Proceedings of the 10th International Colloquium on Paratuberculosis

#109 A sample and efficient method for DNA extraction from Mycobacterium avium subsp.
paratuberculosis cultured in an automated broth culture system
Miguel Angel Salgado, Robert Söderlund, Göran Bölske, David Herthnek, Anna Aspán, Juan Kruze
Universidad Austral de Chile, Chile; National Veterinary Institute (SVA), Sweden
Liquid culture <strong>of</strong> MycobacteriuM. avium subsp. paratuberculosis (Map) has shown advantages over culture on
solid media to detect Map infection. PCR represents a rapid and efficient means <strong>of</strong> confirming Map in broth
culture. There are several commercial kits and proposed methods <strong>of</strong> harvesting Map DNA and removal <strong>of</strong>
inhibitors, most <strong>of</strong> which are costly, laborious and time consuming. This study proposes a simple and efficient
method <strong>of</strong> harvesting DNA from Map cultured in liquid medium (MGIT).
A lysis buffer and proteinase K, toge<strong>the</strong>r with mechanical cell disruption, was used as cell lysis followed by
ethanol and differential centrifugation for removing inhibiting substances from <strong>the</strong> liquid medium. The method
was evaluated comparing its performance on clinical samples with a commercial DNA extraction kit based on
bead beating, proteinase K, and column purification and a protocol described in <strong>the</strong> literature, also based on
mechanical disruption and phenol chlor<strong>of</strong>orm purification. Moreover, spiked samples in serial dilutions were
used for confirming <strong>the</strong> shortest time to detection (TTD) by <strong>the</strong> MGIT reader for a positive sample. Finally,
analytical sensitivity <strong>of</strong> <strong>the</strong> method was calculated spiking clean MGIT tubes with a manually counted bacterial
concentration <strong>of</strong> approximately 1,000 bacteria/ml <strong>of</strong> medium.
The proposed method could accurately confirm more samples than <strong>the</strong> commercial kit and <strong>the</strong> published
protocol by real-time PCR. In addition, it showed lower Ct values and minimal inhibition. At different concentrations
<strong>of</strong> spiked MGIT medium <strong>the</strong> proposed extraction protocol confirmed a positive signal 12 to 17 days after
culture. The analytical sensitivity was assessed to less than 104 Map/ml medium.
These results indicate that <strong>the</strong> new extraction method combines reliability and efficiency with simplicity.
The lack <strong>of</strong> inhibition and <strong>the</strong> high analytical sensitivity suggest an effective Map DNA harvest and an effective
removal <strong>of</strong> inhibitors from both <strong>the</strong> specimen and <strong>the</strong> MGIT medium components.
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