Rapid detection of Vibrio vulnificus in Penaeus (Litopenaeus) vannamei  by loop-mediated isothermal amplification with lateral flow dipstick technique

Shuanghu

doi:10.21077/

Authors

Shuanghu Guangdong ocean university

https://doi.org/10.21077/

Keywords:

Vibrio vulnificus; loop-mediated isothermal amplification; lateral flow dipstick; Litopenaeus vannamei

Abstract

Vibrio vulnificus is a is a highly virulent bacterial pathogen that poses significant risks to both humans and aquatic animals, necessitating the development of rapid and sensitive diagnostic methods. In this study, we designed four specific loop-mediated isothermal amplification (LAMP) primers, including a biotin labelled forward internal primer (FIP) along with a fluorescein isothiocyanate (FITC)-labelled probe targeting the RtxC gene sequence of V. vulnificus and established a novel LAMP combined with lateral flow dipstick (LFD) assay for the rapid visual detection of the pathogen. The optimized LAMP was run at 62°C for 15 min and the LFD could specifically and visually detect V. vulnificus within 5 min. The assay exhibited high specificity, showing no cross-reactivity against 24 non-target bacterial strains. It also exhibited higher sensitivity than conventional PCR methods, with a detection limit as low as 2.0×10-5 ng µl-1 of genomic DNA. We further validated the LAMP-LFD method using Penaeus (Litopenaeus) vannamei samples infected with Vibrio. Notably, the assay accurately detected V. vulnificuswith high accuracy and the presence of host shrimp DNA did not interfere with the detection, highlighting the robustness of the assay for field applications in aquaculture. The developed LAMP-LFD assay offers a rapid, highly specific, sensitive, and reliable visual detection method for V. vulnificus, suited for field based surveillance and disease management in aquaculture.

Keywords: Fluorescein isothiocyanate, LAMP-LFD, RtxC gene, Visual detection, Whiteleg shrimp

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