Duplex qPCR assay for detection and quantification of Anaplasma phagocytophilum and Rickettsia spp

Alena Balážová; Vojtech Baláž; Jaroslav Ondruš; Pavel Široký

doi:10.1016/j.ttbdis.2020.101462

Duplex qPCR assay for detection and quantification of Anaplasma phagocytophilum and Rickettsia spp

Ticks Tick Borne Dis. 2020 Sep;11(5):101462. doi: 10.1016/j.ttbdis.2020.101462. Epub 2020 May 5.

Authors

Alena Balážová¹, Vojtech Baláž¹, Jaroslav Ondruš², Pavel Široký³

Affiliations

¹ Department 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.
² Department 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; CEITEC - Central European Institute of Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. 1946/1, 612 42 Brno, Czech Republic.
³ Department 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; CEITEC - Central European Institute of Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. 1946/1, 612 42 Brno, Czech Republic. Electronic address: sirokyp@vfu.cz.

PMID: 32723653
DOI: 10.1016/j.ttbdis.2020.101462

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.

Keywords: Diagnostics; Ixodes ricinus; Method; Reservoir host; Ticks.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anaplasma phagocytophilum / isolation & purification*
Animals
DNA, Bacterial / analysis
Hydrolysis
Ixodes / microbiology*
Polymerase Chain Reaction / methods*
Rickettsia / isolation & purification*
Sensitivity and Specificity

Substances

DNA, Bacterial