Duplex detection of foodborne pathogens using a SERS optofluidic sensor coupled with immunoassay.

Herein, we developed a surface-enhanced Raman spectroscopy (SERS) optofluidic sensor coupled with immunoprobes to simultaneously separate and detect the foodborne pathogens, Escherichia coli O157:H7, and Salmonella in lettuce and packed salad. The method consists of three steps of (i) enrichment to enhance detection sensitivity, (ii) selective separation and labelling of target bacteria by their specific antibody-bearing SERS-nanotags and (iii) detection of tagged bacterial cells using SERS within a hydrodynamic flow-focusing SERS optofluidic device, where even low counts of bacterial cells were detectable in the very thin-film-like sample stream. SERS-nanotags consisted of different Raman reporter molecules, representing each species, i.e., the detection of Raman reporter confirms the presence of the target pathogen. The anti-E. coli antibody used in this study functions against all strains of E. coli O157:H7 and the anti-Salmonella antibody used in this work acts on a wide range of Salmonella enterica strains. Bacterial counts of 1000, 100, and 10 CFU/ 200 g sample were successfully detected after only 15 min enrichment. Our method showed a very low detection limit value of 10 CFU/ 200 g sample for the bacterial mixture in both lettuce and packed salad, proving the efficiency and high sensitivity of our method to detect multiple pathogens in the food samples. The total analysis time, including sample preparation for simultaneous detection of multiple bacteria, was estimated to be 2 h, which is much less than the time required in conventional methods. Hence, our proposed protocol is considered a promising rapid and efficient approach for pathogen screening of food samples.

Detection of virulence and extended spectrum ß-lactamase genes in Salmonella by multiplex high-resolution melt curve real-time PCR assay.

AIMS: Develop and standardize multiplex high-resolution melt curve (HRM) real-time PCR assays for simultaneous detection of Salmonella virulence and extended spectrum ß-lactamase (ESBL) genes in food. METHODS AND RESULTS: Two sets of multiplex real-time PCR assays targeting six virulence and three ESBL genes with internal amplification control were standardized. The first assay detected hilA, fimH, sipA, blaTEM and blaSHV, and the second detected invA, fimA, stn and blaCMY . The PCR assays were validated with DNA samples from 77 different Salmonella strains. The assay specificity was tested with DNA from 47 non-Salmonella strains. Melt curve analyses showed distinct, well-separated melting peaks of each target gene detected by their respective melting temperatures (Tm ). Different food samples were spiked with 10, 102 and 103  CFU/ml of Salmonella. The optimized assays were able to detect all target genes in concentrations of as low as 10 CFU/ml in 25 g foods within 10 h of enrichment. CONCLUSIONS: Multiplex HRM real-time PCR assays can be used as rapid detection methods for detecting Salmonella in foods. SIGNIFICANCE AND IMPACT OF STUDY: The assays developed in this study will allow for accurate detection of virulence and ESBL genes in Salmonella that are present in low concentrations in food samples.

Antimicrobial effect and toxicity of cellulose nanofibril/silver nanoparticle nanocomposites prepared by an ultraviolet irradiation method.

The objective of this study was to synthesize a novel antimicrobial cellulose nanofibril/silver nanoparticle (CNF/AgNP) nanocomposite by an ultraviolet (UV) irradiation method and evaluate the toxicity of the nanocomposite to human colon cells. AgNPs coated on CNFs have an average size of ˜28 nm and exhibited a surface plasma resonance absorption peak at 402 nm. Coating AgNPs on CNFs interfered with the formation of intra-chain and inter-chain hydrogen bonds of cellulose. Moreover, the CNF/AgNP nanocomposite exhibited significant antimicrobial activities against two important food-borne pathogens, including Escherichia coli O157:H7 and Staphylococcus aureus. No apparent toxicity of the CNF/AgNP nanocomposite to Caco-2 and FHC human colon cells was observed, except when a high content of (≥500 µg/m L) of the nanocomposite was used for which a significant decrease of cell viability was observed. The nanocomposite's toxicity was related to the content, size, and surface charge of UV-synthesized AgNPs on CNFs. These results indicate that the antimicrobial CNF/AgNP nanocomposite prepared by UV irradiation method can be potentially used as an active filler applied in food packaging materials.