Efficiency of DNA Isolation Methods Based on Silica Columns and Magnetic Separation Tested for the Detection of <i>Mycobacterium Avium</i> Subsp. <i>Paratuberculosis</i> in Milk and Faeces

• Main Authors: Marketa Husakova, Petr Kralik Vladimir Babak, Iva Slana
• Format: Article
• Language: English
• Published: MDPI AG 2020-11-01
• Series: Materials
• Subjects: <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i>; magnetic separation; silica columns; Johne’s disease; paratuberculosis; milk
• Online Access: https://www.mdpi.com/1996-1944/13/22/5112

Timely and reliable detection of animals shedding <i>Mycobacterium avium</i> subsp. <i>paratuberculosis </i>(MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using milk and faecal samples artificially contaminated by MAP with a focus on modern instrumental automatic DNA isolation procedures based on magnetic separation. In parallel, an automatic and manual version of magnetic separation and two methods of faecal samples preparation were compared. Commercially available DNA isolation kits were evaluated, and the selected kits were used in a trial of automatic magnetic beads-based isolation and compared with the manual version of each kit. Detection of the single copy element F<i>57</i> was performed by qPCR to quantify MAP and determine the isolation efficiency. The evaluated kits showed significant differences in DNA isolation efficiencies. The best results were observed with the silica column Blood and Tissue kit for milk and Zymo Research for faeces. The highest isolation efficiency for magnetic separation was achieved with MagMAX for both matrices. The magnetic separation and silica column isolation methods used in this study represent frequently used methods in mycobacterial diagnostics.