Detection of , cag A and vac A Helicobacter pylori Virulence Genes in Gastric Biopsies of patients with Gastroduodenal disease using Polymerase chain reaction ( PCR ) technique

The objective of current study was to detect the prevalence of Helicobacter pylori by identifying 16SrDNA and to determine the virulence genes (ureA, cagA and vacA) in biopsy specimens from patients suffering gastroduodenal disease using polymerase chain reaction (PCR). Forty samples were obtained by gastroenterologists during endoscopy from gastric antral of suspected individual attending endoscopy unit at Baqubah Teaching Hospital, Diyala, Iraq, during the period from September 2015 to February 2016. According to the endoscopic finding the patients were allocated into four groups of gastroduodenal diseases and control, which include gastritis (GS), duodenal ulcer (DU), gastric ulcer (GU), gastric cancer (GC), their rates were 30% (12), 20% (8), 17.5% (7), 7.5% (3) and 25% (10), respectively. DNA was extracted from the biopsies and subsequently used for PCR detection of H. pylori and the virulence genes using specific primers. The results shows that 60% of samples were positive for H. pylori, of these positive samples, 91,66%, 66.66%, and 48.83%, were shown to have the virulence genes, ureA, cagA, and vacA, respectively. It is important to mention that cagA shown the highest prevalence rate in gastric cancer cases in comparison with vacA gene. further studies are required to study the link between cagA gene and Detection of, cagA and vacA Helicobacter pylori Virulence Genes in Gastric Biopsies of patients with Gastroduodenal disease using Polymerase chain reaction (PCR) technique Muthanna A. Saleh Al-Mahdawi, Ahmed Alwan Kareem and Ali Ghazi Hamdi 40 Vol: 13 No:4 , October 2017 DOI : http://dx.doi.org/10.24237/djps.1304.311C P-ISSN: 2222-8373 E-ISSN: 2518-9255 gastroduodenal diseases. In conclusion the result of present study provide important information concerning the prevalence of virulence genes of H. pylori.

Although World Health Organization (WHO) classified this pathogen as a class I carcinogen (3) , a low percentage of the general population establish severe outcome.Which may be due to the specific virulence genes which are responsible for the pathogenicity of the bacterium.
Vacuolating cytotoxin which considered as the marker for peptic ulcer disease is encoded by (vacA) gene.The cagA gene "cytotoxin-associated gene" which is considered as the marker for cag pathogenicity island, is associated with peptic ulcer and gastric cancer (4,5) .The ureA gen " encodes for 30 kDa subunit of urease enzyme, that important for bacterial colonization of the human gastric mucosa " is an important H. pylori virulence factor (6) .Recent studies indicate that there are health benefit linked with H. pylori colonization to human stomach including protection from obesity, diarrheal diseases, gastroesophageal reflux diseases, allergic airway diseases and Barrett's esophagus, indicating the dynamic and complex link between human and H. pylori (7) .
The aims of present study was to determine the prevalence of H. pylori, and to detect the virulence genes (ureA, cagA and vacA) using PCR technique.PCR analysis: A specific primer sequences for the gene 16S rRNA (1500bp) was used to confirm the presence of H. pylori in the biopsies specimens(Table 1), anther sequences of primers (Bioneer.Korea)were used for amplification of the genes cagA (1320bp), ureA (411bp) and vacA( 678bp) (Table 1).

Study
Vol   with the following conditions: 5 min of pre incubation at 94ºC, followed by 35 cycles of 30 s at 94 ºC, 30 s at 56 ºC, 30 s at 72 ºC and 3min at 72°C for final extension.PCR products were visualized by electrophoresis on 1.5% agarose gels with ethidium bromide.
Statistics Analysis: A t-test was used to compare groups, Data analysis was performed by using SPSS computing program (Statistical Package for Social Sciences) version 16. (11)
The study results of the ratio for the vacA gene demonstrated that (48.8%) of the H. pylori was positive.Various ratios were associated with pathogenic cases (Table 3).The reason of the difference in vacA gene ratio among gastroduodenal disease explained by the action of this gene which acts on formation of acidic vacuoles in the cytoplasm of gastric epithelial cells by creation of pores in epithelial cell membranes of gastric cell, which allows the anions and be released.The vacA also induces loosening of tight junctions of gastric epithelial cells, and may allow nutrients to cross the mucosal barrier to H. pylori (27) .

Conclusion
H. pylori bacterium can directly detected from biopsy samples obtained from patient with gastroduodenal diseases using PCR.The study showed a various relationship between gastropathological cases and virulence genes ( ureA, cagA and vacA).Only vacA was not detected in gastric cancer biopsy and hence, it should be extensively focusing on the mechanism of action of the ureA, cagA genes in cancer diseases.Therefore, determination of H. pylori virulence genes may reveal information about regarding the risk and clinical outcomes in symptomatic patients with gastroduodenal diseases.
Patients and specimen: Forty individual with dyspeptic symptoms attending endoscopy unit after specialist physician requests./ Baqubah Teaching Hospital in Diyala governorate, Iraq, from the period of September 2015 to February 2016.(23 male and 17 female; mean age, 43.4 ± 1.5 years ) were involved depending on the following study criteria: Vol: 13 No:4 , October 2017 DOI : http://dx.doi.org/10.24237/djps.1304.311CP-ISSN: 2222-8373 E-ISSN: 2518-9255 • Patients with chronic strong epigastric pain, melena, haematemesis, family history of CA stomach were included.• Patients with long-term use of (NSAIDs) drugs, anti-H.pylori therapy or bismuth containing drugs were excluded.A detailed history of each patient was obtained concerning, age, gender, address, job, smoking and alcoholism, chronic disease, treatment, admission diagnosis of all study patients.The endoscopic diagnosis grouped them into five categories "gastritis(GS), gastric ulcer (GU), duodenal ulcer (DU), gastric cancer (GC) and control with non-ulcer dyspepsia (NUD), NUD were defined as individual who had no endoscopic lesions (ulcers or malignancies)".During each endoscopic examination, antral gastric biopsy specimens were excised for total genomic DNA extraction.The study protocol was approved by the ethics and research committees of the hospital, and consent forms were obtained from all patients.Molecular detection of H. pylori Genomic DNA extraction: Biopsy Samples were kept in normal saline and preserved in deepfreeze at −20°C for further analysis.Specimen were thawed, crushed and then genomic DNA was extracted using genomic DNA extraction kit (Geneaid.Chaina).genomic DNA was eluted in 200 μL of 1 × TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), DNA purity and concentration (ng/µL) were checked by absorbance at (260 /280 nm) using Nanodrop spectrophotometer (THERMO.USA).The extracted DNA was kept at -20°C until use.
primers sequince used in this studyThe PCR master mix components (AccuPower ® PCR PreMix Kit) placed in standard AccuPower PCR PreMix Kit that containing all other components which needed for PCR reaction "Taq DNA polymerase, dNTPs, Tris-HCl pH: 9.0, KCl, MgCl2, stabilizer and tracking dye" as shown in Table2.
PCR tubes prepared according to the manufacturer instructions, transferred into spin vortex, centrifuge at 3000rpm for 3 minutes, then placed in PCR Thermocycler (MyGene, Bioneer.Korea).PCR amplification conditions for 16SrRNA gene involved: 3 min of pre incubation at 94ºC, followed by 35 cycles of 30 s at 94 ºC, 30 s at 58 ºC, 2 min at 72 ºC and 10 min at 72°C for final extension.The ureA gen was performed with the following conditions: 5 min of pre incubation at 94ºC, followed by 35 cycles of 1min at 93 ºC, 30 s at 55 ºC, and 1min at 72 ºC and 10 min at 72°C for final extension.The cagA gen was performed with the following conditions: 5 min of pre incubation at 95ºC, followed by 42 cycles of 1min at 95 ºC, 1min at 65 ºC, and 1min at 72 ºC and 5min at 72°C for final extension.The vacA gen was performed