1 was used. A negative control was

selleckchem included for each LAMP run. PCR As a comparison, two sets of PCR reactions were performed, one using LAMP outer primers (F3 and B3) and the other one using the toxR-PCR primers (Table 2) published previously [18]. Each PCR mix in a 25 μl total volume contained 1 × PCR buffer, 0.2 mM of each dNTP, 1.5 mM of MgCl2, 0.5 μM of each forward and reverse primer, 0.625 U of GoTaq Hot Start Polymerase (Promega, Madison, WI), and 2 μl of DNA template. The PCR reactions were conducted using initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at 60°C (50°C for F3/B3 primers) for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 7 min in a Bio-Rad learn more C1000 Thermal Cycler (Hercules, CA). Aliquots https://www.selleckchem.com/products/bmn-673.html (10 μl) of PCR products were analyzed by electrophoresis on 1.5% agarose gel containing ethidium

bromide, and visualized under UV light. Gel images were documented by a Gel Doc XR system (Bio-Rad). LAMP specificity and sensitivity Seventy-five bacterial strains (Table 1) were used to determine the LAMP specificity. DNA templates were made from fresh overnight bacterial cultures and aliquots (2 μl) were subjected to both LAMP and PCR amplifications. Specificity tests were repeated twice. To determine LAMP sensitivity, serial 10-fold dilutions (ca. 108 CFU/ml to extinction) of a mid-log phase V. parahaemolyticus ATCC 27969 culture grown in TSB were prepared in phosphate buffered saline (PBS; BD Diagnostic Systems) and quantified using the standard plating method. DNA templates were prepared from each dilution by the boiling method described above and aliquots (2 μl) were subjected to both LAMP and PCR amplifications. Sensitivity tests were repeated six times and the lower limits of detection

(CFU/reaction) were reported. Standard curves were generated Cediranib (AZD2171) by plotting Ct (cycle threshold; for the real-time PCR platform) or Tt (time threshold; for the real-time turbidimeter platform) values against log CFU/reaction and the linear regression was calculated using the Microsoft Excel Software (Seattle, WA). LAMP testing in experimentally inoculated oyster samples Oyster samples were obtained from local seafood restaurants and determined to be V. parahaemolyticus-negative as described previously [10]. Oyster samples were processed following a previous study with slight modifications [11]. Briefly, 25 g of oyster sample was mixed with 225 ml of alkaline peptone water (APW; BD Diagnostic Systems) and homogenized in a food stomacher (Model 400; Tekmar Company, Cincinnati, OH) for 90 s to generate 1:10 oyster in APW homogenate. Serial 10-fold dilutions of a mid-log phase V. parahaemolyticus ATCC 27969 culture were prepared in PBS as described above. Aliquots (100 μl) of each dilution were inoculated into 900 μl of the 1:10 oyster in APW homogenate.