Dataset Information

Anti-Interference Detection of Vibrio parahaemolyticus from Aquatic Food Based on Target-Cyclized RCA with Dynamic Adapter Followed by LAMP.

ABSTRACT: Vibrio parahaemolyticus (V. parahaemolyticus) is considered the most concerning pathogen for seafood. Like other pathogens in food samples, its gene detection suffers from a problem of background interference when isothermal detection methods are used. The sensitivity and specificity greatly decrease due to large amounts of background genome. Here we describe a novel isothermal detection technology based on target-cyclized rolling circle amplification combined with loop-mediated isothermal amplification (tRCA-lamp). By avoiding unexpected ligation, a short dynamic adapter is employed to increase the sensitivity of target cyclization in the presence of the background genome. At the amplification step, highly specific detection is obtained by linear RCA and simplified LAMP (only two primers are used). Furthermore, visual detection is easily realized with hydroxynaphthol blue (HNB). In the oyster samples, the tRCA-lamp approach can detect V. parahaemolyticus with a detection limit of 22 cfu/g with none necessary to enrich the bacteria and remove the host DNA. This method gets rid of the complicated primer design process and can be extended to the detection of other pathogens in food samples.

SUBMITTER: Zhang B

PROVIDER: S-EPMC8834026 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Publications

Anti-Interference Detection of Vibrio parahaemolyticus from Aquatic Food Based on Target-Cyclized RCA with Dynamic Adapter Followed by LAMP.

Zhang Boying B Sun Wenhua W Ran Lingling L Wang Chenru C Wang Jing J An Ran R Liang Xingguo X

Foods (Basel, Switzerland) 20220126 3

Vibrio parahaemolyticus (V. parahaemolyticus) is considered the most concerning pathogen for seafood. Like other pathogens in food samples, its gene detection suffers from a problem of background interference when isothermal detection methods are used. The sensitivity and specificity greatly decrease due to large amounts of background genome. Here we describe a novel isothermal detection technology based on target-cyclized rolling circle amplification combined with loop-mediated is ...[more]

PMID: 35159502

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Project description:Vibrio parahaemolyticus is a marine and estuarine bacterium that leads to damage of aquatic industry by foodborne outbreaks and possesses an enormous threat to food safety as well as human health worldwide. In the current study, we investigated 905 food samples (ready-to-eat foods, fish, and shrimp) from 15 provinces in China, and aimed to determine prevalence, biological characteristics and genetic diversity of presumptive V. parahaemolyticus isolates. Firstly, 14.17% of 240 fish samples, 15.34% of 365 shrimp samples and 3.67% of 300 RTE food samples were positive for potential V. parahaemolyticus. Secondly, 69 food samples (14.87%) collected in summer were positive for target isolates, while the rate of positive sample of 441 food samples in winter reached 7.26%. Thirdly, we purified 202 V. parahaemolyticus strains for further research. And antimicrobial susceptibility results of strains tested revealed that the highest resistance rate was observed for ampicillin (79.20%). At the same time, 148 (73.27%) of all isolates were classified and defined as multi-drug resistant foodborne bacteria. The results of PCR assay showed that the isolates being positive for the tdh, trh or both genes, were up to 9.90%, 19.80% or 3.96%. Besides, multiplex PCR test showed that the isolates carrying O2 serogroup were the most prevalent. Furthermore, sequence types (STs) of 108 isolates were obtained via multi-locus sequence typing. Not only 82 STs were detected, but also 41 of which were updated in the MLST database. Thus, our findings significantly demonstrated the high contamination rates of V. parahaemolyticus in fish and shrimp and it may possess potential threat for consumer health. We also provided up-to-date dissemination of antibiotic-resistant V. parahaemolyticus which is important to ensure the high efficacy in the treatment of human and aquatic products infections. Lastly, with the identification of 82 STs including 41 novel STs, this study significantly revealed the high genetic diversity among V. parahaemolyticus. All of our research improved our understanding on microbiological risk assessment in ready-to-eat foods, fish, and shrimp.

Project description:Vibrio parahaemolyticus, one of the most common foodborne pathogenic bacteria that forms biofilms, is a persistent source of concern for the food industry. The food production chain employs a variety of methods to control biofilms, although none are completely successful. This study aims to evaluate the effectiveness of quercetin as a food additive in reducing V. parahaemolyticus biofilm formation on stainless-steel coupons (SS) and hand gloves (HG) as well as testing its antimicrobial activities. With a minimum inhibitory concentration (MIC) of 220 µg/mL, the tested quercetin exhibited the lowest bactericidal action without visible growth. In contrast, during various experiments in this work, the inhibitory efficacy of quercetin at sub-MICs levels (1/2, 1/4, and 1/8 MIC) against V. parahaemolyticus was examined. Control group was not added with quercetin. With increasing quercetin concentration, swarming and swimming motility, biofilm formation, and expression levels of target genes linked to flagellar motility (flaA, flgL), biofilm formation (vp0952, vp0962), virulence (VopQ, vp0450), and quorum-sensing (aphA, luxS) were all dramatically suppressed. Quercetin (0−110 μg/mL) was investigated on SS and HG surfaces, the inhibitory effect were 0.10−2.17 and 0.26−2.31 log CFU/cm2, respectively (p < 0.05). Field emission scanning electron microscopy (FE-SEM) corroborated the findings because quercetin prevented the development of biofilms by severing cell-to-cell contacts and inducing cell lysis, which resulted in the loss of normal cell shape. Additionally, there was a significant difference between the treated and control groups in terms of motility (swimming and swarming). According to our research, quercetin produced from plants should be employed as an antibiofilm agent in the food sector to prevent the growth of V. parahaemolyticus biofilms. These results indicate that throughout the entire food production chain, bacterial targets are of interest for biofilm reduction with alternative natural food agents in the seafood industry.

Project description:The demand for minimally processed vegetables (African salad) has increased partly due to its inclusion in ready-to-eat foods. Nevertheless, the associated risk of the presence of emergent foodborne pathogens, such as Vibrio parahaemolyticus might be underestimated. The present study was designed to isolate and characterize foodborne V. parahaemolyticus from minimally processed vegetables using culture-based methods and molecular approach. A total of 300 samples were examined from retail outlets between November 2018 and August 2019 from Southern Nigeria. The prevalence of vibrios from the overall samples based on the colonial proliferation of yellow, blue-green and/or green colonies on thiosulfate citrate bile salts sucrose agar was 74/300 (24.6%). An average of two green or blue-green colonies from respective plates was screened for V. parahaemolyticus using analytical profile index (API) 20 NE. Polymerase chain reaction further confirmed the identity of positive V. parahaemolyticus. The counts of V. parahaemolyticus ranged from 1.5 to 1,000 MPN/g. A total of 63 recovered V. parahaemolyticus were characterized further. The resistance profile of the isolates include ampicillin 57/63 (90.5%), cefotaxime 41/63 (65.1%), ceftazidime 30/63 (47.6%), amikacin 32/63 (50.8%), kanamycin 15/63 (23.8%), and oxytetracycline 16/63 (25.4%). The multiple antibiotic index ranged from 0-0.81. The formation of biofilm by the isolates revealed the following: strong formation 15/63 (23.8%), moderate formation 31/63 (49.2%), weak formation 12/63 (19.1%), and no formation 5/63 (7.9%). A total of 63/63 (100%), 9/63 (14.3%), and 20/63 (31.8%) of the isolates harbored the tox R gene, TDH-related hemolysin (trh) and thermostable direct hemolysin (tdh) determinants respectively. The isolates with O2 serogroup were most prevalent via PCR. Isolates that were resistant to tetracycline, kanamycin, and chloramphenicol possessed resistant genes. The presence of multidrug-resistant vibrios in the minimally processed vegetables constitutes a public health risk and thus necessitates continued surveillance.

Project description:Vibrio parahaemolyticus is a common foodborne pathogen that causes gastroenteritis worldwide. Determining its prevalence and genetic diversity will minimize the risk of infection and the associated economic burden. Multilocus sequence typing (MLST) is an important tool for molecular epidemiology and population genetic studies of bacteria. Here, we analyzed the genetic and evolutionary relationships of 162 V. parahaemolyticus strains isolated in the Guangdong Province, China, using MLST. In the study, 120 strains were isolated from food samples, and 42 strains were isolated from clinical samples. All strains were categorized into 100 sequence types (STs), of which 58 were novel (48 from the food isolates and 10 from the clinical isolates). ST415 was the most prevalent ST among the food isolates, while ST3 was the most prevalent ST among the clinical isolates. Further, 12 clonal complexes, 14 doublets, and 73 singletons were identified in all ST clusters, indicating high genetic diversity of the analyzed strains. At the concatenated sequence level, non-synonymous sites in both, food and clinical isolates, were associated with purifying selection. Of note, the dN/dS ration was greater than 1 for some housekeeping genes in all isolates. This is the first time that some loci under positive selection were identified. These observations confirm frequent recombination events in V. parahaemolyticus. Recombination was much more important than mutation for genetic heterogeneity of the food isolates, but the probabilities of recombination and mutations were almost equal for the clinical isolates. Based on the phylogenetic analysis, the clinical isolates were concentrated in the maximum-likelihood tree, while the food isolates were heterogeneously distributed. In conclusion, the food and clinical isolates of V. parahaemolyticus from the Guangdong Province are similar, but show different evolutionary trends. This may help prevent large-scale spread of highly virulent strains and provides a genetic basis for the discovery of microevolutionary relationships in V. parahaemolyticus populations.

Dataset Information

Anti-Interference Detection of Vibrio parahaemolyticus from Aquatic Food Based on Target-Cyclized RCA with Dynamic Adapter Followed by LAMP.

Publications

Anti-Interference Detection of <i>Vibrio parahaemolyticus</i> from Aquatic Food Based on Target-Cyclized RCA with Dynamic Adapter Followed by LAMP.

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