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Updated: Aug 22, 2025
Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications
Published on: November 5, 2020
Technical Evaluation of qPCR Multiplex Assays for the Detection of Ixodes ricinus-Borne Pathogens
Tal Azagi1, B J A Hoeve-Bakker1, Mark Jonker1
1Centre for Infectious Disease Control, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.
Background:
The extent to which infections with Ixodes ricinus-borne pathogens (TBPs), other than Borrelia burgdorferi s. l. and tick-borne encephalitis virus (TBEV), cause disease in humans remains unclear. One of the reasons is that adequate diagnostic modalities are lacking in routine or research settings.
Methods:
We evaluated the analytical specificity, sensitivity and robustness of qPCR assays for the detection of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Spiroplasma ixodetis, several Babesia species and Spotted Fever Rickettsia species as well as Bartonella species in human samples.
Results:
The qPCRs were found to perform well, given the difficulties of dealing with microorganisms for which confirmed patient materials are scarce or non-existent, a hurdle that was partially overcome by using synthetic controls. Spiking blood samples with the tested microorganisms showed that the detection of the TBPs was not inhibited by the presence of blood. The acceptable sensitivity when multiplexing the different pathogens, the good inter-assay variability and the absence of cross-reactivity make them potentially suitable as human diagnostics.
Conclusions:
The qPCRs evaluated in this study are technically suitable for the laboratory diagnostic assessment of clinical samples for infection with tick-borne pathogens. However, clinical validation and independent confirmation are still needed, pending the availability of sufficient human samples for testing in different laboratories.
