Vet. Res. 35 (2004) 709-713
DOI: 10.1051/vetres:2004038

Determination of an efficient and reliable method for DNA extraction from ticks

Lénaïg Halos^a, Taoufik Jamal^a, Laurence Vial^b, Renaud Maillard^{a, c}, Antonia Suau^d, Arnaud Le Menach^e, Henri-Jean Boulouis^a and Muriel Vayssier-Taussat<sup>a

a</sup>  UMR 956 INRA/AFSSA/ENVA/UVPM, Microbiologie, École Nationale Vétérinaire, 7 avenue du Général de Gaulle, 94703 Maisons-Alfort, France
^b  IRD Montpellier, CEPM, 911 avenue Agropolis, 34090 Montpellier, France
^c  Unité de pathologie du bétail, École Nationale Vétérinaire, 7 rue du Général de Gaulle, 94703 Maisons-Alfort, France
^d  Laboratoire de Biotechnologies, Centre National des Arts et Métiers, 2 rue Conté, 75003 Paris, France
^e  INSERM U444, Hôpital Saint-Antoine, 27 rue Chaligny, 75571 Paris Cedex 12, France

(Received 27 January 2004; accepted 19 May 2004)

Abstract - Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.

Key words: tick / DNA extraction / PCR / nymphal stage

Corresponding author: Muriel Vayssier-Taussat mvayssier@vet-alfort.fr

© INRA, EDP Sciences 2004