PI3K Inhibitor Library parahaemolyticus in oysters in a field setting. Methods Bacterial strains and DNA templates preparation Strains used in this study (Table 1) were maintained in Luria-Bertani broth (BD Diagnostic Systems, Sparks, MD) containing 30% glycerol at -80°C. V. parahaemolyticus ATCC 27969, originally isolated from blue crab hemolymph was used for sensitivity

testing. Additional 35 V. parahaemolyticus clinical and environmental strains and 39 non- V. parahaemolyticus strains were used to evaluate assay 4EGI-1 manufacturer specificity. All Vibrio strains were routinely cultured using trypticase soy agar or broth (TSA or TSB; BD Diagnostic Systems) supplemented with 2% NaCl at 35°C overnight. Non-Vibrio strains were grown on Luria-Bertani agar or blood agar (BD Diagnostic Systems). To prepare DNA template, a single bacterial colony grown on appropriate agar plates was suspended in 500 μl of TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA; Sigma-Aldrich, St. Louis, MO) and heated at 95°C for 10 min in a dry heating block. The crude cell lysate was centrifuged at 12,000 g for 2 min and the supernatant was stored at -20°C until use. LAMP primers and reaction conditions The V. parahaemolyticus toxR gene [GenBank:

L11929] was used as the target for LAMP primer design. Five primers, two outer (F3 and B3), two inner (FIP and BIP), and one buy Dinaciclib loop (Loop) which recognized seven distinct regions of the target sequence were designed using the PrimerExplorer software version 4 (Fujitsu Limited, Japan; http://​primerexplorer.​jp/​e. Oligonucleotide sequences and locations of the primers are shown in Table 2. The primers were synthesized by Invitrogen (Carlsbad, CA). The LAMP reaction mix in a 25 μl total volume consisted of the following: 1 × Thermo buffer, 6 mM of MgSO4, 0.8 M of betaine (Sigma-Aldrich), 4��8C 1.4 mM of deoxynucleotide triphosphate (dNTP), 0.2 μM of each outer primer (F3 and B3), 1.6 μM of each inner primer (FIP and BIP), 0.8 μM of the loop primer, 8 U of Bst DNA polymerase (New England Biolabs, Ipswich, MA), and 2 μl of DNA template. Additionally, 0.4 μM of

SYTO-9 green fluorescent dye (Invitrogen) was added when the LAMP reaction was carried out in a real-time PCR machine as described below. Two platforms were used to run the LAMP reactions. On the first platform, a real-time PCR machine (SmartCycler II System; Cepheid, Sunnyvale, CA) was used and the SYTO-9 green fluorescent dye was added. The assay was conducted at 63°C for 1 h. Fluorescence readings were acquired every 60 s using the FAM channel (excitation at 450-495 nm and detection at 510-527 nm), followed by melting curve analysis from 63°C to 96°C with 0.2°C increment per second. The fluorescence threshold unit was set to be 30. On the second platform, the LAMP reaction was carried out in a Loopamp real-time turbidimeter (LA-320C; Teramecs, Kyoto, Japan) at 63°C for 1 h and terminated at 80°C for 5 min.