Determining the prevalence of Helicobacter pylori infection by molecular identification of 16s rRNA in gastric patients in Hai Duong provincial general hospital

Abstract. In this study, 180 gastric biopsies were taken from patients of Hai Duong Provincial General Hospital who had come to the hospital to be treated for gastritis during the period January 2015 to July 2015. All of the subjects were i) 18-80 years old; ii) they had chronic gastritis, a stomach ulcer or gastric cancer; iii) they did not use antibiotics for 30 days prior to our endoscopy. Endoscopic biopsy samples were taken from the patients at the antrum, body and edge of the gastric ulcer, organizations suspected of infiltrating stomach cancer. These DNA gastric biopsies were extracted, amplified, the 16S rRNA gene was sequenced and the results were compared in Genbank. The results showed that 80 of the 180 (44.44%) gastric patients were Helicobacter pylori positive.

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JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2015-00091 Chemical and Biological Sci. 2015, Vol. 60, No. 9, pp. 148-153 This paper is available online at Received November 22, 2015. Accepted December 17, 2015. Contact Le Thi Phuong, e-mail address: phuongsinh@ymail.com 148 DETERMINING THE PREVALENCE OF Helicobacter pylori INFECTION BY MOLECULAR IDENTIFICATION OF 16S rRNA IN GASTRIC PATIENTS IN HAI DUONG PROVINCIAL GENERAL HOSPITAL Le Thi Phuong Hai Duong Medical Technical University Abstract. In this study, 180 gastric biopsies were taken from patients of Hai Duong Provincial General Hospital who had come to the hospital to be treated for gastritis during the period January 2015 to July 2015. All of the subjects were i) 18-80 years old; ii) they had chronic gastritis, a stomach ulcer or gastric cancer; iii) they did not use antibiotics for 30 days prior to our endoscopy. Endoscopic biopsy samples were taken from the patients at the antrum, body and edge of the gastric ulcer, organizations suspected of infiltrating stomach cancer. These DNA gastric biopsies were extracted, amplified, the 16S rRNA gene was sequenced and the results were compared in Genbank. The results showed that 80 of the 180 (44.44%) gastric patients were Helicobacter pylori positive. Keywords: Gastritis; Helicobacter pylori, infection, molecular identification, 16S rRNA 1. Introduction Helicobacter pylori (H. pylori) is thought to be a common cause or culprit of chronic gastritis and peptic ulceration and it is considered to be the major risk factor for gastric cancer, particularly in developing countries [1, 2]. Many studies have shown that H. pylori is an important factor in precancer and gastric cancer [3]. In Vietnam, a diagnosis of H. pylori presence in a patient‟s stomach is based on endoscopy images combined with the finding of H. pylori in a digestive juice culture and testing Urease for the presence of H. pylori [4]. Because culturing H. pylori from gastric biopsy specimens is a lengthy process and new „super‟ bacteria (non-H. pylori) are in existence, analysis is difficult. It‟s necessary to apply modern biomedicine techniques like PCR - molecular identification of 16SrRNA - to determine the extent to which H. pylori is present in patients and then proceed with an appropriate and direct form of treatment. Determining the prevalence of Helicobacter pylori infection by molecular identification of 16s... 149 2. Content 2.1. Subjects and methods * Subjects 180 patients gave their approval to be examined between January 2015 and July 2015 in Hai Duong Provincial General Hospital. All of these patients had chronic gastritis, gastric ulcer or gastric cancer, and they had not used antibiotics for one month prior to the endoscopy. The patients were interviewed to obtain their medical history, and endoscopic biopsy samples were taken at the antrum, body and edge of the gastric ulcer, organizations suspected of ilfitrating stomach cancer. * Methods Cross–sectional study The specimen test was a rapid biopsy urease test. The 16S rRNA was amplified for fast and accurate detection of H. pylori in specimens. The biopsy samples were stored in liquid nitrogen at -20 oC or -70 oC until DNA extraction. The 16S rRNA gene fragment with 133 base pairs in size was amplified with primer pairs: (F): 5′-AGGGGTAAAATCCGTAGAGAT- 3‟; (R): 5′-CGTTTAGGGCGTGGACTA- 3‟. The composition and volume in the PCR reaction was performed as follows: H2O: 9 µL; Buffer 10x: 2.5 µL; dNTP 10 mM: 2.5 µL; Forward primer (F): 1.25 µL; Reverse primer (R): 1.25 µL; Taq - polymerase (1 U/µL): 1.0 µL; DNA mold 40x: 2.5 µL. The temperature program of the Real time PCR cycle was performed as follows: Table 1. The temperature program of PCR for amplification of a 16S rRNA gene region Stages Steps Temperature ( o C) Duration Initialization Denaturation 95 5 mins. Aggregation Denaturation Pairing Aggregation 94 30 secs. 30 secs. 60 secs. 45 cycles of reduplication 53 72 Finish Finish 72 5 mins. The 16S rRNA was detected by PCR electrophoresis on 3.0% agarose gels with positive control, negative control and DNA marker 100 bp in size. The 16S rRNA gene region was sequenced and compared with the Genbank to determine the prevalence of H. ylori infecti n in gastric patients. 2.2. Results and discussion 2.2.1. DNA extraction and purification 180 gastric biopsy specimens obtained from patients were DNA extracted at the Hai Duong Provincial General Hospital. After DNA extraction, measurement of DNA concentration and purity were performed by absorbance (optical density) and in 1.5% agarose gel electrophoresis. The results are shown in Figure 1. Le Thi Phuong 150 Figure 1. Agarose gel electrophoresis of total DNA products extracted from samples Lanes 1- 6: DNA samples were extracted; Lane 17: 1 kb DNA ladder DNA extraction was performed with phenol/c loroform processing to sequence the 16S rRNA gene. The image of electrophoresis showed bands in most lanes that were bright and clear with molecular size larger than 10 kb, showing a high purity of extracted DNA, less prone that could be used in further ests. 2.2.2. Molecular identification of the 16s rRNA gene of Helicobacter pylori The entire process of determining H. pylori presence was performed as in the following charts: The charts for H.pylori positive detection in gastric patients After PCR reaction completion, 10 µL of PCR products was electrophoresed and compared with positive (+) and negative (-) controls to identify the fragment of DNA 16S rRNA gene. The results are shown in Figure 2. DNA mold 4x : 2,5 µL of samples 2,5 µL of H.pylori specific primers Mastermix PCR (Buffer 10x: 2.5 µL; dNTP 10 mM: 2.5 µL; Taq - polymerase 1 U/µL: 1.0 µL Electrophoretic analysis on agarosel gel Characterization of gene using PCR Mixture design for PCR reaction Determining the prevalence of Helicobacter pylori infection by molecular identification of 16s... 151 Figure 2. Agarose gel electrophoresis of PCR products Lanes 1- 7: samples; Lane 18: negative control; Lane 19: positive control. Figure 2 shows that the study samples were in lanes 1, 2, 3, 4, 5, 6, 7, 10, 14, 15, 16, 17 and there is a presence of DNA fragment on aragose gel electrophoresis with an estimated size of 133 bp. This location of the DNA band was the same as the location of the positive control in lane 19 (the 16S rRNA gene fragment of H. pylori). There were also many DNA bands in lane 18 (the negative control) as the samples and the positive controls. Hence, there was the 16S rRNA sequences of H. pylori in lanes 1, 2, 3, 4, 5, 6, 7, 10, 14, 15, 16, 17. There were no 16S rRNA sequences in DNA samples in lanes 8, 9, 11, 12, 13 or these samples were H. pylori negative. We performed a DNA sequence of 16S rRNA and compared the results with Genbank to determine whether or not the patients carried a DNA fragment of 16S rRNA of H. pylori. 2.2.3. The results of a DNA sequence of the 16S rRNA gene fragment 80 samples that tested positive for H. pylori were taken for sequencing. After the PCR reaction, 20 L of PCR products were purified using a QIAquick PCR Purification Kit (Qiagen - Germany) and were directly sequenced at the Macrogen Clinical Laboratory (Korea). The results are shown in Figure 3. Figure 3. The results of the 16S rRNA gene sequence The results showed the size of the DNA 16S rRNA gene fragment at 133 bp to be consistent with theoretical calculations. The nucleotide sequences of DNA of 16S rRNA gene fragment were identical to the nucleotide sequences in Genbank. This indicates that the patients carried DNA 16S rRNA gene fragment (the samples were H. pylori positive). The result demonstrates that this molecular identification method of 16S rRNA is highly reliable in the detection of H. pylori. Le Thi Phuong 152 2.2.4. The results of the prevalence of H. pylori infection Table 2. The prevalence of patients carrying the DNA 16S rRNA gene fragment (H. pylori infection) Results Numbers of patients Rates % H. pylori negative 100 55.56 H. pylori positive 80 44.44 Total 180 100 In this study, 180 DNA samples were taken from patients with gastric illness and of those 180, 80 (44.44%) samples were found to be H. pylori positive. This prevalence is higher than that in a study done by Phan Tan Tai (2009) [4] that involved 370 gastric patients in the Phu Tan General Hospital with 24.6% H pylori positive, and a 20.6% rate of H. pylori infection in a study done by Lieu Chi Hung (2010) [5] at the Tay Ninh General Hospital. These are considerably lower than other findings, such as 82.65% H. pylori infection in 298 patients in Hue in a study done by Tran Van Huy [6, 7]. The study by Nguyen Duc Toan et al. (2012) [8], Nguyen Van Thinh et al. [7] who examined patients with gastritis and ulcers, and Nijewitch„s study (2002, 2003) [9] in Russia found a H. pylori in 67.9%, 72.8% and 81.1% of their cases respectively [10-12]. This difference is likely due to the difference in sample size, sampling methods and sampling time. In our study thea sample size was 180 patients and samples, whereas other studies have used a sample size that exceeded 300. This accounts for the differring prevalence of H. pylori positive findings in the studies. In addition, scientists have obtained samples in 2002, 2003 and 2005 and it has been said that in those years there was a spike in H. pylori infect on world wide among gastric patients. At this time, some findings indicate that globally, the incidence of H. pylori infection in gastric patients is many times lower than it was in previous years. H. pylori might have been attacked and displaced by a more aggressive microorganism 3. Conclusion A total of 180 gastric patients came to Hai Duong Provincial General Hospital to be examined and treated. All were tested for H. pylori infection using molecular identification in the form of 16S rRNA and DNA sequencing. It was found that 80 of the 180 (44.44%) were infected with Helicobacter pylori and this level of infection is highly significant. REFERENCES [1] Tran Van Huy et al., 2012. The Study of effective treatment of RACM regimens in gastric ulcer patients with Helicobacter pylori infection. Journal of Practical Medicine, 802(1), pp. 53- 9. [2] Lieu Chi Hung, 2000. The study of H.pylori infection in patients of gastroduodenal endoscopy. Ho Chi Minh City Medicine; 4(2): pp. 89-94. [3] Ta Long, 2003. Peptic ulcer disease and Helicobacter pylori. Ha Noi Medical Publishing House. [4] Phan Tan Tai, Huynh Chi Hung, 2009.The incidence of Helicobacter pylori infection in patients of gastroduodenal endoscopy at Phu Tan General Hospital. The summary record of scientific workshop at An Giang General Hospital, 7(3), p.11-21. Determining the prevalence of Helicobacter pylori infection by molecular identification of 16s... 153 [5] Lieu Chi Hung, Ngo Van Long, 2007. Helicobacter pylori infection and gastro- duodenal disease at Tay Ninh General Hospital. Ho Chi Minh City Medicine; 8: pp. 8-10 [6] Nguyen Van Thinh, Nguyen Thi Nguyet and Nguyen Thi Hong Hanh, 2007. Summary of scientific reports – Scientific Conference. The study of basic life science, pp.196-198. [7] Nguyen Van Thinh, Nguyen Van Oai, Ta Long, Tra Van Hop, 2007. An association between Helicobacter pylori infection with intestinal metaplasia - dysplasia of gastric ulcers - duodenum. Internal Medicine, 3, pp.16-20. [8] Nguyen Duc Toan, Ta Long, Nguyen Thi Nguyet, Nguyen Thi Hong Hanh, 2009. The drug resistance of H. pylori in patients with duodenal ulcer in the 6 months early of 2009. Journal of Practical Medicine, 8,pp. 14-18. [9] Nijewitch V. Zaytseva, A. I. Aminova, A. A. Akatova, Ye. Yu. Minchenko, 2009. Peculiarities of Eradication Therapy for Chronic H. pylori-associated Gastroduodenitis in Children Living in Ecologically Unfavorable Conditions. Federal Scientific Center for Medical and Preventive Health Risk Management Technologies, Perm, Russia. Pediatricheskaya farmakologiya [Pediatric Pharmacology]. 2(6): pp.90–93. [10] Janssen M. J., Hendrikse L., De Boer S. Y., Bosboom R., et al., 2006. Helicobacter pylori antibiotic resistance in a Dutch region trends over time . Neth J. Mel. 64, pp. 191-195. [11] Mitui Midori, Ashish Patel, Kristine Leos N., Christophe D., Doern, and Jason Y. Park, 2014. Novel Helicobacter pylori Sequencing Test Identifies High Rate of Clarithromycin Resistance. J . Pediatr Gastroenterol Nutr. 59(1), pp. 6-9. [12] Stephens J. C., Stewart J. A., Folwell A. M., Rathnone B. J., 1998. Helicobacter pylori cagA status, vacA genotype and ulcer diseases. Eur J. Gastroenterol Hepatol, pp. 381-384.
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