Gastric biopsy specimens from each patient were inoculated onto a Mueller–Hinton agar (with 7% horse blood) plate and cultured at 37 °C in an anaerobic jar selleck chemical with a Campypak gas generator. After 3 days, the plates were observed for colony growth, and incubated further for up to 7 days.
Gram stain and biochemical tests for the presence of urease, catalase, and oxidase were performed using a single colony from the plate to confirm the presence of H. pylori. If it is positive for all three enzymes, a single colony was picked from each primary culture plate, inoculated onto a fresh Mueller–Hinton (with Skirrows) agar plate (with 7% horse blood), and cultured under the same conditions described above. After 3–7 days, the plate was flooded with 1 mL Brucella broth and all colonies were scraped off. A part of this bacterial suspension was placed in a freezing medium
(800 μL H. pylori culture in Brucella broth, 100 μL dimethyl sulfoxide, 100 μL fetal bovine serum) and stored at −80 °C. DNA from the H. pylori isolate was extracted using the QIAamp DNA Mini Kit (Qiagen), following the manufacturer’s instructions, and stored at 4 °C until PCR amplification was performed. click here The full product of the cagA gene was determined by PCR using the primers cagA L2(+) and cagA L2(−) (Table 1) (Yamazaki et al., 2005) in a 100 μL reaction mixture containing the following: TaKaRA ExTaq polymerase (5 U mL−1), 10 × ExTaq buffer, dNTP mixture (2.5 mM each), sterile distilled water, and 1 μL of the sample DNA. The regions containing full-length cagA were amplified
by PCR under the following conditions: 94 °C for 1 min; 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 3.45 min; followed by 72 °C for 10 min. PCR products were run on a 1.5% agarose gel (Agarose S) that was stained with ethidium bromide and examined under UV. The PCR products of samples that were cagA+ were purified using Amicon Centricon centrifugal filter devices YM 100MW (Millipore) or the High Pure PCR Product Purification Kit PAK6 (Roche), according to the manufacturer’s instructions. DNA direct sequencing was performed using a Big Dye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems) (3 μL of the purified PCR product in a 20 μL total reaction mixture containing the following: Big Dye, primer, and sterile distilled water). The primers used and their sequences are listed in Table 1 (Yamazaki et al., 2005). The sequencing PCR products were then purified using the Dye Ex 2.0 Spin Kit (Qiagen), according to the manufacturer’s instructions. The purified sequencing PCR products were processed for sequencing performed on the ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems). DNA sequences were analyzed using genetyx v. 7 (Software Development, Tokyo, Japan). To determine the phylogenetic relationship of the 19 Philippine H. pylori strains and other previously reported H.
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