Multiplex PCR method for differentiating highly pathogenic Yersinia enterocolitica and low pathogenic Yersinia
Thi Hien Bui1, Shunsuke Ikeuchi1, Yukiko Sassa O'Brien1
1Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan.
Abstract:
A multiplex PCR method for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as the highly pathogenic Y. enterocolitica, including serotype O8, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis, was developed. Four primer pairs were chosen to detect the genes fyuA, ail, inv, and virF, responsible for the virulence in pathogenic Yersinia species. Under the multiplex PCR conditions, the unique band patterns for the highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis were generated from Yersinia strains. The detection limit of this method was 101-103 CFU per reaction tube. This multiplex PCR method could detect highly pathogenic Y. enterocolitica O8 from the wild rodent fecal samples that were culture-positive. Therefore, the new multiplex PCR method developed in this study is a useful tool for rapid and sensitive diagnosis, distinguishing three pathogenic Yersinia groups.
Insights
A new multiplex PCR method rapidly and sensitively diagnoses three pathogenic Yersinia groups. This tool effectively differentiates highly pathogenic Yersinia enterocolitica O8, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Disease Diagnostics
Background:
- Pathogenic Yersinia species cause significant human and animal diseases.
- Accurate and rapid differentiation of Yersinia groups is crucial for effective treatment and control.
- Existing diagnostic methods may lack the sensitivity or specificity required for certain Yersinia strains.
Purpose of the Study:
- To develop a multiplex PCR assay for the simultaneous detection and differentiation of three pathogenic Yersinia groups.
- To establish a sensitive and rapid diagnostic tool for Yersinia infections.
- To specifically identify highly pathogenic Yersinia enterocolitica, including serotype O8.
Main Methods:
- Development of a multiplex PCR assay utilizing four primer pairs targeting virulence genes (fyuA, ail, inv, virF).
- Optimization of PCR conditions to generate unique band patterns for each Yersinia group.
- Determination of the assay's detection limit using known Yersinia strains.
Main Results:
- The multiplex PCR method successfully generated distinct band patterns for highly pathogenic Y. enterocolitica (O8), low pathogenic Y. enterocolitica, and Y. pseudotuberculosis.
- The assay demonstrated high sensitivity, with a detection limit of 10^1–10^3 CFU per reaction tube.
- The method successfully detected highly pathogenic Y. enterocolitica O8 in culture-positive wild rodent fecal samples.
Conclusions:
- The developed multiplex PCR assay is a valuable tool for the rapid and sensitive diagnosis of pathogenic Yersinia infections.
- This method enables accurate differentiation between key pathogenic Yersinia groups, aiding in epidemiological surveillance and clinical management.
- The assay's ability to detect Y. enterocolitica O8 in environmental samples highlights its potential for broader applications.
