A Simple, Rapid, and Effective Method for the Extraction of Mycobacterium Paratuberculosis DNA from Fecal Samples for Polymerase Chain Reaction

Abstract

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8–16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories ( n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.