Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles.

Loop-mediated isothermal amplification (LAMP) eradicates the need of thermocycler in DNA amplification. Signals are usually obtained via fluorometry or turbidimetry, but such methods need improvement in order to become more effortless and reliable. The authors describe a set of six specific primers targeting the species-specific tlh gene of Vibrio parahaemolyticus which were used in accelerated LAMP reaction. Gold nanoparticles (AuNPs) were functionalized with streptavidin (Avidin-AuNPs), and engineered to signal the LAMP reaction. Two of the loop primers for LAMP were biotinylated and then can produce a DNA that can cause clusterization of Avidin-AuNPs based on the formation of avidin-biotin complex. This leads to a color change of the solution from red to blue. Amplification is completed within 30 min and can be visually detected within 5 min. The detection limit of the method is found to be 8.6 cfu per reaction. This visual detection scheme does not require any fluorescent reagents and detection instruments. Conceivably, the method has a wide scope because such Avidin-AuNPs can be used as nanoprobes for a variety of other LAMP products. This rapid and universal strategy holds promise in point of care testing and food testing, particularly in resource-limited regions. Graphical abstract Six specific primers (two of them are biotinylated) were used to realize the accelerated Loop-Mediated Isothermal Amplification. Streptavidin modified gold nanoparticles (Avidin-AuNPs) cluster on the DNA products, leading to the apparent change of color from red to blue, which is readily identified even by unaided eye.

Correction to: Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles.

The published version of this article, unfortunately, contained error. Modifications have been made to the Abstract, Introduction, Results and discussion, and Acknowledgements section. The original article has been corrected.

[Exposure Level and Risk Impact Assessment of Pesticides and Veterinary Drugs in Aquaculture Environment].

To explore the exposure level of pesticides and veterinary drugs in an aquaculture environment and its impact on the ecological environment, this study took the aquaculture environment in Shanghai as an example, and samples of water, sediment, and inputs from 40 major aquaculture farms were collected from July to September 2022. The types and contents of pesticides and veterinary drugs were screened using high-performance liquid chromatography-electrostatic field orbital ion trap mass spectrometry, and the risk quotient (RQ) method was used to assess the ecological risk of pesticide contamination in water and sediment. The results showed that 13 drugs were screened out from 204 samples (72 samples of water, 72 samples of mud, and 60 samples of input), namely, chlorpromazine, carbendazim, thiophanate, diazepam, florfenicol, simazine, amantidine, diazepam, trimethoprim, ciprofloxacin, ofloxacin, mebendazole, and enrofloxacin. Among them, 12 species were found in water samples with concentrations ranging from 0.016 µg·L-1 to 2.084 µg·L-1. The concentrations of seven species in the mud samples ranged from 0.018 µg·kg-1 to 23.101 µg·kg-1. The results showed that there were four types of inputs, ranging from 1.979 µg·kg-1 to 101.940 µg·kg-1. Seven drugs were found in both water and sediment. The risk quotient (RQ) results showed that there were some high and middle risks in both water and sediment samples of aquaculture farms, and the ecological risks of carbendazim were the highest in both water and sediment samples of aquaculture farms; the RQ values were 3.848 and 1.580, respectively, indicating high risk. It is suggested to strengthen the control and management of exogenous pesticides and veterinary drugs in aquaculture environments to protect the ecosystem health of the aquaculture environment.

Detection of Total and Pathogenic Vibrio parahaemolyticus in Shellfish Growing along the South Yellow Sea and the East China Sea.

This study was conducted to monitor the densities of total and pathogenic Vibrio parahaemolyticus in 300 samples of nine shellfish species harvested from the coasts of the South Yellow Sea and the East China Sea (N 23° to 34°, E 116° to 124°), People's Republic of China, between May and October 2015. Total V. parahaemolyticus densities were measured, and V. parahaemolyticus isolates were biochemically identified with probes for the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh). We found that 202 of the 300 samples were positive for V. parahaemolyticus from all the sites: 58 of the 100 samples from the Fujian province, 71 of the 100 samples from the Zhejiang province, and 73 of the 100 samples from the Jiangsu province. In most (170) of the 300 samples, V. parahaemolyticus densities were 0.3 to 10 most probable number (MPN)/g; five lots exceeded 110 MPN/g, and two lots were estimated at 110 MPN/g. Among the 202 V. parahaemolyticus strains, only one was trh positive. Densities of V. parahaemolyticus in these shellfish were temperature dependent, with highest densities in June and July. Among the nine mollusk species, V. parahaemolyticus was most abundant in the agemaki clam (Sinonovacula constricta). The highest and lowest V. parahaemolyticus prevalences were found in oriental cyclina (Cyclina sinensis, 93.8%) and mussels (Mytilus edulis, 28.1%), respectively. Overall, although V. parahaemolyticus is widely distributed in marine environments, the density of V. parahaemolyticus was low and the prevalence of the main virulence factor was very low in shellfish along the coasts of the South Yellow Sea and East China Sea, which is important from a public health perspective. Data presented here will be useful for correlational research and can be utilized for developing risk management plans that establish food safety guidelines for V. parahaemolyticus in Chinese shellfish.

[Identification of Vibrio campbellii isolated from cultured pacific oyster].

Four bacterial strains were isolated from cultured pacific oyster (Crassostrea gigia) in September 2003 at coast of Fujian province. Their morphological, physiological and biochemical characteristics and 16S rRNA sequence were analyzed. And the relationship between reproduction of the bacterium and NaCl concentrations, pH and temperature were also determined. The results showed that four strains were gram-negative rods with a single polar flagellum, glucose fermented without gas production, oxidase positive, required sodium ions for growth, no pigment, non-luminescence; They grew well on TCBS-plate as green colonies and were sensitive to the vibriostratic agent O/129. Therefore, it was confirmed that the strains belonged to the genus of Vibrio. The sequence analysis of 16S rRNA gene of SXS1 isolation and comparison with that of other related vibrios- showed that SXS1 was very close to Vibrio campbellii. The similarities was 99%. The distribution of V. campbellii in environment and its relationship to the diseases of aquaculture animals were discussed.