Rapid Detection Of Vibrio Parahaemolyticus Research Articles

Immunomagnetic separation-based nanogold enhanced surface plasmon resonance and colloidal gold test strips for rapid detection of Vibrio parahaemolyticus.

Nanogold enhanced surface plasmon resonance (SPR), colloidal gold immunochromatographic test strips (ICTS), and polymerase chain reaction (PCR), combined with immunomagnetic separation (IMS) were established in this study for the rapid detection of Vibrio parahaemolyticus (VP). The sensitivities of SPR, ICTS, and PCR was determined to be 101, 103, and 103CFU/mL for VP, respectively. After separation and enrichment by IMS, the sensitivities of SPR, ICTS, and PCR were 100, 101, and 102CFU/mL for VP, respectively, which were improved by 10-, 100-, and 10-fold compared to the direct detection by SPR, ICTS, and PCR, respectively. When the VP-polluted water samples were directly assessed by SPR, ICTS, and PCR, the results were negative. By contrast, after separation and enrichment for 45min by IMS, the results were all positive. The IMS-SPR, IMS-ICTS, and IMS-PCR detection methods were able to yield results in approximately 1.5h, 55min, and 3.5h, respectively. These combined detection methods have advantages in being high-throughput and easy to operate without the need for sophisticated equipment or specialized skills. These methods might aid in the development of SPR, ICTS, and PCR technologies for simultaneously examining multiple food-borne pathogens in food products.

Point-of-care rapid detection of Vibrio parahaemolyticus in seafood using loop-mediated isothermal amplification and graphene-based screen-printed electrochemical sensor

Vibrio parahaemolyticus is one of the most important foodborne pathogens that cause various life-threatening diseases in human and animals. Here, we present a rapid detection platform for V. parahaemolyticus by combining loop-mediated isothermal amplification (LAMP) and disposable electrochemical sensors based on screen-printed graphene electrodes (SPGEs). The LAMP reactions using primers targeting V. parahaemolyticus toxR gene were optimized at an isothermal temperature of 65 °C, providing specific detection of V. parahaemolyticus within 45 min at the detection limit of 0.3 CFU per 25 g of raw seafood. The LAMP amplicons can be effectively detected using unmodified SPGEs, redox active molecules namely Hoechst-33258 and a portable potentiostat. Therefore, the proposed system is particularly suitable as a point-of-care device for on-site detection of foodborne pathogens.

A label-free multi-functionalized graphene oxide based electrochemiluminscence immunosensor for ultrasensitive and rapid detection of Vibrio parahaemolyticus in seawater and seafood

A label-free electrochemiluminescence (ECL) immunosensor for ultrasensitive and rapid detection of marine pathogenic bacterium Vibrio parahaemolyticus (VP) in seawater and seafood was developed based on multi-functionalized graphene oxide, which was prepared with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and VP antibody (anti-VP) simultaneously immobilized on the surface of magnetic graphene oxide (nanoFe3O4@GO). ABEI and anti-VP acted as the electrochemiluminophore and the capture device for VP respectively. Good conductivity and two-dimensional structure of the nanoFe3O4@GO enabled all the ABEI, immobilized on GO, electrochemically active and thus improved the detection sensitivity. Under optimal conditions, the ECL intensity decreased with increasing logarithmic concentrations of VP in the range of 10–108CFU/mL, with a detection limit of 5CFU/mL for seawater and 5CFU/g for seafood. This ECL immunosensor showed high specificity, stability and reproducibility for the detection of VP. In addition, the ECL immunosensor has been successfully used to determine the concentration of VP in seawater and seafood rapidly, with a recovery of 94.4–112.0% and RSD 4.1–11.7%. Therefore, the developed immunosensor shows great prospect for practical application.

Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loop-mediated isothermal amplification

The objective of this study was to develop a method that combined nanoparticle-based immunomagnetic separation (IMS) with real-time loop-mediated isothermal amplification (LAMP) for the rapid detection of Vibrio parahaemolyticus. Magnetic nanoparticles were functionalized with monoclonal antibodies that were produced against flagella from V. parahaemolyticus to capture and separate the target cells from raw oysters. After optimization, the immunomagnetic nanoparticles (IMNPs) presented a capture efficiency of 87.3% for 105 colony-forming unit (CFU)/mL of V. parahaemolyticus using 2.5μg of IMNPs within 30min. Although a very low level of non-specific binding was seen among 8 non-V. parahaemolyticus Vibrio spp. and 5 non-Vibrio strains, the IMS–LAMP method identified 133 V. parahaemolyticus strains correctly without the amplification from 54 other strains. The detection limit was about 1.4×102CFU/mL in pure culture and was unaffected by the presence of 108CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9×103CFU/g without enrichment. After enrichment for 6–8h, the limit of detectability could be improved to 1.9 to 0.19CFU/g. Hence, the IMS–LAMP assay provided a rapid, simple, and cost-effective method for total V. parahaemolyticus detection. This method will have important implications in the rapid detection of contaminated food in the early stage before distribution.

Ethidium Monoazide-Loop Mediated Isothermal Amplification for Rapid Detection of Vibrio parahaemolyticus in Viable but non-culturable State

Loop-mediated isothermal amplification (LAMP) combined with the ethidium monoazide (EMA) treatment was applied for differentiation from VBNC state and dead state of V. parahaemolyticus. The tlh gene can be amplified remarkably within 1 h when the amount of DNA was 9fg in EMA-LAMP assay. EMA has the ability to penetrate selectively through the damaged cytoplasmic membrane of dead cells and to intercalate into DNA, so the gene of the dead cells could not be amplified. Amplification of DNA from dead bacterial was successfully inhibited by EMA, whereas the DNA derived from V. parahaemolyticus in VBNC state was readily amplified.

Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization

The aim of this study was to explore a new PCR target gene for Vibrio parahaemolyticus, based on the histone-like nucleoid structure (H-NS) gene. Primers for the H-NS gene were designed for specificity to V. parahaemolyticus and incorporated into a PCR assay. The PCR assay was able to specifically detect all of the 82 V. parahaemolyticus strains tested, but did not result in amplification in the 47 other Vibrio spp. and nonVibrio spp. strains. The detection limit of the PCR assay was 0.14 pg purified genomic DNA and 1.8 × 10(5) CFU g(-1) spiked oyster samples from V. parahaemolyticus RIMD2210633. Furthermore, a multiplex PCR assay targeting the hns, tdh and trh genes was successfully developed to detect virulent V. parahaemolyticus strains. The H-NS-based PCR assay developed in this study was sensitive and specific, with great potential for field detection of V. parahaemolyticus in seawater or seafood samples. The H-NS gene was validated as a new specific marker gene in PCR assays for accurate detection and identification of V. parahaemolyticus, which has the potential to be applied in diagnostics and taxonomic studies.

A loop-mediated isothermal amplification method for rapid detection of Vibrio parahaemolyticus in seafood

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 × 102 CFU mL−1 for pure cultures. The detection limit for the seeded seafood samples was 8.9 × 102 CFU g−1. In addition, 42 shares of natural seafood samples were tested and 8 samples were recorded positive for V. parahaemolyticus, while 6 were positive by conventional culture methods. In conclusion, the LAMP assay is an effective and low-cost method with high specificity and sensitivity for rapid detection and identification of V. parahaemolyticus both in culture isolates and seafood samples.

A disposable amperometric enzyme immunosensor for rapid detection of Vibrio parahaemolyticus in food based on agarose/Nano-Au membrane and screen-printed electrode

Recent outbreaks of foodborne illnesses continue to support the need for rapid and sensitive methods for detection of foodborne pathogens. A disposable electrochemical immunosensor for detection of Vibrio parahaemolyticus (VP) based on the screen-printed electrode (SPE) coated with agarose/Nano-Au membrane and horseradish peroxidase (HRP) labeled VP antibody (HRP-anti-VP) has been developed in this paper. Then, the immunosensor was characterized by cyclic voltammetry (CV) and laser scanning confocal microscope (LSCM). The immunosensor was incubated with the one-step immunoassay format involving VP for 30 min at room temperature (25 ± 0.5 °C). The access of the active center of HRP catalyzing the oxidation reaction of thionine by H 2O 2 was partly inhibited by VP, which connected on the surface of the immunosensor by immunoreaction. VP could be quantificationally detected according to the shift of reduction current while CV was used as electrochemical means to detect the products of the enzymatic reaction. Under the optimum conditions of immunoreaction and electrochemical detection, VP was rapidly detectable by sigmoidal curve method and form a linear calibration between 10 5 and 10 9 cfu/ml with an associated detection limit of 7.374 × 10 4 cfu/ml (S/N = 3). The immunosensor had acceptable specificity, reproducibility, stability and accuracy, indicating that the immunosensor could satisfy the need of practical sample detection.

Rapid detection of Vibrio parahaemolyticus in oysters by immunofluorescence microscopy

A method of indirect immunofluorescence microscopy was developed for the rapid detection of Vibrio parahaemolyticus in oysters. Affinity-purified antibodies against two outer membrane proteins (molecular mass 36 and 34 kDa, respectively) of V. parahaemolyticus were used as the primary antibodies to label the vibrio, and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G was used to reveal the antigen-antibody reaction. Of 85 strains of V. parahaemolyticus tested, all produced strong fluorescence under the fluorescence microscope. However, for 63 strains (including 27 vibrios) of other bacteria tested, 6 produced weak to moderate fluorescence. The sensitivity and specificity of the immunostaining technique were 100 and 90.5%, respectively. In the inoculation experiments, as low as 1.7 CFU g of V. parahaemolyticus spiked in oysters could be detected by the immunostaining method after 18-h enrichment in alkaline peptone water containing 3% NaCl. The immunofluorescence microscopy is recommended for rapid screening of V. parahaemolyticus in oysters, a presumptive positive result could be obtained within 24 h.

An Enzyme-Linked Immunosorbent Assay for the Rapid Detection of Vibrio parahaemolyticus

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of Vibrio parahaemolyticus in foods. The assay was based on the detection of two outer-membrane proteins (molecular weights 36,000 and 34,000) of V. parahaemolyticus. Following an 18-h incubation in alkaline peptone salt broth containing 0.1%Teepol, the culture supernatant was added to the microtiter plate coated with antibodies against the two outer-membrane proteins. After washing, the same antibodies absorbed with V. alginolyticus and labeled with horsemdish peroxidase were used as secondary antibodies. The detection limit of the assay for total outer-membrane proteins was 10 ng/ml. For 29 strains of V. parahaemolyticus and 73 strains (including 27 isolates of vibrios) of other bacteria tested, the sensitivity and specificity of the ELISA were 100 and 96%, respectively. Strains producing false positives were V. tubiashii, V. campbellii, and V. vulnificus. Of 23 seafood samples tested, V. parahaemolyticus was detected in 17 and 15 samples, respectively, by the ELISA and by a conventional culture method. V. parahaemolyticus was also detected in samples artificially inoculated with the microorganism at levels less than 10 colony-forming units (CFU) per g. Compared to the conventional culture methods, which may take 4 to 6 days to complete, the ELISA can detect low numbers of V.parahaemolyticus in foods with a total analytical time of only 24 h. The ELISA is recommended as a rapid screening method.

Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods

An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.