157:H7 were detectable in 325 g spiked beef samples. The HRM E. coli O157:H7 detection assay standardized in this study will allow for accurate identification of contaminated food samples and help in preventing foodborne outbreaks caused by this pathogen.

Whole genome sequencing analysis of Salmonella enterica serovar Weltevreden isolated from human stool and contaminated food samples collected from the Southern coastal area of China

Salmonella enterica serovar Weltevreden is the most common non-typhoid Salmonella found in South and Southeast Asia. It causes zoonoses worldwide through the consumption of contaminated foods and seafood, and is considered as an important food-borne pathogen in China, especially in the Southern coastal area. We compared the whole genomes of 44 S. Weltevreden strains isolated from human stool and contaminated food samples from Southern Coastal China, in order to investigate their phylogenetic relationships and establish their genetic relatedness to known international strains. ResFinder analysis of the draft genomes of isolated strains detected antimicrobial resistance (AMR) genes in only eight isolates, equivalent to minimum inhibitory concentration assay, and only a few isolates showed resistance to tetracycline, ciprofloxacin or ampicillin. In silico MLST analysis revealed that 43 out of 44 S. Weltevreden strains belonged to sequence type 365 (CC205), the most common sequence type of the serovars. Phylogenetic analysis of the 44 domestic and 26 international isolates suggested that the population of S. Weltevreden could be segregated into six phylogenetic clusters. Cluster I included two strains from food and strains of the “Island Cluster”, indicating potential inter-transmission between different countries and regions through foods. The predominant S. Weltevreden isolates obtained from the samples from Southern coastal China were found to be phylogenetically related to strains from Southern East Asia, and formed clusters II–VI. The study has demonstrated that WGS-based analysis may be used to improve our understanding of the epidemiology of this bacterium as part of a food-borne disease surveillance program. The methods used are also more widely applicable to other geographical regions and areas and could therefore be useful for improving our understanding of the international spread of S. Weltevreden on a global scale.

In Vitro Isothermal Nucleic Acid Amplification Assisted Surface-Enhanced Raman Spectroscopic for Ultrasensitive Detection of Vibrio parahaemolyticus.

Detection of pathogenic microorganisms is of great importance for public health and food safety. Traditional protocols can hardly meet the continuously increasing demand in sensitivity and specificity of pathogen detections. In this study, we adopted Vibrio parahaemolyticus (V. parahaemolyticus, Vp) as the model analyte, and developed an antibody-Vp-aptamer heterosandwich-based surface-enhanced Raman scattering (SERS) method in conjunction with in vitro isothermal amplification for sensitive detection of V. parahaemolyticus. The rolling circular amplification (RCA) products provided enormous sites for assembling the Au@Ag nanoparticles and forming excess "hot-spot" sites for Raman measurement. By using this enhanced Raman signal strategy in the detection, a limit of detection (LOD) as low as 1 cfu/mL was successfully achieved for ultrasensitive detection of V. parahaemolyticus. In addition, we have applied this method to artificially contaminated food samples. The detection data indicated that this method is able to determine the concentrations of V. parahaemolyticus in the spiked food samples with satisfactory sensitivity and specificity and, thus, this developed ultrasensitive SERS scheme is well suited for the urgent need in pathogen detection and demonstrated great potential in food safety, environment monitoring, and a clinical setting.

Evaluation of microplate immunocapture method for detection of Vibrio cholerae, Salmonella Typhi and Shigella flexneri from food

BackgroundImproved methods with better separation and concentration ability for detection of foodborne pathogens are in constant need. The aim of this study was to evaluate microplate immunocapture (IC) method for detection of Salmonella Typhi, Shigella flexneri and Vibrio cholerae from food samples to provide a better alternative to conventional culture based methods.ResultsThe IC method was optimized for incubation time, bacterial concentration, and capture efficiency. 6 h incubation and log 6 CFU/ml cell concentration provided optimal results. The method was shown to be highly specific for the pathogens concerned. Capture efficiency (CE) was around 100% of the target pathogens, whereas CE was either zero or very low for non-target pathogens. The IC method also showed better pathogen detection ability at different concentrations of cells from artificially contaminated food samples in comparison with culture based methods. Performance parameter of the method was also comparable (Detection limit- 25 CFU/25 g; sensitivity 100%; specificity-96.8%; Accuracy-96.7%), even better than culture based methods (Detection limit- 125 CFU/25 g; sensitivity 95.9%; specificity-97%; Accuracy-96.2%).ConclusionThe IC method poses to be the potential to be used as a method of choice for detection of foodborne pathogens in routine laboratory practice after proper validation.

Determination of Listeria monocytogenes numbers at less than 10 cfu/g

AbstractListeria monocytogenes is a foodborne pathogen that causes a relatively rare foodborne disease called listeriosis, with a high mortality rate of 20%-30% and an undefined dose response. Current European Union regulations permit up to 100 colony-forming units (cfu)/g in food at the end of its shelf life, where the food has been shown not to support the growth of this pathogenic bacterium. Therefore, enumeration of L. monocytogenes at low numbers in food is important. The objective of this study was to reduce the detection limit of L. monocytogenes in food by a factor of 10. The International Organisation for Standardisation (ISO) 11290-2 method for enumeration of L. monocytogenes in food recommends spreading 0.1 mL of a 1:10 dilution of the food on the surface of an agar plate (detection limit 100 cfu/g), or 1.0 mL spread in equal parts on the surface of three agar plates (detection limit: 10 cfu/g). The pour-plate method (using 1 or 10 mL of an appropriate dilution) was compared to the spread-plate method using the ISO-approved chromogenic medium Agar Listeria according to Ottaviani and Agosti (ALOA). Using the pour-plate method, the colony morphology and halo formation were similar to the spread-plate method from pure cultures and inoculated foods. Using the pour-plate method in a 140 mm Petri dish, 10 mL of a 1:10 dilution of food allowed determination of numbers as low as 1 cfu/g. Applying this method, L. monocytogenes in naturally contaminated food samples were enumerated at numbers as low as 1-9 cfu/g.

Improvement of Polymyxin–Egg Yolk–Mannitol–Bromothymol Blue Agar for the Enumeration and Isolation of Bacillus cereus in Various Foods

A modified polymyxin-egg yolk-mannitol-bromothymol blue agar (mPEMBA) was developed by supplementing polymyxin-egg yolk-mannitol-bromothymol blue agar (PEMBA) with trimethoprim to improve the selectivity for and recoverability of Bacillus cereus from naturally and artificially contaminated food samples. The number of B. cereus in mPEMBA was significantly higher than in PEMBA, indicating better recoverability (P < 0.05) in red pepper powder (PEMBA 0.80 ± 0.22 log CFU/g versus mPEMBA 1.95 ± 0.17 log CFU/g) and soybean paste (PEMBA 2.19 ± 0.18 log CFU/g versus mPEMBA 3.09 ± 0.13 log CFU/g). In addition, mPEMBA provided better visual differentiation of B. cereus colonies than PEMBA, which is attributable to the reduced number of competing microflora. We conclude that the addition of trimethoprim to PEMBA could generate a synergistic effect to improve selectivity for B. cereus .