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 × 10 3 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.

Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and mariculture. Rapid and accurate detection technologies are critical for effective control of its outbreak and spreading. Conventional technologies and polymerase chain reaction (PCR)‐based approaches have limited usage because of the requirement of laboratory instruments and trained personnel. Using the isothermal recombinase polymerase amplification (RPA) technology, several detection assays have been developed with added convenience. Combining the lateral flow strip (LFS) test with RPA can further simplify the detection. In this study, an improved RPA assay using LFS for visual detection of V. parahaemolyticus was developed. Primers were designed targeting the virulence genes and screened for amplification efficiency, nonspecific amplification, and primer–dimer formation. Probes were designed for the best primer pairs, and the weakness of LFS tests, being easily affected by primer‐dependent artifacts, was overcome by sequence modifications on primers and probe. The RPA–LFS assay took 25 min at 35 to 45 °C, and showed excellent specificity. It detected as low as one colony forming unit (CFU) of V. parahaemolyticus per reaction without DNA purification, or 10 CFU/10 g spiked food samples with 2 hr of enrichment. The detection limit was better than the currently available RPA‐based detection methods. Application of the RPA–LFS assay for simulated samples or real clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. The RPA–LFS assay provided a rapid, accurate, and convenient V. parahaemolyticus detection method suitable for on‐site detection in resource‐limited conditions. Practical Application This research developed a rapid and visual detection technology for Vibrio parahaemolyticus that is not dependent on complicated equipment. The detection process takes 25 min and the result is read with the naked eye. A detection kit can be developed based on this technology for on‐site detection of V. parahaemolyticus in resource‐limited regions for food safety management and mariculture.

During a survey of keratinolytic fungi in China, three Cunninghamella strains were isolated. Phylogenetic analyses of ITS and ITS+LSU+EF-1α sequence data showed that these strains constitute a new species related to C. blakesleeana, C. bigelovii, C. multiverticillata and C. phaeospora. The new species differs from C. multiverticillata and C. phaeospora in the shape and size of its teminal and lateral vesicles and can be distinguished from C. blakesleeana and C. bigelovii by the absent of zygosporangia, and the shape and size of it sporangioles. The results of phylogenetic and morphological analyses indicate that the three strains are a new species of Cunninghamella. Descriptions and illustrations of this novel species are provided in this paper.

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