Selective turn-on fluorescence detection of Vibrio parahaemolyticus in food based on charge-transfer between CdSe/ZnS quantum dots and gold nanoparticles (original) (raw)
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Annals of Microbiology, 2012
Vibrio parahaemolyticus is an important human pathogen responsible for foodborne gastroenteritis worldwide. In this paper, a loop-mediated isothermal amplification (LAMP) method was developed for detection of V. parahaemolyticus in seafood. A set of four primers, two outer and two inner, was designed specifically to recognize the thermolabile hemolysin gene (tlh) of V. parahaemolyticus. The LAMP assay was capable of detecting a minimum of 900 fg test tube −1 for V. parahaemolyticus genomic DNA and 2.4×10 2 CFU mL −1 for pure cultures. The detection limit for the seeded seafood samples was 8.9×10 2 CFU g −1 .
Journal of microbiological methods, 2016
Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2×10(3)CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and...
International Journal of Food Microbiology, 2011
Thermostable direct hemolysin (TDH) TDH-related hemolysin (TRH) Monoclonal antibody (mAb) Enzyme-linked immunosorbent assay (ELISA) Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus.
LWT, 2019
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Detection of environmental Vibrio parahaemolyticus using a polyclonal antibody by flow cytometry
Environmental Microbiology Reports, 2010
The aim of this study was to detect and quantify Vibrio parahaemolyticus using flow cytometry (FCM) in combination with a polyclonal antibody developed in our laboratory. Experiments were carried out using V. parahaemolyticus cells in pure and mixed bacteria culture suspensions in either artificial or natural seawater. Using FCM, V. parahaemolyticus cells labelled with the polyclonal antibody and a secondary fluorescein isothiocyanate-conjugated antibody were detected and rapidly quantified at low cell densities (10 3 cells ml -1 ) in both the pure and mixed cultures. To determine the specificity of our antibody, its crossreactivity with other ATCC bacterial strains and some environmental Vibrio spp. and Gram-positive isolates was also assessed. Significant immunoreactivity levels above background were obtained for V. harvey 64, V. parahaemolyticus 704 and V. alginolyticus 1407, although the intensities were significantly less than for V. parahaemolyticus Conero. The experiments carried out in natural seawater confirmed the antibody specificity towards V. parahaemolyticus Conero even if a lower proportion of labelled cells was observed. The application of FCM in combination with a primary polyclonal antibody appears to be a promising technique for the detection and quantification of V. parahaemolyticus cells in aquatic environments. . (1999) Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tlh, tdh and trh. Expression and characterization of the periplasmic cobalamin binding protein of Photobacterium damselae subsp. piscicida. J Fish Dis 32: 745-753. Virion stability is important for the circulative transmission of tomato yellow leaf curl sardinia virus by Bemisia tabaci, but virion access to salivary glands does not guarantee transmissibility. J Virol 83: 5784-5795.
A review of the current status of cultural and rapid detection of Vibrio parahaemolyticus
International Journal of Food Science & Technology, 2012
Vibrio parahaemolyticus is ubiquitous in estuarine environments and can be commonly found in seafood products. This bacterial pathogen continues to emerge as an important cause of foodborne illness, and several foodborne disease outbreaks caused by V. parahaemolyticus have been linked to the consumption of contaminated seafood, in particular those consumed raw such as oysters. In response to these outbreaks, especially during the 1990s, several cultural, immunological-based and molecular detection methods have been developed, which allow for rapid detection and quantification of total and pathogenic V. parahaemolyticus. The development of molecular methodology has allowed for clinical and environmental isolates of V. parahaemolyticus to be subtyped, thus providing the framework for risk-based strategies aimed at controlling foodborne outbreaks cause by this pathogen. It is important that the detection and typing methods strive to accomplish detection and differentiation of the pathogenic strains from environmental (non-pathogenic) ones, as well as to detect the presence of the organism and not just the presence of V. parahaemolyticus produced toxins, which can also be produced by closely related species. This review covers the current status of detection and typing methodology for identification and characterisation of V. parahaemolyticus from seafood.
Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters
Food Science and Biotechnology, 2021
This study was undertaken to develop enhanced selective media for detection of Vibrio parahaemolyticus in oysters. Primarily, tryptic soy agar (TSA) was supplemented with 4.5-5% NaCl, 0.1-0.5% oxgall, and/or 1-2% sodium citrate, and adjusted to pH 8-9. A total of 21 Vibrio spp., 24 indicators, and 26 food-borne isolates were streaked on the modified media, followed by 24 h of incubation at 37°C. While all the indicators and isolates failed to grow on TSA containing 5% NaCl, 0.5% oxgall, and 2% sodium citrate (TSA OSS1 ; pH 9), V. parahaemolyticus was culturable on this selective medium. Particularly, the ability of TSA OSS1 to quantify V. parahaemolyticus in oysters was superior to thiosulphate citrate bile salts sucrose (TCBS) agar. V. parahaemolyticus distinctly produced its white-yellowish, round, and edgepointed colony on TSA OSS1. TSAO SS1 with high selectivity potentials over TCBS may be a promising alternative for detection of V. parahaemolyticus in seafoods or natural reservoirs.
A quantum-dot-based fluoroassay for detection of food-borne pathogens
Journal of Photochemistry and Photobiology B: Biology, 2017
Evaluation of the distribution capability of food-borne pathogens existing in food products by taking the advantage of quantum dots (QDs) for their photoluminescence properties was carried out. Bacteria namely Escherichia coli (E. coli) labelled with CdSe-QDs were examined both on an Agar nutrient and ground fish substrates in order to observe their growth rate in different environments in the Lab. A sample with an appropriate concentration ratio 10 7 CFU/ml of bacteria/CdSe-QDs was empirically selected from the samples which were grown on the Agar containing plates. The selected sample was also tested on a ground fish substrate as a real food sample. The bacterial growth was observed under the irradiation of UV light and the growth patterns were investigated for 3 successive days. The growth patterns indicated that E. coli can stay alive and can be distributed on food products so that the growth can be easily monitored. This approach makes bacterial growth on food products detectable so that it can be used as a bacteria-QD assay for an easy detection of food borne pathogens grown on a food sample.
International Journal of Food Microbiology, 2009
Vibrio parahaemolyticus is a marine bacterium with a worldwide distribution and is frequently associated with human outbreaks of infection. Detection and isolation of V. parahaemolyticus from natural sources is often problematical because of limitations in the analytical procedures. We evaluated a combination of conventional and molecular protocols previously described for the investigation of V. parahaemolyticus, with the aim of identifying the best procedures for improved detection of this organism in environmental matrixes. A total of 259 samples of zooplankton (103), mussels (48) and seawater (108) were investigated by an Absence-Presence method (A/P), whereas 118 samples of zooplankton (70) and mussels were analyzed by the Most Probable Number (MPN) method. All samples were processed by a two-step enrichment procedure, firstly with APW broth and then with SPB as selective secondary broth. Detection of V. parahaemolyticus was by direct-PCR and by plate culture on TCBS and CHROMagar Vibrio, after sample enrichment in APW and SPB. With the A/P method, V. parahaemolyticus was detected in 23.6% samples by direct-PCR, whereas only 11.2% samples were positive with the plate culture method. With the MPN method, V. parahaemolyticus was detected in 54.2% and 27.1% of the samples by direct-PCR and plate culture respectively; this indicated the existence of 31% false negative results with the A/P method. No significant differences between the use of a single (APW) or two-step enrichment (APW+SPB) were observed by direct-PCR with A/P or MPN, although a significant higher presence of V. parahaemolyticus was detected by plate culture in both protocols with the two-step enrichment procedure. In conclusion, direct-PCR after sample enrichment in APW broth was the most successful method for detection of V. parahaemolyticus with the A/P procedure and enumeration by MPN. Better detection was obtained with MPN than with the A/P protocol. Conversely, the plate culture procedure showed better results with the two-step enrichment protocol in which CHROMagar Vibrio was used as the selective agar.