Development of a Real-Time Resistance Measurement for Vibrio parahaemolyticus Detection by the Lecithin-Dependent Hemolysin Gene

Abstract

The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg2+), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg2+) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1–2 hours, with a minimum bacterial density of 10 CFU.mL−1 and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing.

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

“…4 the sample pretreatment method, LOD of this method for seafood is estimated roughly to be 5 CFU/g. It is obvious that this method exhibits advantages in terms of detection sensitivity, which is nearly as same as or much lower than those reported values of 1.7 CFU/g [12], 10 CFU/mL [13], 7.374 × 10 4 CFU/mL [25], 100 CFU/mL [23] and 73 CFU/mL [18], due to the following reasons: (1) ECL method has inherent ultrasensitivity; (2) owing to the good conductivity, large surface area and two-dimensional structure of multi-functionalized graphene oxide, a large amount of electrochemiluminophore ABEI was chemically bound to the GO sheets, similar to direct immobilization on the electrode surface. Thus all of them were electrochemically active and emitted high and stable ECL signal.…”

“…This bacterium has emerged as an important cause of seafood-associated disease outbreaks throughout the world, and is a significant concern for seafood safety [6,7]. Methods available to investigate the presence and quantify VP mainly include most probable number (MPN) [8,9], enzyme-linked immunosorbent assay (ELISA) [10,11], immunofluorescence [12], loop-mediated isothermal amplification (LAMP) [13][14][15][16][17][18], other polymerase chain reaction (PCR) based assays [19][20][21][22][23], DNA probe [24] and sensor-based electrochemical techniques [25]. However, these methods mostly suffer from one or more of the drawbacks such as complex sample pretreatment steps, poor sensitivity, long analytical time, high analytical costs and expensive instrumentation.…”

A highly sensitive and specific electrochemical sensing method for robust detection of Escherichia coli lac Z gene sequence

“…The detection limit was confirmed to be around 30 fM (S/N ¼3). This detection limit is much lower than those obtained from two above-mentioned sensors with no amplification reported (Wang et al, 1997;Loaiza et al, 2007), even lower than those obtained from the enzymeamplification sensor (Jiang et al, 2013) or GOx-Thi-Au@SiO 2 nanocomposites and DNAzyme amplification sensor (Li et al, 2015). For the linear range, as shown in the inset, an excellent linear relationship was observed between the current signal value (CSV) of MB and the target concentration within a definite range from 0.01 nM to 10 nM which is used for the quantitative analysis of target DNA.…”

“…Though a detection limit of 0.1 ng/mL for target DNA was achieved, enzyme-amplification was still used in the sensor system. The MDA was also not tested for this sensor (Jiang et al, 2013). One year later, Li et al (2015) developed a novel sensitive E-DNA biosensor for the detection of E. coli O157:H7.…”

Christmas-tree Derived Amplification Immuno-Strategy for Sensitive Visual Detection of Vibrio parahaemolyticus Based on Gold Label Silver Stain Technology

“…[5][6][7] Although conventional culturing ensures the accuracy of determinations, and are optimum methods for VP identification, there still exist some shortcomings, such as long-time consumption and cumbersome practical application. Various approaches have been developed to obtain better performances of VP analysis, including enzyme-linked immunesorbent assay (ELISA), [8][9][10] DNA probe, 11 most probable number (MPN), 12,13 polymerase chain reaction (PCR), [14][15][16][17][18] loop-mediated isothermal amplification (LAMP), [19][20][21][22][23] and electrochemistry (EC). 24,25 Despite each of these new approaches having a combination of excellent sensitivity, accuracy and specificity, they still suffer drawbacks involving high analytical cost, need for expensive equipment, and professional trained personnel.…”