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      An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models

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          Abstract

          Vibrio alginolyticus is one of the most common pathogenic marine Vibrio species, and has been found to cause serious seafood-poisoning or fatal extra-intestinal infections in humans, such as necrotizing soft-tissue infections, bacteremia, septic shock, and multiple organ failures. Delayed accurate diagnosis and treatment of most Vibrio infections usually result to high mortality rates. The objective of this study was to establish a rapid diagnostic method to detect and identify the presence of V. alginolyticus in different samples, so as to facilitate timely treatment. The widely employed conventional methods for detection of V. alginolyticus include biochemical identification and a variety of PCR methods. The former is of low specificity and time-consuming (2–3 days), while the latter has improved accuracy and processing time. Despite such advancements, these methods are still complicated, time-consuming, expensive, require expertise and advanced laboratory systems, and are not optimal for field use. With the goal of providing a simple and efficient way to detect V. alginolyticus, we established a rapid diagnostic method based on loop-mediated Isothermal amplification (LAMP) technology that is feasible to use in both experimental and field environments. Three primer pairs targeting the toxR gene of V. alginolyticus were designed, and amplification was carried out in an ESE tube scanner and Real-Time PCR device. We successfully identified 93 V. alginolyticus strains from a total of 105 different bacterial isolates and confirmed their identity by 16s rDNA sequencing. We also applied this method on infected mouse blood and contaminated scallop samples, and accurate results were both easily and rapidly (20–60 min) obtained. Therefore, the RT-LAMP assay we developed can be conveniently used to detect the presence of V. alginolyticus in different samples. Furthermore, this method will also fulfill the gap for real-time screening of V. alginolyticus infections especially while on field.

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          Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects.

          Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3'-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.
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            Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh.

            Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.
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              Cholera toxin transcriptional activator toxR is a transmembrane DNA binding protein.

              The toxR gene encodes a transcriptional activator controlling cholera toxin, pilus, and outer-membrane protein expression in V. cholerae. Nucleotide sequence and mutational analysis has identified the toxR gene product as a 32,527 dalton protein. Hydropathicity analysis of the derived amino acid sequence of ToxR predicts a transmembrane structure. The properties of hybrid proteins composed of N-terminal fragments of ToxR fused to the periplasmic enzyme alkaline phosphatase provide additional evidence for the transmembrane topology of the ToxR protein. These fusion proteins also allowed the localization of the transcriptional activation and DNA binding domains of the ToxR protein to its cytoplasmically located N-terminal portion. DNA binding assays and a deletion analysis of the cholera toxin promoter support a model for transcriptional activation that involves ToxR binding to a tandemly repeated 7 bp DNA sequence 56 bp upstream of the transcriptional start point.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                06 May 2016
                2016
                : 7
                : 651
                Affiliations
                [1] 1Central Laboratory, Navy General Hospital Beijing, China
                [2] 2Medical Administrative Department, Navy General Hospital Beijing, China
                [3] 3Department of Biochemistry and Molecular Biology, Medical College, Qingdao University Qingdao, China
                [4] 4College of Light Industry and Food Sciences, South China University of Technology Guangzhou, China
                [5] 5Institute of Food Safety and Nutrition, Jinan University Guangzhou, China
                Author notes

                Edited by: Abd El-Latif Hesham, Assiut University, Egypt

                Reviewed by: Maria De Los Angeles Serradell, CCT La Plata-CONICET, Instituto de Ciencias de la Salud-UNAJ, Argentina; Jinshui Zheng, Huazhong Agricultural University, China

                *Correspondence: Lijun Zhou, hzzhoulj@ 123456126.com ; Lei Shi, leishi@ 123456scut.edu.cn

                These authors have contributed equally to this work.

                This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2016.00651
                4858747
                27199971
                a12addb3-b8e1-4147-9366-67b17872473f
                Copyright © 2016 Fu, Li, Wang, Liu, Yan, Shi and Zhou.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 13 January 2016
                : 18 April 2016
                Page count
                Figures: 5, Tables: 3, Equations: 0, References: 58, Pages: 10, Words: 0
                Funding
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Award ID: 31400107
                Award ID: 81273311
                Award ID: 31170008
                Categories
                Microbiology
                Methods

                Microbiology & Virology
                vibrio alginolyticus,toxr gene,real-time lamp,16s rdna sequencing,rapid detection

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