Comparison of three methods for extraction of Mycobacterium avium subspecies paratuberculosis DNA for polymerase chain reaction from broth-based culture systems.
Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subspecies paratuberculosis (MAP) extracted with 3 different methods (MagMAX, DNeasy(R), and phenol-chloroform) after growth in a broth-based culture system. Of the 304 samples tested, bacterial DNA was detected in 197 (65%) of samples after MagMAX, 156 (51%) after phenol-chloroform, and 123 (40%) after DNeasy extractions. By acid-fast stain, 177 (58%) of the samples yielded acid-fast-positive bacilli, of which 4 were PCR negative by the 3 extraction methods. The results demonstrated that the amplifiable MAP DNA, as evidenced by the number of PCR-positive cultures and amplicon intensity on ethidium bromide-stained agarose gel, was best for MagMAX, intermediate for phenol-chloroform, and least for DNeasy. When subjected to real-time polymerase chain reaction, the MagMAX extracts produced the best results, thereby making it an excellent kit for the efficient extraction of MAP DNA from the broth-based culture system.
Okwumabua
Ogi
O
University of Wisconsin, WVDL, 445 Easterday Lane, Madison, WI 53706, USA. ogi.okwumabua@wvdl.wisc.edu
Shull
Eileen
E
O'Connor
Mike
M
Moua
Tou Vue
TV
Danz
Tonya
T
Strelow
Kathy
K
D000818:Animals; D001431:Bacteriological Techniques; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000302:isolation & purification; D010283:Paratuberculosis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial