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Title:   Author  item
                                            Thacker, Tyleritem
                                            HARRIS, BETH - Animal And Plant Health Inspection Service (APHIS)item
                                            Palmer, Mitchellitem
                                            Waters, Wade
  Submitted to: BioMed Central (BMC) Veterinary Research
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/17/2011
                                    Publication  Date: 8/25/2011
                                    Citation: Thacker, T.C., Harris, B., Palmer, M.V., Waters, W.R. 2011. Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR. BioMed Central (BMC) Veterinary Research [serial online]. 7(50). Available: http://www.biomedcentral.com/1746-6148/7/50.Interpretive Summary: Culturing bacteria from diagnostic samples is the primary test for bovine tuberculosis in the US. Culture can take up to 3 months. A rapid simple PCR based test may provide a quick method of detecting Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis, taking days rather than months to complete. One of the most significant impediments to this approach is the presence of mycobacteria other than M. bovis that may interfere with the accuracy of the test. To overcome this problem we developed a new real-time PCR assay, IS6110T. IS6110T was tested against 11 mycobacterial species that are commonly cultured from diagnostic samples. IS6110T was significantly more specific than previously published assays. Diagnostic samples were obtained from 30 M. bovis infected and 18 non-M. bovis infected animals. IS6110T in this small sample had a specificity of 100% and a sensitivity of 66.7%.
                                Technical Abstract: Background: Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the US. Detection of M. bovis by PCR in tissue homogenates may provide a simple, rapid method to complement diagnostic 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 environment mycobacteria commonly isolated from diagnostic tissues submitted to the National Veterinary Service Laboratory. A real-time PCR assay was developed (IS6110_T) that had increased specificity over other IS6110 based assays. 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 had a specificity of 100% and a sensitivity of 66.7%.
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 US and compliments current testing strategies.


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