DNA Extraction Procedures

Detection of MAP can be described as a ‘triple threat' the combination of a complex, chronic disease, an extremely resilient organism and sample matrices that are some of the most difficult to work with in a molecular biology setting.

An overarching principle is that the DNA extraction step is critically important and can significantly impact the detection of MAP in a PCR diagnostic test (Kawaji et al., 2007; Schwalm et al., 2018). The DNA extraction method used must be optimized for the sample type. Various methodologies have been applied, from spin column extraction methods, magnetic bead technologies, to ‘old school' organic sol­vent phase extraction methodologies, immuno- magnetic separation (IMS) (Grant et al., 2002; Khare et al., 2004), peptide-mediated magnetic separation (Stratmann et al., 2002) and hybridi­zation capture techniques (Marsh et al., 2000). Although these show some promise in their ability to approach the sensitivity required to align with MAP culture, they have largely fallen out of favour due to the risk of sample cross­contamination and the need for methods that are less time-consuming, more conducive to a diagnostic setting and cost efficient. The various extraction methods will be considered below in relation to specific sample types.

A factor that is independent of the sample type, however, is the need to achieve sufficient lysis of the MAP bacterium. While DNA can be extracted from many bacterial species using chemical lysis alone, MAP can require the inclu­sion of both a mechanical and a chemical lysis step to obtain complete lysis (Odumeru et al., 2001; Fang et al., 2002; Herthnek and Bolske, 2006; Timms et al., 2015). Mechanical lysis by a bead-beating process, with silica/zirconia beads and a homogenization instrument, is able to lyse MAP and allow release of the DNA. The addition of this single step to extraction protocols has had a major impact on the diagnostic sensitivity of PCR-based mycobacterial detection methods (Amaro et al., 2008).

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