Comparison of four rapid DNA extraction techniques for conventional polymerase chain reaction testing of three Mycobacterium spp. that affect birds.
Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.
Tell
Lisa A
LA
School of Veterinary Medicine, Department of Medicine and Epidemiology, Tupper Hall, Room 2108, University of California, Davis, CA 95616, USA.
Foley
Janet
J
Needham
Martha L
ML
Walker
Richard L
RL
D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods / Q000662:veterinary; D002645:Chickens; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D009164:Mycobacterium Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D009162:Mycobacterium avium; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology
D004269:DNA, Bacterial