**Antimicrobial susceptibility testing**

The susceptibilities of the *H. pylori* isolates to clarithromycin were examined by an agar dilution method according to *CLSI* (Clinical and Laboratory Standard Institute) protocol (32). Resistance breakpoint for clarithromycin was defined as the >4 µg/liter.

#### **Conventional PCR assays**

A PCR assay targeted at the *ureC* (*glmM*) gene of *H. pylori* was performed with specific primer (Table 4) in an eppendorf mastercycler gradient (eppendorf, Germany). Briefly, the 23 µl PCR mixture, containing 1 µl of extracted DNA, 200 mM (each) deoxynucleoside triphosphates (dNTPs) (dNTP Mix, CinnaGen, Iran), 0.2 mM (each) primer (CinnaGen, Iran), 1.5 mM MgCl2, and 1 U of *Taq* polymerase (CinnaGen, Iran) in PCR buffer (CinnaGen, Iran), was held for 5 min at a denaturation temperature of 95°C, followed by 30 cycles of 1 min each at a denaturation temperature of 95°C, an annealing temperature of 58°C, and an elongation temperature of 72°C and by 5 min at 72°C. The amplified fragment was visualized after electrophoresis on a 1.5% agarose gel stained with ethidium bromide.

#### **PCR-RFLP analysis**

A 1,400-bp fragment of the *23S rRNA* gene was amplified with primers Cla-18 and Cla-21 [44] (Table 3). PCR amplification of DNA was performed in a final volume of 24 µl PCR mixture, containing 2 µl of extracted DNA, 200 mM dNTPs, 0.2 mM (each) primer, 1.5 mM MgCl2, and 1 U of *Taq* polymerase in PCR buffer. Amplification was carried out in an eppendorf mastercycler gradient over 30 cycles, each for 1 min at 95°C, 1 min at 62°C, and 1 min at 72°C. These cycles were performed after a denaturation of 5 min at 95°C and a final elongation step at 72°C for 5 min. The amplicon was digested with *BsaI* for 1 h at 37°C and *MboII* (Fermentas GMBH, Germany) for 1 h at 37°C to detect the restriction site occurring when the mutation was A to G transition at 2143 or 2142, respectively (Figure 5). The restriction products were analyzed by electrophoresis on a 2% agarose gel.

Fig. 5. Schematic diagram for detection of nucleotide alterations of *23S rRNA* by PCR-RFLP assays