Alkaline peptone broth

Shrimp, oyster, fish flesh (pomfret), and crab were purchased from a local market and verified to be free of V. vulnificus by quantitative PCR (Kumar et al., 2006 (link)). Food samples were homogenized thoroughly using a handheld grinder (3rd Gen. TGrinder, Tiangen Biotech Co., Ltd.). Ten grams of the food homogenate was made to 100 ml with alkaline peptone broth (Sinopharm Chemical Reagent Co., Ltd., Beijing, China) and spiked with desired amounts of V. vulnificus. If no enrichment was needed, 1 ml of the solution was boiled at 100°C for 10 min and centrifuged at 5,000 g for 5 min. One microliter of the supernatant was used as the template for real-time RPA detection. If enrichment was needed, the spiked food samples were incubated at 30°C with 200-rpm shaking. A total of 1 ml of the enrichment solution was collected at different time points and centrifuged at 800 g for 10 min to remove food debris. Bacterial cells were pelleted with 5,000 g centrifugation for 5 min, resuspended with 200 μl of water, and boiled at 100°C for 10 min. Also, 1 μl of the boiled resuspension was used as the template for real-time RPA detection.

Shrimp were purchased from a local market and verified to be free of V. alginolyticus by PCR [40 (link)]. The PCR primers are listed in Table 1. To test the detection limit in the spiked shrimp sample, 1 mL of serially diluted V. alginolyticus culture from 2.5 × 10⁴ CFU/mL to 2.5 CFU/mL was mixed with 1 g of shrimp homogenate (shrimp were ground in liquid nitrogen) and 9 mL alkaline peptone broth (Sinopharm Chemical Reagent Co., Ltd., China). Genomic DNA was purified using the TIANamp Genomic DNA Kit (Tiangen Biotech) in a 50 μL volume. One microliter of the purified DNA was used for RPA-LFD. For application simulation, the shrimp was cut into small pieces, and 300 mg of the pieces was ground in liquid nitrogen for each sample. Into several randomly selected homogenate samples, 3 μL of the 10⁶ CFU/mL V. alginolyticus culture was mixed. DNA was purified from the homogenate samples by the TIANamp Genomic DNA Kit (Tiangen Biotech) in a 50 μL volume. One microliter of the purified DNA was used for RPA-LFD or PCR detection.

Spiked food samples were used to determine the sensitivity of the biosensor in food matrix. Shrimp was selected as the representative food sample. After purchased from a local supermarket, the shrimp samples were confirmed to have no contamination of V. cholerae or V. vulnificus using qPCR [21 (link),22 (link)]. Homogenization of the shrimp samples were conducted with the handheld 3rd Gen. TGrinder (Tiangen Biotech Co Ltd.). To 100 mL of alkaline peptone broth (Sinopharm Chemical Reagent Co Ltd., Beijing, China), 10 g of the homogenate was added and suspended thoroughly, and the suspension was spiked with 10⁰, 10¹, or 10² CFU of the mix of V. cholerae and V. vulnificus. For enrichment, the suspension was put in a shaker and incubated with 200 rpm shaking under 30 °C. At each planned time points, 1 mL of the enrichment suspension was collected and centrifuged at 5000× g for 5 min. The pelleted bacterial cells were resuspended with 200 µL of water. DNA was released by boiling at 100 °C for 10 min, and 1 µL of the boiled resuspension was used as the template for RPA-LFS.