Determination of an efficient and reliable method for DNA extraction from ticks
2004
Halos , Lenaïg (INRA , Maisons-Alfort (France). UMR 0956 Biologie Moléculaire et Immunologie Parasitaires et Fongiques) | Jamal , Taoufik (INRA , Maisons-Alfort (France). UMR 0956 Biologie Moléculaire et Immunologie Parasitaires et Fongiques) | Vial , Laurent (Institut de Recherche pour le Développement, Montpellier(France). Centre d'Etudes sur le Polymorphisme des Microorganismes (CEPM)) | Maillard , Renaud (INRA , Maisons-Alfort (France). UMR 0956 Biologie Moléculaire et Immunologie Parasitaires et Fongiques) | Suau , Antonia (Conservatoire National des Arts et Métiers, Paris(France). Laboratoire de Biotechnologies) | Le Menach , Arnaud (Institut National de la Santé et de la Recherche MédicaleAssistance Publique - Hôpitaux de Paris, ParisParis(France). U 444Hôpital Saint-Antoine) | Boulouis , Henri-Jean (INRA , Maisons-Alfort (France). UMR 0956 Biologie Moléculaire et Immunologie Parasitaires et Fongiques) | Vayssier-Taussat , Muriel (INRA , Maisons-Alfort (France). UMR 0956 Biologie Moléculaire et Immunologie Parasitaires et Fongiques)
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.
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