Saline peptone water

Saline peptone water, Brain Heart Infusion (BHI), Tryptic Soy Broth (TSB) supplemented with Yeast, Plate Count Agar (PCA), Tryptone Sulphite Neomycin (TSN), Dextrose Sabouraud (DS), and Mannitol Egg Yolk Polymyxin (MYP) mediums were obtained from Merck KGaA, (Darmstadt, Germany). Acetonitrile; amphotericin B solution (250 μg/mL); 2,2′-Azinobis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS); Fetal Bovine Serum (FBS); glacial acetic acid; hydrochloric acid; 6-hydroxy 2,5,7,8-tetramethyl-2-carboxylic acid (Trolox); L-glutamine solution (200 mM); MEM (Minimum Essential Medium); methanol; penicillin (10,000 U/mL); pure phenolic compounds (caffeic acid, catechin, epicatechin, epicatechin gallate, ethyl gallate, gallic acid, gentisic acid, p-coumaric acid, procyanidin B1, procyanidin B2, protocatechuic acid, syringic acid, t-resveratrol, t-piceid, and vallinic acid); streptomycin solution (P/S; 100 mg/mL); and 2,4,6-Tris (2-pyridyl)-S-triazine (TPTZ) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Folin–Ciocalteu (FC) reagent, 70% (v/v), iron (III)-chloride acid (FeCl3), iron (II)-sulphate (FeSO₄), sodium acetate (NaC₂H₃O₂), and sodium carbonate (Na₂CO₃) were purchased from Panreac Quimica S.L.U. (Barcelona, Spain). Malvidin-3-glucoside was obtained from Estrasynthese (Genay, France).

The multiplex PCR specificity was checked by examining the ability of the test to detect and distinguish non-target bacterial strains (Table 1) among these three target pathogens. In addition, to archive the method sensitivity, reference strains of E. coli O157:H7 (HCMUS), S. enteritidis ATCC13076 and L. monocytogenes ATCC19111 were grown on LB agar overnight. Small inocula were grown in LB broth for 10 h at 37 °C with shaking, followed by serial dilutions in saline peptone water (Merck, Germany). Each dilution was spread on LB agar to determine the bacterial counts and also prepare DNA by the boiling method as described. The DNA mixture template was made by mixing the five DNA templates with equal concentrations, decimally diluted with each bacterium representing 10⁵, 10⁴, 10³, 10², 10¹ and 1 colony-forming unit (CFU) and subjected to multiplex PCR.