Comparison of methods of DNA extraction from ixodid ticks

Viktoriya  Levytska; Andriy  Mushinsky; Dorota Dwużnik; Ewa J.  Mierzejewska; Anna Bajer

doi:10.32845/bsnau.vet.2020.1.2

Viktoriya Levytska State Agrarian and Engineering University in Podilya https://orcid.org/0000-0003-3100-009X
Andriy Mushinsky State Agrarian and Engineering University in Podilya https://orcid.org/0000-0003-2850-2355
Dorota Dwużnik Faculty of Biology, University of Warsaw https://orcid.org/0000-0002-4432-3712
Ewa J. Mierzejewska Faculty of Biology, University of Warsaw https://orcid.org/0000-0002-1334-6110
Anna Bajer Faculty of Biology, University of Warsaw https://orcid.org/0000-0002-9631-0761

DOI: https://doi.org/10.32845/bsnau.vet.2020.1.2

Keywords: ticks, DNA extraction, Babesia spp, Rikettsia spp, Borrelia spp

Abstract

Ixodid ticks are ectoparasites of animals and humans that carry a wide variety of pathogenic microorganisms. Ticks can cause pathological conditions such as paralysis, fever, toxicosis and allergies, as well as a large number of infectious and invasive diseases. The aim of our study was to compare three methods of DNA extraction, to test their effectiveness and practicality in obtaining material from ticks and to determine their effect on the results of PCR-based studies. During 2018, questing ticks were collected from vegetation in Khmelnytsky region. Three different methods were used for DNA extraction: crushing of ticks with scissors and lysis in ammonium hydroxide, crushing with scissors followed by DNA extraction with a commercial kit Genomic Mini AX Tissue Spin (A&A Biotechnology, Gdynia, Poland), homogenization of ticks with programmable cryogenic grinder SPEX Sample Prep Freezer Mill 6875 followed by extraction of DNA with Genomic Mini AX Tissue Spin.

A total of 72 ticks (60 D. reticulatus and 12 I. ricinus) from vegetation were examined by PCR for the presence of pathogens from genera/complex Babesia, Rickettsia and Borrelia burgdorferi sensu lato (s.l.).

Following the first method of DNA extraction, 4.2% of ticks tested positive for Babesia spp., and DNA of Rickettsia spp. and Borrelia burgdorferi s.l. was detected in 12.5% and 33.3% of ticks, respectively. Following the second method of DNA extraction, 4.2% of ticks tested positive for Babesia spp., and DNA of Rickettsia spp. and Borrelia burgdorferi s.l. was detected in 54.2% (13 positive specimens) and 33.3% of ticks, respectively. The third method also revealed 4.2% of ticks positive for DNA of Babesia spp. Rickettsia DNA was detected in 7 D. reticulatus and 2 I. ricinus ticks, (37.5%). DNA of Borrelia spp. was identified in 37.5% of ticks. Thus, the combined method of mechanical homogenization of ticks in combination with a commercial kit for DNA isolation, offers maximum efficiency in terms of speed, number and size of samples to be studied.

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