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Design a Diagnostic Kit for Molecular Identification of Helicobacter pylori
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| Author:
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HANEEN B AL-WAELI, DHAFAR N AL-UGAILI, ZAINAB SH KHALAF
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| Abstract:
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Helicobacter pylori a Gram-negative spiral-shaped bacterium that affects up to 50% of the population worldwide. It has been considered one of the main risk factors that cause acute and chronic gastritis, peptic ulcer disease, gastric carcinoma, and low grade gastric mucosa associated lymphoid tissue lymphomas. This study aimed to design a rapid diagnostic kit for the identification of Helicobacter pylori clinical samples and compare it with the conventional methods. A total of eighty samples (50 biopsy and 30 saliva samples) were collected from patients suffering different gastric diseases, 48 of them were females which represents (60%) and 32 were males with a ratio of (40 %). The clinical biopsy samples were collected in Gastrointestinal Endoscopy Unit at Al–Kindi Teaching Hospital in Baghdad during the period from January 2019 to May 2019. Also, 30 saliva samples were collected from patients that suffer from stomach pain after gave positive result by urea breath test (UBT), the saliva samples were collected at privet laboratory Al-Moaz laboratory. In biopsy samples; two biopsy specimens were taken from each patient, one biopsy for rapid urease test and other biopsy for molecular detection by RT-PCR. Depending on the endoscopic diagnosis, the patients were grouped into: 28 of patients (56%) had gastritis followed by peptic ulcer (PU) includes duodenal ulcer (DU) in 4 patients (8%), gastric ulcer (GU) in11 patients (22%), gastroesophageal reflux disease (GERD) in 4 patients (8%), gastric pain without ulcer in 2 patients (4%) and gastric cancer (GC) in 1 patients (2%). The results revealed that 43 out of 50 patients that represent (86%) patient were positive for the rapid urease test (RUT) when biopsy taken from the antrum of the stomach. In saliva samples 26 out of 30 patients gave positive result by urea breath test. The molecular analysis conducted to detect Helicobacter pylori DNA in 50 of biopsy and 8 of saliva samples using RT-PCR technique using 23SrRNA primer and probe specific for Helicobacter pylori. Primers designed for 23SrRNA gene Forward and reverse oligonucleotides were derived from the conserved region located between from 1372910 to 1375882 (2973 bp); Helicobacter pylori 51. Two biopsy samples numbered 37 and 38 were used during the optimization step for the primer and the probe applied to detect Helicobacter pylori. The results clarified that the amplification of 23SrRNA were positive for 48 (96%) of the gastric biopsy sample by using RT- PCR, while only 2 of 8 saliva samples was positive for 23SrRNA (25%). The number of bacterial load was quantified by interpolation of the corresponding Ct values in the standard curve to measurement who samples have low and high copy number for 23SrRNA Helicobacter pylori gene.
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| Keyword:
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Helicobacter pylori, Gastrointestinal Diseases, Real Time PCR, Rapid Urease Test, 23SrRNA.
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| EOI:
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| DOI:
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https://doi.org/10.31838/ijpr/2021.13.01.731
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| Download:
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