Abstract
BackgroundCulture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA. Detection of M. bovis by PCR in tissue homogenates may provide a simple rapid method to complement bacterial culture. A significant impediment to PCR based assays on tissue homogenates is specificity since mycobacteria other than M. bovis may be associated with the tissues.ResultsPreviously published IS6110 based PCR diagnostic assays, along with one developed in house, were tested against environmental mycobacteria commonly isolated from diagnostic tissues submitted to the National Veterinary Services Laboratory. A real-time PCR assay was developed (IS6110_T) that had increased specificity over other IS6110 based assays. Of the 13 non-tuberculous mycobacteria tested with IS6110_T only M. wolinskyi was positive. Thirty M. bovis infected tissue homogenates and 18 control tissues were used to evaluate the potential for the assay as a diagnostic test. In this small sample, IS6110_T detected 20/30 samples from M. bovis infected animals and 0/18 control tissues.ConclusionsThe IS6110_T assay provides a PCR based assay system that is compatible with current diagnostic protocols for the detection of M. bovis in the USA and compliments current testing strategies.
Highlights
Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA
These non-M. bovis mycobacteria may interfere with PCR diagnostic tests performed on these tissues if the test does not have sufficient specificity
Specificity of PCR primers The following mycobacteria are routinely isolated from tissues submitted for M. bovis diagnostic culture at the National Veterinary Services Laboratories: M. smegmatis, M. terrae, M. goodii, M. fortuitum, M. kansasii, M. wolinskyi, M. simiae, M. peregrinum, M. intracellulare, M. chelonae, M. avium subsp avium, and M.avium subsp paratuberculosis
Summary
Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA. Detection of M. bovis by PCR in tissue homogenates may provide a simple rapid method to complement bacterial culture. A significant impediment to PCR based assays on tissue homogenates is specificity since mycobacteria other than M. bovis may be associated with the tissues. Detecting the presence of M. bovis DNA in tissues prepared for culture may provide a simple, rapid diagnostic method. Mycobacteria other than M. bovis are routinely isolated from tissues submitted for diagnostic culture. These non-M. bovis mycobacteria may interfere with PCR diagnostic tests performed on these tissues if the test does not have sufficient specificity. Most PCR primer pairs published to date were designed for identification of mycobacteria that have been isolated by culture
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