**7.3 Analysis 1**

### **Patients**

263 consecutive patients with dyspeptic symptoms attending the endoscopy center of the gastroenterology department of the Hajar Hospital, Shahrekord, Iran, were enrolled in this study from July to December 2008. Patient-reported symptoms and endoscopic findings of pathologist were recorded at the time of the consultation by the pathologist help, and these data were obtained retrospectively for analysis. The number of participants who were ineligible or declined participation in the study were not recorded.

For the purpose of analysis, three global variables were created: 1) patient-reported included age, gender and symptoms, 2) clinical signs, and 3) clarithromycin resistance data. Patientreported symptoms included pain, anorexia, heaviness after meal, early satiety, nausea, vomiting and flatulence. Clinical signs were included gastric ulcer, gastric cancer, non-ulcer disease, gastic erosion, nodularity, gastritis and duodenit. All patients read and signed an 'informed consent' form at the beginning of endoscopy declared their satisfaction for application of their anonymous data for research purpose.

Three biopsy specimens were taken from antrum and corpus of each patient, using a disinfected endoscope. Biopsy samples were placed in 0.1 ml of sterile saline solution and sent to the Biotechnology research center of Shahrekord Azad University. A rapid test for the detection of urease activity was performed by Gastro Urease kit (Bahar-afshan, Iran) according to manufacturer's instructions. DNA was isolated from each tissue with a DNA extraction kit (DNPTM, CinnaGen, Iran) according to the manufacturer's instruction and immediately used for molecular analysis.

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 111

200 biopsy samples were obtained over a 6-month period (June 2009 to November 2009) from 200 dyspeptic patients referred for endoscopy at Hajar hospital in Iran. All patients read and signed an 'informed consent' form at the beginning of endoscopy declared they are satisfied with application of their anonymous data for research purpose. Every patient history sheet was examined in detail and clinical findings including demographic data were recorded. The mean age of the patients was 52.5 years (range, 17 to 88 years), and 48.1% of the patients were men. Rapid urease test was performed with a Gastro urease kit (baharafshan, Iran). DNA was isolated from each tissue with a DNA extraction kit (DNPTM, CinnaGen, Iran) according to the manufacturer's instruction and immediately used for molecular analysis. Conventional PCR for detection of *ureC* gene and PCR-RFLP analysis for

Fig. 6. PCR-RFLP patterns obtained after digestion with *Bsa*I or *Mbo*II. *BsaI* cuts the PCR product of the wild-type sequence into two fragments of 1,000 and 400bp and that of the A2143G sequence into three fragments of 700, 400, and 300bp. *MboII* cuts the PCR product into two fragments of 700bp only when A2142G is present in the sequence: 1)A2142G positive control, 2)A2142G negative control, 3)A2142G positive strain, 4)A2143G positive

The real-time PCR was performed by using primer pair HP23S-1 and HP23S-2 and modified probes Pwt, P44G, P43G and P43C that previously reported by Pina et al [33] and newly designed probe P42G according to *23S rRNA* gene sequence (GenBank accession no. U27270) for identification of wild type, A2144G, A2143G, A2143C and A2142G genotypes

The real-time (TaqMan) PCR mixture was prepared until reaching a final solution of 25 µl containing 2 µl of extracted DNA, 200 mM dNTPs, 0.2 mM (each) primer HP23S-1 and HP23S-2, 0.1 mM probe Pwt, 0.2mM probe P44G, 0.1 mM probe P43G, 0.1 mM probe P43C and 0.75 mM probe P42G (Bioneer,Tokyo, Korea) (Table I), 1.5 mM MgCl2, and 1 U of *Taq*

detection of point mutations were performed as in analysis 1.

control, 5)A2143G negative control, 6)A2143G positive strain

**Real-time (TaqMan) PCR assay** 

respectively (Table 3).

**7.4 Analysis 2 Sample collection** 
