Establishment of a Real-Time Recombinase Polymerase Amplification for Rapid Detection of Pathogenic Yersinia
Hongjian Zhang1, Meng Zhao1, Siyun Hu1
1College of Agriculture and Animal Husbandry, Qinghai University, Xi'ning 810016, China.
Abstract:
Yersinia enterocolitica is a zoonotic proto-microbe that is widespread throughout the world, causes self-limiting diseases in humans or animals and even leads to sepsis and death in patients with severe cases. In this study, a real-time recombinase polymerase amplification (RPA) assay for pathogenic Y. enterocolitica was established based on the ail gene. The results showed that the RPA detection for Y. enterocolitica could be completed within 20 min at an isothermal temperature of 38 °C by optimizing the conditions in the primers and Exo probe. Moreover, the sensitivity of the current RT-RPA was 10-4 ng/μL, and the study found that the assay was negative in the application of the genomic DNA of other pathogens. These suggest the establishment of a rapid and sensitive real-time RPA method for the detection of pathogenic Y. enterocolitica, which can provide new understandings for the early diagnosis of the pathogens.
Insights
A new real-time recombinase polymerase amplification (RPA) assay rapidly detects pathogenic Yersinia enterocolitica using the ail gene. This sensitive method offers a faster alternative for early pathogen diagnosis.
Area of Science:
- Microbiology
- Molecular Biology
- Diagnostic Assays
Background:
- Yersinia enterocolitica is a globally prevalent zoonotic bacterium causing various illnesses, from self-limiting infections to severe sepsis.
- Accurate and rapid detection is crucial for managing Yersinia enterocolitica infections and preventing severe outcomes.
Purpose of the Study:
- To develop a rapid and sensitive real-time recombinase polymerase amplification (RPA) assay for the detection of pathogenic Yersinia enterocolitica.
- To establish an assay based on the conserved ail gene for improved specificity.
Main Methods:
- Development of a real-time RPA assay targeting the ail gene of Yersinia enterocolitica.
- Optimization of reaction conditions, including primers and Exo probe, for isothermal amplification at 38 °C.
- Evaluation of assay sensitivity and specificity using known concentrations of genomic DNA and DNA from other pathogens.
Main Results:
- The real-time RPA assay successfully detected Yersinia enterocolitica within 20 minutes at a constant temperature of 38 °C.
- The assay demonstrated high sensitivity, with a detection limit of 10⁻⁴ ng/μL.
- The assay showed high specificity, yielding negative results with genomic DNA from other common pathogens.
Conclusions:
- A rapid, sensitive, and specific real-time RPA method for detecting pathogenic Yersinia enterocolitica has been successfully established.
- This assay provides a valuable tool for the early diagnosis of Yersinia enterocolitica infections, potentially improving patient outcomes.
- The optimized assay offers a significant advancement in pathogen detection technology.
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