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.
6 trang |
Chia sẻ: thanhle95 | Lượt xem: 473 | Lượt tải: 0
Bạn đang xem nội dung tài liệu Determining the prevalence of Helicobacter pylori infection by molecular identification of 16s rRNA in gastric patients in Hai Duong provincial general hospital, để tải tài liệu về máy bạn click vào nút DOWNLOAD ở trên
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.