Optimized method for extraction of Vibrio genomic DNA
The genomic DNA of V. vulnificus, V. splendidus, V. parahaemolyticus, and V. angularis was extracted using the boiling and column extraction methods. The purity and concentration of each sample were evaluated using an ultra-micro spectrophotometer (Table 2). As shown in Figure 1, nucleic acid purity index values (A260/A280) of ³1.5 or above were achieved by both boiling and column extraction, with no significant difference between the two methods (P > 0.05, Mann–Whitney test).
LAMP fluorescence amplification curves were generated for the extracted DNA after the addition of the SYTO-9 fluorescent dye (Figure 2), and changes in fluorescence intensity of the product were observed under UV light after the addition of MnCl2-calcein (Figure 3). Typical LAMP fluorescence amplification curves were generated using V. vulnificus DNA extracted by both methods (Figure 2), with no difference between the Ct values obtained for each group. Typical changes in the fluorescence intensity were also observed using the visual dye method (Figure 3). These findings indicate that the residual carbohydrates produced in the sample extracted using the boiling DNA cleavage method do not affect the LAMP reaction. Furthermore, this method has the advantages of rapid extraction, low cost and convenience. Therefore, we selected the boiling method for extraction of Vibrio genomic DNA in this study.
Screening of LAMP primers
Six primers designed for specific detection of the gyrB gene sequence of V. vulnificus (Table 1) were screened by LAMP amplification curve analysis (with the SYTO-9 fluorescent dye) using V. vulnificus, and V. parahaemolyticus as positive and negative controls, respectively (Figure 4). The results showed that V.vulnificus and V.parahaemolyticus were amplified with the first, second, third and sixth primer sets (Figure 4A, 4B, 4C and 4F). V.vulnificus were not amplified in the fourth primer (FIG. 4D). The peak time of V.vulnificus with the fifth primer (FIG. 4E) was 9min, and the S-type amplification curve was typical, while V.parahemolyticus was not amplified. Therefore, based these results, we selected the fifth set of primers for detection of V. vulnificus due to its earlier peak Ct value and high specificity for subsequent experiments.
LAMP assay specificity
The specificity of the selected primers was then evaluated for detection of eight Vibrio strains (V. vulnificus, V. splendidus, V. mimicus, V. metschnikovii, V. furnissii, V. fluvialis, V. alginolyticus, and V. parahemolyticus) using the fluorescence amplification curve (SYTO-9 fluorescent dye) and color change (MnCl2-calcein) methods of LAMP amplification as shown in Figures 5 and 6, respectively. V. vulnificus was amplified specifically, while no amplification of the other Vibrio strains was detected. Furthermore, the results obtained using the two detection methods were consistent. These findings indicate that these primers allow specific detection of V. vulnificus using the LAMP method.
LAMP assay sensitivity
The sensitivity of the LAMP assay for detection of V. vulnificus using the optimized primers was evaluated using serial dilutions of the bacterial genomic DNA as templates. Using the LAMP reaction fluorescence amplification curve (with SYTO-9) method, the fluorescence amplification curves were consistent were stable at concentrations of V. vulnificus genomic DNA ³10 fg/μL, while the amplification was inconsistent and unstable at concentrations of £1 fg/μL (Figure 7). Using the color change ( MnCl2-calcein) method, V. vulnificus amplification products were detected at concentrations of genomic DNA ³10 fg/μL, but not at concentrations of £1 fg/μL (Figure 8). Thus, both LAMP methods can be used to detect V. vulnificus with a sensitivity of 10 fg/μL.
Analysis of actual samples
The LAMP assay established in this study was then evaluated for the analysis of aquatic product samples and water samples. Among 655 samples of aquatic products, 59 samples (9.01%) were positive for V. vulnificus (Table 3). Among 558 environmental water samples, 48 samples (8.60%) were positive for V. vulnificus (Table 4). Furthermore, consistent results for the detection of V. vulnificus in aquatic product and environmental water samples were obtained using the fluorescence amplification curve (with SYTO-9 fluorescent dye) and color change (with MnCl2-calcein) methods.
Validation of the LAMP results by real-time fluorescent PCR  revealed 100% consistency between the two methods. Furthermore, V. vulnificus samples cultured in vitro were detected with 83.76% positivity (P = 0.00002). The results of this study showed that the rate of V. vulnificus detection in aquatic products and environmental water samples using biochemical methods was significantly lower than that of achieved using the LAMP method. This discrepancy can be accounted for by the slow growth of many Vibrio isolates in vitro, which limits detection using biochemical methods.
Detection of V. vulnificus in different kinds of samples
We also analyzed the detection V. vulnificus rates of 655 aquatic product samples comprised of pools of DNA obtained from different numbers of biological samples using the LAMP assay (Table 3). The number of positive samples was The highest positive detection rate was obtained for the pool of 35 shellfish samples (18.52%; 35/189), indicating that V. vulnificus is enriched in shellfish. Furthermore, the positive detection rate of V. vulnificus in shellfish samples was significantly higher than that in seawater fish samples (χ2 =10.461, P < 0.01), freshwater fish samples (χ2 = 9.221, P < 0.01) and freshwater shrimp and crab samples (χ2 =7.895, P < 0.01). There was no significant difference in the positive detection rates of cephalopod samples (χ2 = 21.271, P < 0.01), and sea shrimp and crab samples (χ2 =1.524, P > 0.05).
Similar analysis of the 558 environmental water samples (Table 5) showed that the positive V. vulnificus detection rates for seawater, river water and aquaculture water were 10.23%, 2.04%and 5.00%, respectively. Furthermore, the positive rate of V. vulnificus detection in seawater samples was significantly higher than that in river water samples (χ2 = 6.737, P < 0.01), whereas there was no significant difference in the positive rate between the aquaculture and river water samples (P > 0.05).
Detection of V. vulnificus in samples collected at different times of year
Studies have shown that the positive detection rate of V. vulnificus, which is a thermophilic bacterium, increases as the water temperature rises throughout the year, with the highest detection rate in summer . In our analysis of samples collected at different times of year, the highest positive V. vulnificus detection rate (29.79%) was observed between June and August, which was 29.79% (Table 5).
Detection of V. vulnificus in samples obtained at different stages of the sales process
Most farmers’ markets in China operate based on open management and sales models. Compared with farmers’ markets, the conditions in supermarkets will be more standardized, with better sanitation and less cross-contamination between goods. In accordance with this, we found that the average rate of V. vulnificus contamination of samples from farmers’ markets was higher (30.01%; 68/206) than that in supermarkets (7.41%; 14/189) (Table 6).