Comparison of Three Methods for Extraction of Mycobacterium Avium Subspecies Paratuberculosis DNA for Polymerase Chain Reaction from Broth-Based Culture Systems

Abstract

Abstract. 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 TM , DNeasyH, 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 phenolchloroform, 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.

Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

“…It can be used for confirmation and characterization of strain type and other phenotypic properties (e.g., antibiotic resistance), in the mycobacterial field particularly but also more broadly. Magnetic bead methods have been used for the isolation of bacterial and viral nucleic acids from various substrates (13,14,(24)(25)(26)(27). Okwumabua et al (13) provide proof that a magnetic bead DNA isolation method can be used to isolate MAP DNA in broth media.…”

“…For PCR-based methods, the type of organism and the media components can significantly affect the success of DNA isolation and the presence of PCR inhibitors in the extract. Some simple cleanup procedures may not be applicable for all cultures, as identified in MAP cultures containing egg yolk (7,13). One method for the differentiation of nontuberculous mycobacteria from M. tuberculosis in clinical cultures involves protein extraction followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (23).…”

“…A mechanical lysis step such as bead beating has been found to enhance the recovery of DNA from mycobacteria in various matrices (9)(10)(11)(12). Recently, a magnetic-bead-based DNA extraction technique was successfully applied to egg yolk-containing broth cultures to overcome issues of PCR inhibition (13).…”

Growth Rate Retardation and Inhibitory Effect of para-JEM® BLUE on Mycobacterium Avium Subspecies Paratuberculosis

“…b Thereafter, acid-fast staining is performed on all cultures, and the identity of MAP organisms is confirmed by the polymerase chain reaction (PCR) method by using specific primers. 3 and discussed in 2003 at the annual meeting of the American Association of Veterinary Laboratory Diagnosticians in San Diego, California, that addition of the para-JEM BLUE to the culture broth slowed down the growth of certain isolates of MAP up to 1 week compared with the broth lacking the culture reagent. Based on that observation, the Wisconsin Veterinary Diagnostic Laboratory (WVDL; Madison, Wisconsin) made a choice to extend the recommended 42 days incubation time to 49 days to compensate for this negative effect.…”

“…All the signal-positive samples were acidfast stained, and all the acid-fast-positive samples were confirmed by PCR by using MAP-specific primers. 3 All signal-negative samples were acid-fast stained after 49 days of incubation, followed by the MAP-specific PCR assay, regardless of acid-fast results. The median difference in days-to-positive between MAP-positive cultures incubated in broth with and without para-JEM BLUE were compared by using the Wilcoxon signed rank test.…”