Rapid and Visualized Detection of Virulence-Related Genes of Vibrio cholerae in Water and Aquatic Products by Loop-Mediated Isothermal Amplification

Abstract

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 developed for the first time based on loop-mediated isothermal amplification (LAMP) for detection of the important virulence-related genes ace, zot, cri, and nanH for toxins and the infectious 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°C for 40 min. Positive results were inspected by the production of a light green color under visible light or green fluorescence under UV light (302 nm). Limit of detection of the LAMP method ranged from 1.85 to 2.06 pg per reaction of genomic DNA or 2.50 × 100 to 4.00 × 102 CFU per reaction for target genes of cell culture of V. cholerae, which was more sensitive than standard 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 absence from all water samples from various sources. However, high occurrences of the nanH gene were observed in intestinal 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. HIGHLIGHTS

Absolute quantification of viable Vibrio cholerae in seawater samples using multiplex droplet digital PCR combined with propidium monoazide

“…In simulated seawater samples, the LODs of PMA-ddPCR for serogroups O1 and O139 were 150.66 and 147.57 CFU/mL, respectively, which were better than the qPCR sensitivity (on the order of 10 3 CFU/mL). The method was also superior to the previous detection methods, such as 250 ~ 400 CFU/reaction of LAMP (Chen et al, 2022) and 1.0 × 10 4 CFU/mL of monoclonal antibodies immunochromatography (Chen et al, 2014). However, when V. cholerae concentration is high (more than 10 7 CFU/mL), producing excessive positive droplets, the DNA template must be diluted to avoid exceeding the ddPCR detection limit.…”

Development of a simple, rapid, and sensitive molecular diagnostic assay for cholera

“…Several LAMP-based assays were developed previously in the laboratories for the diagnosis of cholera [9,[33][34][35][36][37][38][39][40][41][42][43]. The stool is a complex sample to extract DNA and amplify because of the presence of inhibitors.…”

Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2

“…However, as seen in lane 6 ( Figure 3 D and Figure 4 D, respectively), the color changes we not as vivid. SYBR Green is a DNA intercalating dye with double-stranded DNA and showed color changes from orange to green [ 47 , 48 ]. This method is rapid in the sense that positive detection was observed with the naked eye without the need for agarose gel electrophoresis.…”