In addition, M. bovis cells are likely to be in an altered physiological state once outside the mammalian host, as pathogens can enter a resilient, but quiescent state, in order to survive the biotic and abiotic stresses of the environment, as demonstrated for Vibrio cholerae. Approaches such as immunomagnetic capture circumvent the need for cultivation but are currently neither reliable nor suited to high throughput sample screening. We have recently developed a real-time PCR assay for bTB that could be an ideal screening surveillance tool of use for improving farm biosecurity. The reliability of such a test however depends on efficient extraction of M. bovis DNA from GI 254023X environmental samples. DNA extraction from soils can be hindered by the presence of humic and fulvic acids, which have similar physico-chemical properties to DNA making the two difficult to separate. Faeces contain biliary salts, urea, haemoglobin and heparin in addition to other compounds, depending on the diet of the animal, which can affect DNA amplification by PCR. The waxy cell wall of mycobacteria, and the possibility of spore formation under conditions of stress may further hinder lysis and DNA recovery. Published DNA extraction protocols for soils address PCR inhibition to varying extents by including refinement steps such as column chromatography or chemical flocculation, however these methods are laborious, time consuming, expensive and therefore inappropriate for high throughput processing. Here we report a blinded multi-operator randomised trial to evaluate four commercial DNA extraction kits and one previously published manual KRM-III method for their comparative ability to recover and detect M. bovis target DNA in soil and faecal samples. The test kits were UltracleanTM, PowersoilTM, QIAamp Stool mini kit, and FastDNAH Spin Kit; the manual method was adapted from the one published by Griffiths. The specific aims were: to measure the analytical sensitivity and the extraction efficiency of these methods in extracting known quantities of M. bovis DNA from spiked substrates, to determine the reproducibility of each method by replication with multiple operators; to quantify the loss of sensitivity that may be due to carry over of contaminants using a novel inhibition control PCR assay, and to analyse cost benefits ratio and  hands-on time for each method.