Detection Of Vibrio Parahaemolyticus Research Articles

Rapid and simultaneous detection of virulence factors of Vibrio parahaemolyticus by multiplex microfluidic-LAMP (MMLAMP)

Vibrio parahaemolyticus (VP) is an essential foodborne bacterium who can cause gastroenteritis. However, current detection methods generally still have the difficulty for quickly screening VP strains and accurately detecting VP. In this work, a sensitive and specific multiplex microfluidic-LAMP (MMLAMP) method was constructed to rapidly detect VP using a centrifugal microfluidic chip combined with LAMP. The system enables simultaneous detection of two hemolysin genes (tlh and trh) and one VP regulatory factor (toxR) in a single chip improving the accuracy for detecting VP. The MMLAMP system detected 2.42 × 104 copies/μL of the plasmid (tlh-pUC57), 4.44 × 103 copies/μL of the plasmid (trh-pUC57) and 4.86 × 104 copies/μL of the plasmid (toxR-pUC57) in less than 35 min, respectively. Meanwhile, MMLAMP system was successfully applied for VP detection in artificially contaminated samples, the LOD is 220 CFU/g, which is 10 times lower than that of qPCR within the same detection time. The system also demonstrated 100 % specificity and good precision (C.V. < 5 %) in detecting the three targets of the VP, providing a new approach for point-of-care testing (POCT) of VP.

Polydopamine-enhanced vertically-ordered mesoporous silica film anti-fouling electrochemical aptasensor for indicator-free Vibrio parahaemolyticus discrimination using stable inherent Au signal

Vertically-ordered mesoporous silica film (VMSF) has proven to be an effective anti-interference and anti-fouling layer in the field of analysis. However, because VMSF is not stable enough in complex samples, its application in sensing field is limited. We herein use mussel-inspired multi-functional agent polydopamine (PDA) to enhance the stability of VMSF on electrode. Owing to the amino-functionalized VMSF combined with amino groups on PDA, superhydrophilicity was achieved which was proved to enhance excellent anti-fouling property. In addition, the electrodeposition of Au nanoparticles (AuNP) in the nanochannel of VMSF serving as an internal electrochemical signal shows excellent electrochemical signal stability, which provides a prerequisite for maintaining the further detection accuracy in complex samples. Finally, we validated the detection performance of the anti-fouling sensing strategy using a foodborne pathogen, Vibrio parahaemolyticus (VP), as a model target. Specifically, the aptamer was modified by covalent bond on the PDA-integrated VMSF interface surface for the recognition of VP. Using shrimp homogenate as a real sample, the anti-fouling detection performance of the aptasensor was verified. The linear range of VP detection in real samples was 103-107 CFU/mL, and the detection limit was 103 CFU/mL. The results show that the anti-fouling aptasensor has excellent anti-interference and stability. It is worth emphasizing that this strategy (using inherent Au signal) enables rapid electrochemical analysis of VP without outer indicators, greatly reducing the difficulty of constructing an electrochemical VP detection strategy.

An LF-NMR homogeneous immunoassay for Vibrio parahaemolyticus based on superparamagnetic 2D nanomaterials

In this work, a sensitive low field nuclear magnetic resonance (LF-NMR) homogeneous immunoassay, also called magnetic resonance switch (MRSw) sensor, for Vibrio parahaemolyticus (VP) was developed. Superparamagnetic 2D nanomaterial was designed and used as the magnetic probe of MRSw sensor. It was GO@SPIONs&Ab, a composite nanomaterial with many superparamagnetic Fe3O4 nanoparticles (SPIONs) providing a magnetic signal and VP antibody (Ab) specifically recognizing the target VP evenly distributed on the surface of GO. The presence of VP controllably changed the aggregation state of the probe, eliminating the uncertainty of MRSw sensor type, and thus then achieving a regular variation of transverse relaxation time T2 and ensuing quantitative detection of VP. Triple signal enhancement of the MRSw sensor was obtained due to the application of the designed 2D probe, by increasing the number of SPIONs, improving the magnetic intensity and susceptibility, and forming a synergistic effect. Under optimized experimental conditions, VP could be detected with satisfied sensitivity, selectivity, precision, accuracy, and stability, even in turbid real samples. LOQ for VP was 10 CFU/mL. This detection principle is widely applicable, providing an idea for the construction of highly sensitive MRSw sensors.

Dual-mode colorimetric-electrochemical biosensor for Vibrio parahaemolyticus detection based on CuO2 nanodot-encapsulated metal-organic framework nanozymes

Rapid and accurate detection of Vibrio parahaemolyticus (VP) in a variety of testing environments is critical for preventing the spread of foodborne illnesses. Therefore, a dual-mode colorimetric-electrochemical biosensor was prepared for the rapid and sensitive detection of VP. Phenylboronic acid-decorated CuO2 nanodot-mediated organic metal-organic framework (CP@MOF) nanozymes were constructed to produce colorimetric and electrochemical signals. Furthermore, an electrode surface was fabricated using tetrahedral DNA nanostructure-functionalized poly(amidoamine) dendrimer-Au nanoparticles to specifically bind VP. Under acidic environments, the CP@MOF nanozymes disintegrated, enabling the simultaneous release of Cu2+ and H2O2. This in turn causes a Fenton-type reaction, generating a hydroxyl radical, which then oxidizes 3,3′,5,5′-tetramethylbenzidine, thereby enabling visual detection of VP. Moreover, the produced copper ions can be used for the electrochemical detection of VP. This dual-mode biosensor achieved an accuracy of 88.7 %, which was higher than that of single-mode assays (81.1 %). Hence, this dual-mode assay may facilitate the effective on-site detection of pathogens in shrimp samples.

On-site marine pathogen (Vibrio parahaemolyticus) rapid colorimetric determination based on modified-free aptamer and metal-organic frameworks with simple washing step

Nowadays, microbial visual sensing platforms based on aptamers and metal-organic frameworks (MOFs) have been reported, but most of them require complex modification processes, which undoubtedly increases the difficulty of detection. Here, we constructed modified-free magnetic-aptamer MOF (Fe3O4-Ap@ZIF-8) and signal-aptamer MOF (Pt-Ap@ZIF-8), which effectively avoided complex biological modification. The rapid sensing strategy was adopted in this method to avoid the complicated washing steps and greatly shorten the detection time. The performance of this method was verified by taking Vibrio parahaemolyticus (VP) as the mode target. The limit of detection (LOD) of VP is 102 CFU/mL. Furthermore, this strategy can achieve rapid visual analysis of other target objects only by changing different aptamers, and has broad application prospects in the fields of rapid detection of food pollutants, rapid medical diagnosis, environmental monitoring, etc.

Faraday cage-type aptasensor for dual-mode detection of Vibrio parahaemolyticus.

AFaraday cage-type aptasensor has been developedfor dual-mode detection of a common bacterial pathogen Vibrio parahaemolyticus (VP) by electrochemiluminescence (ECL) and differential pulse voltammetry (DPV), using a multi-functionalized material Pb2+-Ru-MOF@Apt2 as signal unit. Therecognition aptamer Apt2 recognizes VP; specifically, ruthenium-based metal organic framework (Ru-MOF) and lead ions (Pb2+) embedded produce anECL signal at a working potential from 0 to 1.5V and DPV signal from - 0.3 to - 0.8V vs. Ag/AgCl. SinceRu-MOF is a two-dimensional conductive material signal unit overlapped onto the electrode surface to form a Faraday cage-type aptasensor. Thus, electrons could be easily exchanged between electrode and signal tags without being hindered by micron-size VP, resulting in a high detection sensitivity with a detectionlimit of 1.7CFUmL-1. In addition, dual-mode detection was achieved, improving the accuracy and reliability of rapid field detection. Stability and selectivity were alsosatisfactory. The tests of real samples indicate that this Faraday cage-type aptasensor is suited forrapid detection of VP and analog pathogens and shows great potential in food safety. Graphical abstract.

A Faraday cage-type immunosensor for dual-modal detection of Vibrio parahaemolyticus by electrochemiluminescence and anodic stripping voltammetry

Vibrio parahaemolyticus (VP), as a kind of harmful bacterium to human beings, its rapid and accurate detection is particularly important. Herein, we developed a simple and efficient dual-modal Faraday cage-type immunosensor for ultrasensitive detection of VP based on electrochemiluminescence (ECL) and anodic stripping voltammetry (ASV). After being captured by capture unit on magnetic glassy carbon electrodes, VP was recognized by detector unit, achieving ECL and ASV dual-modal detection. Compared with traditional sandwich-type immunosensors, this proposed immunosensor has the advantages of significant signal amplification effects, due to its distinctive Faraday cage-type structure. Benefited from it, all Ru(bpy)22+ and AgNPs labeled on the detector unit could participate in the electrode reaction and generate stronger ECL and electrochemical signals respectively. Under the optimized conditions, the linear range was from 102 to 107 CFU mL−1 with a limit of detection (LOD) of 33 CFU mL−1, and the selectivity, stability and reproducibility were satisfactory. In addition, the results of two detection methods can mutually validate each other to avoid false positive results. Moreover, this Faraday cage-type immunosensor with dual-modal detection can also be used for detection of VP in seawater samples with recoveries in the range of 96.1–107.9%, which would be a promising tool for pathogenic bacteria analysis.

Detection of Vibrio parahaemolyticus From Marine Crabs in and Around Puducherry and Its Public Health Significances

ABSTRACT Food borne illnesses have major social and economic impacts. The WHO estimates that worldwide food borne and waterborne diarrheal diseases together kill about 2.2 million people annually and in India around 6 per cent of the population. Vibrio parahaemolyticus has recently been recognized as one of the most important food borne pathogens as the leading causal agent of human acute gastro enteritis. This study was conducted to detect the presence of V. parahaemolyuticus from marine crabs sold in puducherry and human stool samples and was isolated as per the standard procedure. A total of 35 marine crab samples and 50 human stool samples were screened for presence of V. parahaemolyticus. Out of 35 samples, 14 samples (40 per cent) and out of 50 stool specimens, only 2 (4 per cent) were positive for V. parahaemolyticus by conventional methods. This isolates were further subjected to PCR. Out of 18 isolates, 14 isolates (87.5 per cent) from crabs and 100 per cent from stool specimens of V. parahaemolyticus were confirmed and subjected to biofilm production assay by CRA plate method. In that 14 isolates, 9 were positive for biofilm production and none of the isolates from human stool samples were positive for biofilm production. The antibiotic resistance profiling of the V. parahaemolyticus isolates does not show high resistance. This can be a major concern as microorganisms can be associated with chronic and recurrent human infections. This study clearly indicates the need for proper handling and processing of seafood especially in marine crabs. It is also important that in kitchen when ever seafood is prepare, care shall be taken to avoid cross contamination.

Sensitive detection of multiple pathogens using a single DNA probe

A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10−9–2.0×10−13M with a detection limit of 5.3×10−12M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4–45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP.

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.

Detection of Vibrio parahaemolyticus in food samples using in situ loop-mediated isothermal amplification method

A novel in situ loop-mediated isothermal amplification (in situ LAMP) technique for rapid detection of the food-borne Vibrio parahaemolyticus strains had been developed and evaluated in this study. The sensitivity of the in situ LAMP assay was detected to be 10CFU/reaction via test in serial 10-fold dilutions of V. parahaemolyticus cells, and high specificity had also been obtained through confirmation with 14 reference gram-positive and -negative strains. Application of the established in situ LAMP assay had been performed on 58 strains previously isolated from seafood samples, including 48 V. parahaemolyticus and 10 non-V. parahaemolyticus strains. Of 48 V. parahaemolyticus strains, 48, 45 and 34 strains were detected as positive by in situ LAMP, regular LAMP and PCR, respectively, with the detection rate and negative predictive value (NPV) found to be 100% vs 93.8% vs 70.8% and 100% vs 76.9% vs 41.7%. In addition, none of the tested non-V. parahaemolyticus strains showed positive result, indicating a 100% positive predictive value (PPV) for all of 3 assays. Compared with regular LAMP methods and PCR-based methods, the in situ LAMP assay is advantageous on rapidity, high specificity, less time consumption and ease in operation, and may provide a novel, useful and practical detection platform for pathogens in food safety laboratories.

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.

Electrochemical detection of a Vibrio parahaemolyticus sequence-specific gene based on a gold electrode modified with a single stranded probe

An electrochemical DNA biosensor for specific-sequences detection of Vibrio parahaemolyticus (VP) was fabricated. A single-stranded 20-mer oligonucleotide (ssDNA) and 6-mercapto-1-hexanol (MCH) were immobilized via a thiol linker on gold disk electrodes by self-assembling. The ssDNA underwent hybridization in a hybridization solution containing complementary or non-complementary or single base pair mismatched DNA sequences of VP. Examination of changes in response to these three target DNAs showed that the developed biosensor had a high selectivity and sensitivity.

Detection of Vibrio parahaemolyticus in tropical shellfish by SYBR green real-time PCR and evaluation of three enrichment media

A rapid, sensitive and highly reproducible SYBR green based real-time PCR assay was developed for detection of tdh positive pathogenic Vibrio parahaemolyticus. Minimum detection limit was 0.1 pg of pure V. parahaemolyticus genomic DNA with typical R(2) values >0.99 and coefficient of variation (CV) values ranging from 1.2 to 4.2 on three different days. The method was also used to evaluate the effect of three different enrichment media alkaline peptone water (APW), sodium taurocholate (ST) broth and salt polymyxin broth (SPB) on detection of V. parahaemolyticus. Crude lysates were directly used for real-time PCR. Without enrichment, the detection limit of pure cultures was 10(1) CFU/ml for ST broth and 10(2) CFU/ml in APW and SPB but for shrimp homogenates spiked with pure culture, the minimum detection limit was 10(2) CFU/ml for all three broths with a linear detection range of 10(2)-10(6). Without enrichment, detection in ST broth was more efficient than APW and SPB. After 6 h enrichment, limit of detection was found to be 1 CFU in all three media. However, for iced shrimp, the limit of detection was 10(2) after 6 h enrichment. No significant difference was seen between different enrichment media with respect to tdh gene detection of V. parahaemolyticus. The methodology developed here can be useful for rapid detection of tdh positive V. parahaemolyticus by laboratories involved in monitoring programmes for pathogenic V. parahaemolyticus.