Vibrio cholerae can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Continuous testing of V. cholerae contamination in water and aquatic products is imperative for food safety control and human health. In this study, a rapid and visualized method was for the first time developed based on loop-mediated isothermal amplification (LAMP) for detection of very important virulence-related genes ace , zot , cri , and nanH for toxins and infection process of V. cholerae . Three pairs of molecular probes targeting each of these genes were designed and synthesized. The one-step LAMP reaction was conducted at 65 o C for 40 min. Positive results were simply inspected by the production of light green color under visible light or green fluorescence under UV light (302 nm). Limit of detection (LOD) of the LAMP method ranged from 1.85-2.06 pg/reaction of genomic DNA or 2.50-4.00×10 2 CFU/reaction for target genes of cell culture of V. cholerae , which was more sensitive than standard polymerase chain reaction (PCR). Inclusivity and exclusivity of the LAMP method were 100% for all target genes. The method showed similar high efficiency to a certain extent in rapid testing of spiked or collected specimens of water and aquatic products. Target genes were detected by the absence from all water samples from various sources. However, high occurrences of nanH gene were observed in intestine samples derived from four species of fish and one species of shellfish, indicating a risk of potentially toxic V. cholerae in commonly consumed aquatic products. The results in this study provide a potential tool for rapid and visualized detection of V. cholerae in water and aquatic products.
Simple Visualized Detection Method of Virulence-Associated Genes of Vibrio cholerae by Loop-Mediated Isothermal Amplification
