Cultures were incubated for 7 days at 37°C under microaerophilic conditions. Grown bacteria were identified as H. pylori by typical morphology, Gram staining results and positive reactions to oxidase, catalase, and urease activities. The cagA and vacA status as a virulence factors have been determined in all strains by PCR method. All strains were harvested by suspension in Brucella broth (Difco) supplemented with 10% fetal bovine serum (BB, Euroclone) and 30% glycerol
and stored in liquid nitrogen until used. DNA extraction from H. pylori isolates DNA was extracted from H. pylori isolates Selonsertib mw using the QIAamp DNA Mini Kit (Qiagen, Milan, Italy) according to the manufacturer’s instructions. Briefly, one colony was harvested learn more from an agar plate and added to an appropriate volume of phosphate-buffered saline homogenized by vortexing. Twenty microliters of a proteinase K solution (20 mg/mL) and 200 μL of buffer AL provided in the kit were then added, followed by incubation at 56°C for
10 min. Next, 200 μL of ethanol (96%) were added. The mixture was then loaded onto the QIAamp spin PI3K inhibitor column provided in the kit and centrifuged at 6000 g for 1 min. The QIAamp spin column was placed in a 2-mL collection microtube, and the tube containing the mixture was discarded. The column material was washed (500 μL each) with the first washing buffer (buffer AW1) and with the second washing buffer (buffer AW2) provided in the kit. Finally, the DNA was eluted with 150 μL of a third buffer (buffer AE) provided in the kit. Oligonucleotide primers The primers targeting the vacA gene (region m and region Branched chain aminotransferase s) and cagA genes  used in the PCR assay for the analysis of H. pylori isolates, are reported in Table 1. The primers were synthesised by MWG-Biotech AG (Mannheim, Germany). Table 1 Primers used for cytotoxin-associated gene ( cagA ) and vacuolating cytotoxin gene ( vacA ) typing of H. pylori Gene target Primer designation
Nucleotide sequence Amplicon size (bp) vacAS-F VacAS-F 5’-ATGGAAATACAACAAACACAC-3’ 259 (type s1) VacAS-R 5’-CTGCTTGAATGCGCCAAAC-3’ 286 (type s2) vacA midregion VacAM-F 5’-CAATCTGTCCAATCAAGCGAG-3’ 567 (type m1) VacAM-R 5’-GCGTCAAAATAATTCCAAGG-3’ 642 (type m2) cagA CagA-F 5’-GATAACAGGCAAGCTTTTGAGAGGGA-3’ 393 CagA-R 5’-CCATGAATTTTTGATCCGTTCGG-3’ PCR conditions The amplification was performed using a PCR SprintThermal Cycler (Hybaid, Ashford, UK) and carried out in 50 μL reaction volume containing 200 μmol/L (each) dNTP, 0.1 μmol/L (each) primer, 1X PCR buffer, 50 mmol/L KCl, 10 mmol/L Tris–HCl pH 8.8, 0.1% Triton X-100, 50 mmol/L MgCl2, 2 U of Taq DNA polymerase and 5 μL of template DNA or water for the negative control.