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PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene
J G Smith1, L Kong, G K Abruzzo
1Department of Enzymology, Merck Research Laboratories, Merck and Co., Inc., Rahway, New Jersy 07065-0900, USA.
Abstract:
Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods.
Insights
New PCR primers targeting the 16S ribosomal gene offer a sensitive and specific method for detecting Helicobacter pylori (H. pylori) colonization in mouse models, overcoming limitations of traditional techniques.
Area of Science:
- Microbiology
- Molecular Biology
- Animal Models
Background:
- Traditional methods for detecting Helicobacter pylori (H. pylori) in animal models, such as culture, urease testing, and histopathology, often lack sensitivity and specificity.
- These limitations hinder the accurate assessment of H. pylori colonization in research settings, impacting the development of effective therapeutic strategies.
- There is a critical need for advanced detection methods to reliably study H. pylori infection dynamics in animal models.
Purpose of the Study:
- To develop and validate sensitive and specific PCR primers for detecting H. pylori in conventional mouse models.
- To establish a reliable molecular method for assessing H. pylori colonization over extended periods.
- To improve the accuracy and efficiency of H. pylori detection in research settings.
Main Methods:
- Design and synthesis of PCR primers targeting the 16S ribosomal gene sequence of H. pylori.
- Testing primer specificity against closely related bacterial species to rule out cross-reactivity.
- Application of the developed PCR assay for detecting H. pylori colonization in conventional mice at various time points post-inoculation.
- Semi-quantitative analysis of H. pylori load using PCR methodology.
Main Results:
- The developed PCR primers demonstrated high sensitivity, detecting as few as 0.2 cells of pure H. pylori.
- No cross-reactivity was observed with closely related bacterial species, confirming primer specificity.
- H. pylori colonization was successfully detected in conventional mice up to 4 weeks post-inoculation.
- Detection of H. pylori persisted in 100% of mice at 6 months and 60% at 1 year post-inoculation.
- The PCR method allowed for semi-quantitative detection of approximately 10^3 to 10^4 H. pylori cells per stomach.
Conclusions:
- The developed PCR primers and methodology provide a sensitive and specific tool for detecting H. pylori colonization in conventional mice.
- This molecular approach overcomes the limitations of traditional detection methods, enabling more accurate assessment of H. pylori infection.
- The established PCR assay facilitates reliable long-term monitoring of H. pylori colonization in mouse models, crucial for research and therapeutic development.