Bovine TB: Research project summary
Project SE3008: Detection and enumeration of Mycobacterium bovis from clinical and environmental samples
Project duration: 5 years 9 months
The project aim was to develop methods for the detection and enumeration of Mycobacterium bovis (M. bovis) in clinical samples, such as cattle tissues, nasal mucus, blood, badger excreta and environmental samples. The development of such assays will allow rapid screening of samples from infected cattle and monitoring of the environment and badger populations for the presence of M. bovis.
The project has developed a number of direct molecular methods for detection, quantification and genotyping of M. bovis from different samples using PCR as a foundation technique. Initial work was performed to find the best method of extracting M. bovis DNA from various sample types prior to the use of PCR. The best DNA extraction method was found to be NucliSens isolation kit (bioMerieux). This extraction method has been coupled with a sensitive PCR screening method for the Mycobacterium tuberculosis (M. tb) complex (IS1081, able to detect 1 genome copy). Also a series of genotyping PCRs for specific deletion events or regions of difference within the M. tb complex were developed which allow M. bovis species confirmation. The sensitivity and specificity of the PCRs was tested against reference strains, spiked samples and on clinical and environmental samples collected in the field. Additional methods for confirmation of species were also developed (to distinguish M. bovis from M. bovis caprae, M. africanum, M. microti etc). The detection limits for all the confirmatory methods was 10 genome copies.
Testing of clinical samples highlighted two shortfalls of the molecular
approach. Firstly the M. tb complex specific PCR is only 70%
as sensitive as the gold standard of culture, when the more specific PCRs
were compared with culture the sensitivity dropped to 50%. Secondly, the
success rate of spoligotyping applied directly to the same extracts was
50% compared with cultures. Improvements were made on these sensitivities
towards the end of the project such that the M. tb complex PCR
sensitivity could be increased to 90%. Such improvements suggest that
this method, with further development and evaluation, could be applied
as a screening method for the rapid identification of positive cases.
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Page last modified:
July 7, 2008