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Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus-specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate-labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP-LFD technique for V. alginolyticus was 1.8 × 10(2) CFU/mL while that of PCR was 1.8 × 10(3) CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 10(3) CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP-LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP-LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity.

Decapod iridescent virus 1 (DIV1), White spot syndrome virus (WSSV), and Enterocytozoon hepatopenaei (EHP) pose serious threats to the shrimp farming. To date, early detection remains an important way to control the occurrence and diffusion of these pathogens. Here, we developed for the first time, a loop‐mediated isothermal amplification (LAMP)‐based microfluidic chip detection system, which could detect DIV1, WSSV, and EHP simultaneously. The limits of detection (LoD) of the system were 10 copies/reaction for EHP and DIV1, and 102 copies/reaction for WSSV. The entire detection procedure could be completed rapidly in 40 min at 63°C with 100% specificity and had no cross‐reaction with other common shrimp pathogens. This newly established method was further validated using 94 Penaeus vannamei clinical samples, which were comparable to a typical qPCR assay and revealed good stability and reproducibility. These results illustrate that this LAMP microfluidic chip detection system allows rapid triplex pathogen analysis and could satisfy the demands of the field and routine diagnoses in aquaculture. [ABSTRACT FROM AUTHOR]

Rapid and user‐friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on‐site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual‐sample microfluidic chip integrated with a real‐time fluorogenic loop‐mediated isothermal amplification assay (dual‐sample on‐chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on‐chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10–1 pg/μl for genomic DNA of tested bacteria and 10–4 to 10–5 pg/μl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time‐to‐positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on‐chip LAMP assay provides an effective dual‐sample and multiple pathogen analysis, and thus would be applicable to on‐site detection and routine monitoring of multiple pathogens in aquaculture. [ABSTRACT FROM AUTHOR]

Background: Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry because of its strong pathogenicity, quick onset after infection and high mortality rate in aquatic animals. Fast, simple and specific methods are needed for on-site detection to effectively control outbreaks and prevent economic losses. The detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR)-based molecular approaches have been developed, but their application is limited due to the requirement of complicated thermal cycling machines and trained personnel. Results: A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted the virulence gene toxR, which is reported to have good coverage for V. alginolyticus pathogenic strains. To ensure the specificity of the method, the primer-probe set of the RPA system was carefully designed to recognize regions in the toxR gene that diverge in different Vibrio species but are conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening of amplification performance, primer-dimer formation and false positive signals. The RPA-LFD method was confirmed to have high specificity for V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species or other pathogenic bacteria and was able to detect as little as 1 colony forming unit (CFU) per reaction without DNA purification, or 170 fg of genomic DNA, or 6.25 × 103 CFU/25 g in spiked shrimp without any enrichment. The method finishes detection within 30 min at temperatures between 35 °C and 45 °C, and the visual signal on the dipstick can be directly read by the naked eye. In an application simulation, randomly spiked shrimp homogenate samples were 100% accurately detected. Conclusions: The RPA-LFD method developed in this study is fast, simple, highly specific and does not require complicated equipment. This method is applicable for on-site detection of V. alginolyticus pathogenic strains for the mariculture industry. [ABSTRACT FROM AUTHOR]

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 104/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers' specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples. [ABSTRACT FROM AUTHOR]

Summary: Loop‐mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro‐organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro‐organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real‐time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro‐organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease. [ABSTRACT FROM AUTHOR]

The article reviews several articles on topics related to marine organisms including management of fish species, fish production and the development of assays that can provide rapid diagnostics on the presence of marine disease.