Comparison of four methods for isolation of Mycobacterium avium subsp. paratuberculosis DNA from tissue samples | Primerjava stirih metod osamitve DNA Mycobacterium avium subsp. paratuberculosis iz vzorcev tkiva
2003
Pislak, M., Ljubljana Univ. (Slovenia). Veterinary Fac. | Ocepek, M. | Zabavnik-Piano, J. | Pogacnik, M.
The PCR reaction (polymerase chain reaction) is a rapid and specific method for detection the IS900 (insertion sequence IS900), which is a DNA sequence specific for Mycobacterium avium subsp. paratuberculosis. However, the efficacy of the method is dependent on the qualitative isolation of DNA from tissue samples. In this paper, four methods of isolation of the Mycobacterium avium subsp. paratuberculosis DNA from 30 intestinal and lymph-node samples, which were taken from 11 seropositive small ruminants that originated from paratuberculosis-positive flocks, were compared. The isolation of genomic DNA was performed by: 1. using the commercial kit (High Pure PCR Template Preparation Kit, Roche, Mannheim); 2. treatment the samples with HPC (hexadecylpyridinium chloride) and then performing the isolation using the same commercial kit; 3. the method described by Gwozdz; and 4. treatment of the samples with HPC and then performing the isolation with the method described by Gwozdz. Using the first method of isolation we obtained 22 positive samples, with the second method 24 samples were positive, with the third method 13 samples were positive and with the fourth method 16 samples were positive. The results indicate that the second method, treatment of the samples with HPC and subsequent DNA isolation with the commercial kit (Roche), was the most efficient. This method was also the fastest and the easiest to perform.
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