Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium.
OBJECTIVE
To compare three methods for DNA extraction from Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium.


RESULTS
The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS6110 in M. bovis and M. tuberculosis, and of a 1700 bp fragment of FR300 region in M. avium avium.


CONCLUSIONS
Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex.


CONCLUSIONS
The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis.
Amaro
A
A
Laboratório Nacional de Investigação Veterinária, Lisboa, Portugal.
Duarte
E
E
Amado
A
A
Ferronha
H
H
Botelho
A
A
D001431:Bacteriological Techniques / Q000379:methods* / Q000592:standards; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis*; D009162:Mycobacterium avium / Q000235:genetics / Q000302:isolation & purification*; D009163:Mycobacterium bovis / Q000235:genetics / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods
D002593:Cetrimonium Compounds; D004269:DNA, Bacterial; D000077286:Cetrimonium