A novel tube-in-tube filter system for refinement of DNA preparation for detection of Mycobacterium using Real-Time PCR

Reinhard Sting, Lisa Schneider-Bühl, Chemisches und Veterinäruntersuchungsamt (CVUA) Stuttgart

Eduard Woizenko, HAMILTON Bonaduz AG

A novel tube-in-tube filter system, the AutoLys M 1.0 tube with a 1 µm pore size non-DNA binding and RNase and DNase free filter designed and produced by HAMILTON Bonaduz AG could be successfully integrated in the procedure of DNA extraction from fecal samples for testing on Mycobacterium avium subsp. paratuberculosis (MAP) in Real-Time PCR. The results obtained indicate an increase in sensitivity of MAP detection in feces. The implementation of this filter tube system means a decisive step towards standardization and high-throughput detection of MAP in fecal samples as an alternative to time and labor intensive culture.

In the diagnosis of Johne's disease, isolation of the pathogen agent Mycobacterium avium subsp. paratuberculosis (MAP) by cultivation has been increasingly superseded by direct detection of MAP using Real-Time PCR (Leite et al., 2013; Okwumabua et al., 2010; Park et al., 2014; Selim and Gaede, 2012). The reasons for this development include the long lasting cultivation of MAP and also improved methods of DNA preparation and PCR protocols for sensitive and specific pathogen detection (EFSA, 2004; Logar et al., 2012).

However, sensitive detection of MAP in feces using PCR is still cumbersome and challenging due to inhibitors, low pathogen numbers, clustering of the MAP bacteria in feces and high stability of MAP cells hampering DNA extraction (Bull et al., 2003; EFSA, 2004; Plain et al., 2014; Wilson, 1997). A recently published study (Sting et al., 2014) describes the reduction of inhibitors and concentration of the MAP bacteria by the integration of a filtration step in the extraction procedure of MAP DNA from fecal suspensions leading to increased detection rates.

The aim of the present study is to facilitate, standardize and refine this filtration procedure by using a novel filter tube system as a decisive step in the extraction of DNA from fecal samples for testing in MAP PCR.

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