A rapid method for the extraction and detection of Mycobacterium avium subspecies paratuberculosis from clinical specimens
Challans, J.A.; Stevenson, K.; Reid, H.W.; Sharp, J.M.
Veterinary Record 134(4): 95-96
1994
ISSN/ISBN: 0042-4900 PMID: 8178419 Document Number: 431403
A modified method for the extraction and detection of M. avium subspecies paratuberculosis from lymph nodes of red deer is described. After extraction the DNA was amplified using the polymerase chain reaction (PCR) and the amplified product was detected using gel electrophoresis, and two hybridization procedures one using non-isotopic (digoxigenin-labelled) and the other radiolabelled ([ alpha -32P]deoxycytidine triphosphate-labelled) DNA. The range of sensitivities for the 3 detection methods, calculated by repeating the experiments 6 times, were 35 to 45 mycobacteria/g of sample, 18 to 22 mycobacteria/g sample and 5 to 10 mycobacteria/g of sample, respectively. Preliminary results from a survey for mycobacterial infections in culled free-living deer using the method revealed that 48 of 50 lymph nodes for which mycobacteria were cultured were also positive by PCR. In addition 34 samples which were culture-negative were found to be positive by PCR. Inconsistent results were found when the technique was applied to faeces, owing to the presence of inhibitors. Further work is in progress to overcome this problem and to facilitate the introduction of the PCR for diagnosis of Johne's disease.