Quantitative real-time RT-PCR was performed using one-step SYBR Green PCR Master Mix (Takara) as per the manufacturer’s instructions. qRT-PCR was performed for the genes of interest and 16S rRNA this website in an iCycler IQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). From threshold cycle (CT) values, the relative levels of expression of the genes of interest were calculated using the 2−ΔΔCT method , with 16S rRNA as an internal reference. The experiments were repeated at least thrice, and statistical significance of the observed differences
was calculated using the two-sample t-test. p-value of <.05 was considered significant. The genes whose expression was examined and the sequences of the primers used for each gene are given in Table S1. To introduce deletion mutation into the fur gene of H. pylori, two pairs of primers designated P1-P2 and P3-P4 (Table S1) were used to PCR amplify the up-and downstream regions, respectively, of the fur gene producing 306-bp and 310-bp products flanking the region Apoptosis antagonist to be deleted. A chloramphenicol resistance gene (cat) was also PCR-amplified using primers designated C1 and C2. Primers P2 and P3 contained
21 base complementary regions at the 5′ region to C1 and C2, respectively (Table S1). A template mixture containing 200 ng to 400 ng of each of the three purified PCR products was then PCR-amplified using primers P1 and P4 in a single reaction, to generate a linear construct in which the up- and downstream fragments of fur were fused to the cat gene in the center. PCR conditions used were as follows: 10 cycles of 94 °C for 1 minute, 42 °C for 5 minutes and 72 °C for 4 minutes, followed by 40 cycles of 94 °C for 1 minute, 50 °C for 1 minute and 72 °C for 2.5 minute, and finally 72 °C for 10 minutes. The resulting allelic replacement construct was confirmed by sequencing
and introduced into H. pylori 26695 by natural transformation. Briefly, H. pylori cells from 48-hour confluent plates were suspended in 0.2 mL of BHI broth, about 3 μg PCR-amplified DNA was added, and the culture was spotted on GC agar plates. After incubation for 12 hours, the transformed cells were spread over the plates and incubated for a further 24 hours. The cells were then scraped onto selective GC agar plates containing chloramphenicol (20 μg per mL) and incubated for 3 to 7 days. Mutation Methocarbamol was confirmed by sequencing using primers P5 and P6. Two independent experiments were performed, and one mutant from each experiment was selected. Expression of the major virulence genes cagA, vacA, and ureA was examined in H. pylori strain 26695 following adherence to AGS cells and compared with that in unadhered bacteria grown under identical conditions without cell line. RNA was isolated from unadhered and adhered bacteria at different times after adherence, and expression of cagA, vacA and ureA was quantitated by real-time RT-PCR.