The
A variety of tests are now available to diagnose
141 samples were collected from 131 patients with several diagnoses of gastrointestinal pathologies. Among them, 99 patients who were involved in this study had Gastritis, 29 had ulcers, 5 had Gastritis and ulcers, 3 had esophagitis, and 5 had other gastrointestinal diseases. The patients were 48 years old on average; their ages varied from 4 to 90 years old. 81 were males and 50 females (Table
Patient characteristics.
Total ( |
|
---|---|
Patients | |
Sex (male/female) | 81/50 |
Age years (median) | 48 years (range 4–90) |
Disease | |
Gastritis | 99 (70.2%) |
Ulcers | 29 (20.6%) |
Gastritis + ulcers | 5 (3.5%) |
Esophagitis | 3 (2%) |
Other* | 5 (3.5%) |
*Inflammation, duodenitis, and splenomegaly.
The methods used for the detection of
DNA extraction from the endoscopic biopsies fastened in paraffin followed the method described by Davis et al., 1995 [
At least two, 5 to 10 mm, ribbons of paraffin were placed in a 1.5 mL Eppendorf tube. One mL of xylene was added to the samples. They were shaken, allowed to rest for 3 to 5 minutes, and then centrifuged for 5 minutes, discarding the xylene afterwards. After three washes in 100%, 95%, and 70% ethanol, respectively, the samples were dried at room temperature. Next, the material was resuspended in a solution of Proteinase K, 50 mM Tris, 0.5% SDS, and sterile water. 430
Firstly, a fresh 3 to 7 mm biopsy section was placed in a 1.5 mL sterile tube with 190
The polymerase chain reaction followed the method described by Saiki and col. [
For each amplification reaction, 0.5 to 0.7
The reactions that followed were found to be optimal. The samples were heated to 94°C for 60 s to denature the DNA, cooled to 57°C for 90 s to allow the primers and the DNA to reanneal, and then heated to 72°C for 120 s for primer extension. By the final cycle, the extension period was 7 min. A total of 40 cycles were performed. The amplified product was detected by direct gel analysis. 5
Automated sequencing (Abi Prism 377) for the ureC region of
After the amplification was confirmed, the PCR product was submitted to digestion with the restriction enzymes HhaI and MboI for fragmentation of Urease-C [
The fragments which were produced were submitted to electrophoresis in a 2% gel agarose 1000 (Gibco-BRL), stained with ethidium bromide, visualized under ultraviolet light and photographed in Polaroid System. The patterns which were found were compared and analyzed with the computational program Bio 1D (Analysis of Restriction—PCR-RFLP—Restriction Fragment Length Polymorphism), version 99 (Vilber Loumart) (Figures
Digestion patterns with the enzyme
Digestion patterns with the enzyme
Approximately 10
Automatic sequencing was performed using the program Abi Prism, model 377, version 3.4, and Abi 100, version 3.2. Sequencing allowed for the identification of the studied DNA region (Primers P1 and P2). Figure
A total of 141 endoscopic biopsy samples from 131 patients were studied for
Comparison between PCR and urease test in fresh biopsy samples.
Urease test | PCR | |
---|---|---|
Positive | 64 | 64 |
Negative | 18 | 18 |
Total | 82 | 82 |
100% agreement.
Fifty-nine paraffin biopsies, all found to be positive through a histological examination, were submitted to the DNA extraction procedure and Beta-Globin PCR to prove the quality and the presence of DNA in the extracted samples. Only 14/59 (23.7%) samples were positive for the Beta Globin gene, but in two of them
Comparison between PCR and histology test in paraffin biopsy samples.
Histology | PCR | |
---|---|---|
Positive | 59 | 12 |
Negative | 0 | 47 |
| ||
Total | 59 | 59 |
| ||
Positive |
— | 14 |
Negative |
— | 45 |
| ||
Total | 0 | 59 |
*Two paraffin samples were positive for the
Among the 141 fresh endoscopic biopsy samples, 58 were tested using the RFLP technique to detect the different
Use of the Restriction Fragment Length Products (RFLP) technique for genotyping the positive
Restriction enzyme | Frequency (%) | Disease | Median age (years) |
---|---|---|---|
HhaI-1 | 1/58 ( 1.7) | Gastritis + ulcers | 41 |
HhaI-2 | 5/58 (8.6) | 3 Gastritis; 2 ulcers | 29 |
|
|
|
|
|
|
|
|
HhaI-5 | 3/58 (5.2) | 1 Gastritis; 2 ulcers | 54 |
HhaI-6 | 6/58 (10.3) | 3 Gastritis; 2 ulcers; 1 Gastritis + ulcers | 39 |
HhaI-7 | 1/58 (1.7) | 1 ulcers | 19 |
HhaI-8 | 4/58 (6.9) | 1 Gastritis; 2 ulcers; 1 esophagitis | 48 |
HhaI-9 | 2/58 (3.4) | 1 Gastritis; 1 ulcers | 37 |
HhaI-10 | 2/58 (3.4) | 2 ulcers | 72 |
HhaI-11 | 8/58 (13.8) | 6 Gastritis; 1 inflamation; 1 ulcers | 54 |
MboI-1 | 2/58 (3.4) | 2 Gastritis | 72 |
|
|
|
|
MboI-3 | 8/58 (13.8) | 3 Gastritis; 3 ulcers; 1 inflamation | 49 |
|
|
|
|
MboI-5 | 4/58 (6.9) | 1 Gastritis; 1 ulcers; 1 esophagitis; 1 Gastritis ulcers | 27 |
MboI-6 | 2/58 (3.4) | 1 Gastritis; 1 ulcers | 37 |
MboI-7 | 4/58 (6.9) | 1 Gastritis, 1 splenomegaly; 2 ulcers | 50 |
MboI-8 | 1/58 (1.7) | 1 Gastritis | 45 |
MboI-9 | 5/58 (8.6) | 2 Gastritis; 2 ulcers; 1 Gastritis + ulcers | 47 |
MboI-10 | 2/58 (3.4) | 2 Gastritis | 28 |
MboI-11 | 1/58 (1.7) | 1 Gastritis | 31 |
MboI-12 | 1/58 (1.7) | 1 Gastritis | 38 |
Gastric cancer is one of neoplasms that cause the majority of deaths not only in Brazil but all over the world. The type of cancer caused by
The
It should be noticed that the route of fecal-oral transmission appears to be the biggest problem in the prevalence of infection, making
The present study analyzes the effectiveness of the molecular biology method PCR in the detection of
The polymerase chain reaction (PCR) for the diagnosis of
The genotyping of
In the present study we standardized PCR with material obtained from the fresh endoscopic biopsies samples of patients attending Gastrocentro (the Center of Digestive Tract Studies), Medical School, UNICAMP. Some of the gastric biopsy samples were collected in paraffin and some were not. With regard to the standardization of the DNA extraction technique from the paraffin biopsy samples, several difficulties were found, because the samples contained a small amount of tissue fragments and many of the paraffin samples did not amplify the
PCR was used because it is more specific and faster when compared to other methods; the product of PCR can be processed with restriction enzymes to verify
As an internal control of the reaction was used in all samples (human
The efficiency of
The extraction of DNA from fresh samples had excellent results. Among the 82 analyzed samples, 64 were positive and 18 negative, with 100% in agreement with PCR.
In the present study, we used the PCR-RFLP method for the differentiation of
This data suggests that genotyping using PCR-RFLP can be useful as a fast procedure for the specific identification of
Several studies have confirmed that PCR-RFLP analysis of the ureC gene can differentiate clinically isolated
In our study we used two types of restriction enzymes in the amplified products of 820 pb of the
This study also revealed a high level of genetic diversity isolated in the different
The obtained genotyping patterns were compared using the computational program Bio 1D. We found 11 patterns with the
We believe that the genotyping of
Percentage agreement was calculated to compare
Authors have no conflict to declare.
The authors thanks the patients who agreed to participate in this study.