, jhp0918_2 AAGCTGAAGCGTTTGTAACG     2005 jhp0918_3 GCTAGAAAGATCA

, jhp0918_2 AAGCTGAAGCGTTTGTAACG     2005 jhp0918_3 GCTAGAAAGATCAACGGAAC 216 – This study * jhp0918_4 CACTTGTCTGGCTCTCAT       *The primers were self-designed by applying Primer 3 and the restriction enzyme was selected based on the reference of Fedratinib Shibata et al, 2005. Figure 1 MMP and TIMP selleck compound genotyping were done by PCR-RFLP and visualized by electrophoresis on 4% agarose

gel. The listed genotype patterns were (A) for MMP-3 -1612 as 5A5A or 6A6A; (B) for MMP-7 -181 as AA, AG, or GG; (C) for MMP-9 exon 6 as AA, AG, or GG; (D) for TIMP-1 372; as CC, TC, or TT; and (E) for TIMP-2 -418 as CG or GG. Quality control for genotyping was achieved by including in each amplification a negative PCR control sample and three positive control samples for each SNP analyzed (homozygous

for allele 1, heterozygous, and homozygous for allele 2). At least 10% of the samples were run twice in separate assays to reveal 100% concordance of the genotype designation for all of the polymorphisms. For the positive controls, the genopositive products were confirmed by direct sequencing. Detection of dupA gene by PCR The dupA-positive H. pylori was determined by positive PCR amplifications of at least 2 regions (jhp0917 and jhp0918) of the gene using two specific primer pairs (Figure 2) for strains J99 and 26695 as templates (Table 1) [6]. DNA 2 μl were HDAC activation added to 50-μl reaction mixture, containing 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP (Protech, Taiwan), 0.2 μM primers and 1 U Taq DNA polymerase (Fermentas, USA). PCR was performed with a thermal cycler (2720 thermal cycler; Applied Biosystems). The mixture was stored Progesterone at 4°C and the PCR products were separated by electrophoresis on 2% agarose gel. The gels were stained with ethidium bromide and visualized under UV illumination. Figure 2 The sites of primer pairs for PCR on the dupA regions, including for the jhp0917 and the jhp0918 regions, applying the standard H. pylori isolates (J99) as reference. Statistical analysis Statistical analysis was performed with the SPSS software (SPSS 12, Chicago, IL, USA). The χ2 test, Fisher’s exact test or Mann-Whitney U test were

used as appropriate to validate the dupA prevalence rates, histological severity, or SNP genotypes of MMPs and TIMPs between patients with and those without ulcers. A p < 0.05 was taken as significant. All test significances were validated by two-tailed analysis. The odds ratios (OR) and 95% confidence intervals (CI) were used to estimate the risk to get gastroduodenal ulcer in these H. pylori-infected subjects. Results Demographic features of the study subjects Of the 470 H. pylori-positive dyspeptic patients who provided blood samples for SNP analysis, 276 were females and 194 males, with median age of 47.2 years (range, 16-87 years). Endoscopic diagnoses included 265 with gastritis, 118 with duodenal ulcers, and 87 with gastric ulcers. Their demographics and histology after H. pylori infection were listed in Table 2.

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