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Detection and enumeration of Mycobacterium bovis from clinical and enviromental samples - SE3008

The Krebs Review has recommended the development of a Mycobacterium bovis-specific PCR-based assay for the detection of bacteria in badger carcasses, excreta and environmental samples. The development of such an assay would allow rapid screening of samples from badger carcasses and monitoring of the environment and badger populations for the presence of M. bovis.

The objective of this proposal is to develop PCR-based methods which allow detection and fingerprinting of M. Bovis from badger carcasses, clinical samples and excreta. Extraction and enrichment techniques will be developed for the purification of M. bovis DNA from contaminating materials present in the clinical samples which inhibit the PCR assay. A two-stage approach to M. bovis detection will be applied to these enriched samples. In the first stage we will apply an M. bovis specific PCR assay to the samples as a low-cost screening asssay. For samples that are PCR-positive for M. bovis, molecular typing will be performed by spoligotyping and fluorescent VNTR (Variable Number of Tandem Repeat) analysis. In addition, we propose to develop the PhaB (phage amplified biologically) assay to determine the levels of M. bovis excreted from sputum, urine and faeces of M. bovis-infected animals in order to discriminate between infected and infectious animals.
01 VLA IC 31/08/99 Design and optimise an M. bovis-specific PCR for the detection of M. bovis DNA. Design and optimise a PCR assay for the detection of badger DNA.

02 VLA IC 30/06/00 Develop an economical and reproducible method for recovery of M. bovis DNA from visible lesion tissue, pooled lymph nodes, tracheal washings, faecel samples, blood urine and latrine samples.

03 VLA IC 30/09/00 Assess the success of PCR amplification and apply DNA fingerprinting by spoligotyping and Fluorescent VNTR to PCR-positive samples.

04 VLA IC 31/03/01 Validate methodologies on field samples from Woodchester Park and culling experiment (if in progress).

05 VLA PHLS 31/03/01 Develop and validate PhaB assay for enumeration of M. bovis in clinical and environmental samples.
Project Documents
• Final Report : Detection and enumeration of Mycobacterium bovis from clinical and environmental samples   (2002k)
Time-Scale and Cost
From: 1999

To: 2004

Cost: £548,808
Contractor / Funded Organisations
Veterinary Laboratories Agency
Animal Health              
Bovine Tuberculosis              
Plants and Animals              
Fields of Study
Animal Health