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Published on: March 30, 2015
Duplex qPCR assay for detection and quantification of Anaplasma phagocytophilum and Rickettsia spp
Alena Balážová1, Vojtech Baláž1, Jaroslav Ondruš2
1Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. 1946/1, 612 42 Brno, Czech Republic.
Abstract:
Anaplasma phagocytophilum and Rickettsia spp. are vector-borne zoonotic bacteria, which are clinically important especially in immunocompromised patients. There are large gaps in the current knowledge of their geographic distribution and prevalence in both their vectors and hosts. Our aim was to develop reliable and easy detection method for both these pathogens. We made a new hydrolysis probe based duplex Real-Time PCR assay based on previous studies. We optimized the assays and tested them to provide reliable recommended procedures with a sensitivity to a minimum of 10 target DNA copies per sample. The assays were designed to be specific for A. phagocytophilum and in the same reaction detect multiple species of rickettsiae. We designed gBlock quantification standards that provide the option to identify differences in pathogen load among different samples in subsequent studies.
Insights
A new duplex Real-Time PCR assay reliably detects Anaplasma phagocytophilum and Rickettsia species. This method enhances understanding of these important zoonotic bacteria in vectors and hosts.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Anaplasma phagocytophilum and Rickettsia spp. are significant vector-borne zoonotic bacteria.
- Knowledge gaps exist regarding their geographic distribution and prevalence in vectors and hosts.
- These pathogens pose risks, particularly to immunocompromised individuals.
Purpose of the Study:
- To develop a reliable and user-friendly detection method for Anaplasma phagocytophilum and Rickettsia spp.
- To establish a duplex Real-Time PCR assay for simultaneous detection of these pathogens.
- To create a sensitive assay for quantifying pathogen load.
Main Methods:
- Development of a hydrolysis probe-based duplex Real-Time PCR assay.
- Optimization and validation of the assay for sensitivity and specificity.
- Design of gBlock quantification standards for pathogen load assessment.
Main Results:
- The developed assay demonstrated high sensitivity, detecting a minimum of 10 target DNA copies per sample.
- The assay is specific for Anaplasma phagocytophilum and can detect multiple Rickettsia species concurrently.
- Quantification standards were created for future pathogen load analysis.
Conclusions:
- The new duplex Real-Time PCR assay provides a reliable and sensitive method for detecting Anaplasma phagocytophilum and Rickettsia species.
- This assay will aid in studying the geographic distribution and prevalence of these zoonotic pathogens.
- The developed method facilitates research into pathogen load differences in various samples.
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