title: Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  Previously 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, USA. tyler.thacker@ars.usda.gov
authors.forename: Tyler C
authors.initials: TC
authors.lastname: Thacker
authors.forename: Beth
authors.initials: B
authors.lastname: Harris
authors.forename: Mitchell V
authors.initials: MV
authors.lastname: Palmer
authors.forename: Wade R
authors.initials: WR
authors.lastname: Waters
mesh_terms: D000818:Animals; D002417:Cattle; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D009163:Mycobacterium bovis / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014380:Tuberculosis, Bovine / Q000175:diagnosis / Q000382:microbiology*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial