Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease

Abstract

A quadruplex loop-mediated isothermal amplification (LAMP) method was developed to detect four Vibrio species, including Vibrio ichthyoenteri, Vibrioparahaemolyticus, Vibrioscophthalmi, and Vibrio vulnificus, simultaneously. Four sets of species-specific primers were designed with different restriction sites contained in the inner primers. The quadruplex LAMP method could distinguish four Vibrio species via the subsequent restriction enzyme analysis. The sensitivity of the quadruplex LAMP method were 102–103 times higher than the sensitivity of conventional PCR. V. scophthalmi, V. vulnificus, V. parahaemolyticus and V. ichthyoenteri could be detected in the different tissues of the infected fish by the quadruplex LAMP method simply and conveniently through using SYBR Green I to facilitate visual inspection of the LAMP products. The method we developed in this study could be a simple and convenient diagnostic tool for field detection of Vibrio infection in fish.

Rapid and Sensitive Detection of Vibrio natriegens in Portunus trituberculatus with the Loop-Mediated Isothermal Amplification Test

“…However, they are time-consuming and labor intensive. As the gold standard for Vibrio species detection, polymerase chain reaction (PCR), and real-time PCR are also impractical for in situ detection, due to the long operation time, and expensive, non-portable, instruments (Wang et al, 2017;Zhou et al, 2016;Garrido et al, 2012). In order to simply, rapidly, and accurately identify V. natriegens, a gyrB-LAMP method was developed.…”

“…In aquaculture, LAMP has been used widely for Vibrio detection in aquatic animal disease. It shows high specificity, sensitivity, and rapidity under isothermal conditions with the presence of Bst DNA polymerase (Wang et al, 2017;Zhou et al, 2016).…”

Antimicrobial and Immunoregulatory Activities of TS40, a Derived Peptide of a TFPI-2 Homologue from Black Rockfish (Sebastes schlegelii)

“…This experiment was based on the method previously reported [58]. The washed L. anguillarum were resuspended to 1 × 10 10 CFU/mL by PBS.…”

Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip

“…Moreover, the Chinese National Food Safety Standard (CNFSS): Limit of Pathogen in Foods (GB 29921-2013) has defined that the highest safety limit value ( M ) of VP is 1000 colony-forming units (CFU)/mL. Consequently, to reach the detectable level, all three methods above and even some cutting-edge technologies have to enrich the target bacteria beforehand. ,− However, pre-enrichment inevitably raises the time and cost of detection. Thus, overcoming the above obstacles demands for a new method to rapidly and quantitatively detect VP.…”