These spotting buffers were validated with 18 different bacterial strains using antibodies and a phage with known specificities (Supplementary Table 1). in humans and animals, which are commonly found in many types of food such as pork, eggs, poultry, seafood, unpasteurized dairy products, and vegetables (Jackson et al., 2013; Gu et al., 2018). The standard methods for detecting are based on culturing techniques including pre-enrichment, selective-enrichment, and confirmation with biochemical checks, following procedures layed out from the International Business for Standardization (ISO 6579) or Bacteriological Analytical Manual (BAM). These methods can detect low figures or injured viable immune response from an antigen. The success of antibody production depends on the antigen characteristics such as types of immunogens, antigenicity, and antigen dosing. On the other hand, antibodies can be produced by phage display technology, which can determine binders to antigens no matter their immunogenic properties, therefore permitting the selection of binders against self-antigens, toxic, unstable, and non-immunogenic antigens (Frenzel et al., 2016). This technology also facilitates genetic executive of the binding sites to improve affinity and specificity. Its advantages over the traditional antibody production method possess fostered applications ranging from epitope mapping (Spillner et al., 2003; Youn et al., 2′,3′-cGAMP 2004), the detection of bacteria and viruses (Ferrer and Harrison, 1999; Yang et al., 2003; Morton et al., 2013b; Karoonuthaisiri et al., 2014; Wang et al., SELL 2014; Niyomdecha et al., 2018), protein domains (Christ and Winter season, 2006), and small molecules (Zhao et al., 2005; Qi et al., 2008). Given the power of phage technology, this study targeted to (1) develop a bacterial microarray method to speed up the process of screening and selecting phage clones 2′,3′-cGAMP expressing specific antibody fragments and (2) utilize the selected phage clones for developing a rapid lateral flow detection method for live Enteritidis. Materials and methods Bacteria, antibodies, and phage clones All bacteria in Table 1, except for spp., were inoculated from a single colony grown in a LB agar plate and cultured in 10 mL of 2xYT medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl) at 37C, 250 rpm for 16C18 h. spp. were cultured in 10 mL of Campylobacter Enrichment Broth (CEB) supplemented with 20 mg/L cefoperazone, 20 mg/L vancomycin, 20 mg/L trimethoprim, and 25 mg/L natamycin (#X132, Lab M, UK) at 41.5C, in microaerophilic conditions (5% CO2 and 10% O2) for 48 h. TABLE 1 Bacteria strains used in this project. Choleraesuis1, 6,7:c:1,5DMST 5580Dublin1, 9,12:g,pDMST 30404Enteritidis1, 9,12:g,mATCC 13076Hadar1, 8,z10:e,n,xDMST 10634Infantis1, 6,7:r:1,5DMST 26426Mbandaka1, 6,7:z10:e,n,z15DMST 17377Senftenberg1, 1,3,19:g,s,tDMST 17013Typhimurium1, 4,12:i:1,2ATCC 13311Virchow1, 6,7:r:1,2DMST 32758 O157:H7CDMST 12743 (5 109 colony forming units (CFU)/mL for each serovar) and the phage library (5 1011 plaque forming unit, pfu/mL) were mixed in PBS (total volume 1 mL) in the blocked tube, 20 rpm at RT for 1 h. Unbound phages were removed by centrifuging at 3,200 g for 10 min. The pellet of phage-bound bacterial cells was washed five occasions by resuspending in PBS made up of 0.1% Tween 20 and separation of phage-bound bacterial cell pellet by centrifugation at 3 200 g for 10 min. To elute phages from the bacterial target, a trypsin answer (1 mL of 100 g/mL Trypsin in Tris-buffered saline calcium chloride) was added, and the suspension was incubated at RT for 1 h. The eluted phages were used to infect a mid-log phase culture of TG1 TR strain (OD600 = 0.5) at 37C for 1 h. The non-infecting phages were separated by centrifugation at 3,200 g for 5 min. To enumerate the phage-infected TG1, the pellet was resuspended in 1 mL 2′,3′-cGAMP of 2xYT medium, and the bacterial cell suspension was serially diluted and plated on TYE ampicillin glucose agar plates (10 g/L bacto-tryptone, 5 g/L yeast extract, 8 g/L NaCl, 100 mg/L ampicillin and 40 g/L glucose). Open in a separate window Physique 1 Schematic of the process for the development of a lateral flow strip test assay for live detection consisting of three actions: biopanning and individual phage clones amplification, high-throughput screening, and lateral flow development. (A) Suspension biopanning was performed against a mixture of nine serovars adapted from Lee et al. (2007). (B) Phage clones were amplified in a 96-well plate and screened by a bacterial microarray method. (C) A bacterial microarray in a 96-well plate format was developed and constructed. Each well was spotted with target and nontarget bacteria. (D) Phage supernatant was tested with a bacterial microarray. (E) Lateral flow strip test was developed using a gold nanoparticle-labeled.