Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa
Background and Aim: Helicobacter pylori is an important pathogen in humans and animals involved in chronic gastritis, leading to the development of gastric cancer. Urease produced by H. pylori is an enzyme that promotes bacterial colonization and can be used clinically as a biomarker of H. pylori in...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Veterinary World
2021-07-01
|
| Series: | Veterinary World |
| Subjects: | |
| Online Access: | http://www.veterinaryworld.org/Vol.14/July-2021/36.pdf |
| _version_ | 1818607493302976512 |
|---|---|
| author | Chularat Hlaoperm Kiattawee Choowongkomon Chantima Pruksakorn Jatuporn Rattanasrisomporn |
| author_facet | Chularat Hlaoperm Kiattawee Choowongkomon Chantima Pruksakorn Jatuporn Rattanasrisomporn |
| author_sort | Chularat Hlaoperm |
| collection | DOAJ |
| description | Background and Aim: Helicobacter pylori is an important pathogen in humans and animals involved in chronic gastritis, leading to the development of gastric cancer. Urease produced by H. pylori is an enzyme that promotes bacterial colonization and can be used clinically as a biomarker of H. pylori infection as part of a rapid urease test (RUT). A test with high specificity (95-100%) would be more convenient and faster than histopathology, bacterial culture, and polymerase chain reaction (PCR). The aim of this study was to develop a simple, cheap, and fast kit for detecting H. pylori infection in the gastric mucosa of canines, which can be used in clinical practice for diagnosing infection with this bacterium.
Materials and Methods: The RUT assays developed were prepared using 1% agar, 1% sodium phosphate monobasic, and 1% urea followed by the addition of 3% methyl red indicator. The cutoff value of sensitivity of the RUT assay was established using the urease of H. pylori ATCC 43504 and color change was monitored for 24 h. Comparisons of the sensitivity to H. pylori ATCC 43504 were made between the developed RUT assays and the Hp Fast™ commercial kit. Then, the limit of detection for H. pylori ATCC 43504 number was analyzed by the SYBR Green real-time PCR assay to measure the copy number of the ureC gene. Gastric biopsy samples from the antrum, body, and fundus of the stomach were collected from eight canines presenting with vomiting and gastroenteritis. Analyses were performed on fresh samples using the developed RUT assays and the Hp Fast™ commercial kit, which were read within 24 h; then, the results were confirmed with SYBR Green real-time PCR. The specificity of the RUT assays was tested with a number of different bacteria, including Staphylococcus pseudintermedius, Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus spp., Escherichia coli, and Salmonella spp.; H. pylori ATCC 43504 was used as a positive control.
Results: The results showed that the developed assays were sensitive to the urease enzyme at 0.1 mg/mL. The lowest detection limit of this assay for H. pylori ATCC 43504 was found to be 102 copies at 30 min. The sensitivity of detection of H. pylori in gastric biopsies of canines occurred in a minimum of 30 min. The RUT showed similar results to the Hp Fast™ commercial kit. In the developed RUT, the color change of the test from red to yellow could be clearly distinguished between the color of the positive test and the negative one; however, in the commercial Hp Fast™, it was difficult to observe the gel color change in the negative pH range of 5.8 and the positive pH of 6.5. The developed RUT was specific for H. pylori and did not detect any of the other tested bacteria. The test kit can also be stored for 6 months at 4°C.
Conclusion: The sensitivity of the developed assays allowed the detection of urease enzyme at a minimum concentration of 0.1 mg/mL. Our RUT could also detect H. pylori from one in eight canine specimens at a minimum of 102 copies within 30 min. This RUT is specific to H. pylori as it did not detect any of the other tested bacteria. |
| first_indexed | 2024-12-16T14:27:37Z |
| format | Article |
| id | doaj.art-701d74c69bd6499892aab6fd195e883d |
| institution | Directory Open Access Journal |
| issn | 0972-8988 2231-0916 |
| language | English |
| last_indexed | 2024-12-16T14:27:37Z |
| publishDate | 2021-07-01 |
| publisher | Veterinary World |
| record_format | Article |
| series | Veterinary World |
| spelling | doaj.art-701d74c69bd6499892aab6fd195e883d2022-12-21T22:28:19ZengVeterinary WorldVeterinary World0972-89882231-09162021-07-011471977198710.14202/vetworld.2021.1977-1987Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosaChularat Hlaoperm0https://orcid.org/0000-0002-4387-3026Kiattawee Choowongkomon1https://orcid.org/0000-0002-2421-7859Chantima Pruksakorn2https://orcid.org/0000-0003-3245-5707Jatuporn Rattanasrisomporn3https://orcid.org/0000-0002-2426-0961Graduate Program in Animal Health and Biomedical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand; Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand.Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.Graduate Program in Animal Health and Biomedical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand; Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand.Background and Aim: Helicobacter pylori is an important pathogen in humans and animals involved in chronic gastritis, leading to the development of gastric cancer. Urease produced by H. pylori is an enzyme that promotes bacterial colonization and can be used clinically as a biomarker of H. pylori infection as part of a rapid urease test (RUT). A test with high specificity (95-100%) would be more convenient and faster than histopathology, bacterial culture, and polymerase chain reaction (PCR). The aim of this study was to develop a simple, cheap, and fast kit for detecting H. pylori infection in the gastric mucosa of canines, which can be used in clinical practice for diagnosing infection with this bacterium. Materials and Methods: The RUT assays developed were prepared using 1% agar, 1% sodium phosphate monobasic, and 1% urea followed by the addition of 3% methyl red indicator. The cutoff value of sensitivity of the RUT assay was established using the urease of H. pylori ATCC 43504 and color change was monitored for 24 h. Comparisons of the sensitivity to H. pylori ATCC 43504 were made between the developed RUT assays and the Hp Fast™ commercial kit. Then, the limit of detection for H. pylori ATCC 43504 number was analyzed by the SYBR Green real-time PCR assay to measure the copy number of the ureC gene. Gastric biopsy samples from the antrum, body, and fundus of the stomach were collected from eight canines presenting with vomiting and gastroenteritis. Analyses were performed on fresh samples using the developed RUT assays and the Hp Fast™ commercial kit, which were read within 24 h; then, the results were confirmed with SYBR Green real-time PCR. The specificity of the RUT assays was tested with a number of different bacteria, including Staphylococcus pseudintermedius, Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus spp., Escherichia coli, and Salmonella spp.; H. pylori ATCC 43504 was used as a positive control. Results: The results showed that the developed assays were sensitive to the urease enzyme at 0.1 mg/mL. The lowest detection limit of this assay for H. pylori ATCC 43504 was found to be 102 copies at 30 min. The sensitivity of detection of H. pylori in gastric biopsies of canines occurred in a minimum of 30 min. The RUT showed similar results to the Hp Fast™ commercial kit. In the developed RUT, the color change of the test from red to yellow could be clearly distinguished between the color of the positive test and the negative one; however, in the commercial Hp Fast™, it was difficult to observe the gel color change in the negative pH range of 5.8 and the positive pH of 6.5. The developed RUT was specific for H. pylori and did not detect any of the other tested bacteria. The test kit can also be stored for 6 months at 4°C. Conclusion: The sensitivity of the developed assays allowed the detection of urease enzyme at a minimum concentration of 0.1 mg/mL. Our RUT could also detect H. pylori from one in eight canine specimens at a minimum of 102 copies within 30 min. This RUT is specific to H. pylori as it did not detect any of the other tested bacteria.http://www.veterinaryworld.org/Vol.14/July-2021/36.pdfdoghelicobacter pylorirapid urease test |
| spellingShingle | Chularat Hlaoperm Kiattawee Choowongkomon Chantima Pruksakorn Jatuporn Rattanasrisomporn Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa Veterinary World dog helicobacter pylori rapid urease test |
| title | Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa |
| title_full | Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa |
| title_fullStr | Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa |
| title_full_unstemmed | Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa |
| title_short | Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa |
| title_sort | development of an easy to use urease kit for detecting helicobacter pylori in canine gastric mucosa |
| topic | dog helicobacter pylori rapid urease test |
| url | http://www.veterinaryworld.org/Vol.14/July-2021/36.pdf |
| work_keys_str_mv | AT chularathlaoperm developmentofaneasytouseureasekitfordetectinghelicobacterpyloriincaninegastricmucosa AT kiattaweechoowongkomon developmentofaneasytouseureasekitfordetectinghelicobacterpyloriincaninegastricmucosa AT chantimapruksakorn developmentofaneasytouseureasekitfordetectinghelicobacterpyloriincaninegastricmucosa AT jatupornrattanasrisomporn developmentofaneasytouseureasekitfordetectinghelicobacterpyloriincaninegastricmucosa |