Growth rate retardation and inhibitory effect of para-JEM(R) BLUE on Mycobacterium avium subspecies paratuberculosis.

The effect of para-JEM(R) BLUE on Mycobacterium avium subspecies paratuberculosis (MAP) inoculated into broth-based culture media was evaluated by using 84 fecal samples with known MAP status. Results showed that growth of the organism in samples inoculated into the broth without the para-JEM BLUE was detectable 1-35 days (average of 6 days) earlier in 35 of the samples (42%) compared with the same samples inoculated in broth with para-JEM BLUE. Four additional samples (5%) that were MAP positive in the culture broth that lacked the para-JEM BLUE gave negative results when the reagent was included. Of the remaining 45 samples, growth of MAP was detected 1-4 days (average of 3 days) earlier in 4 of the samples (5%) inoculated in the broth with para-JEM BLUE compared with the same samples inoculated in the broth without the para-JEM BLUE, whereas 41 samples (49%) yielded equivalent results with respect to time-to-growth detection and negative growth, regardless of whether para-JEM BLUE was present in the culture broth. However, exclusion of para-JEM BLUE from the broth increased the number of samples that produced false-positive instrument signals compared with the number that produced false-positive signals when the reagent was added. Modification of the sample processing step had no measurable effect. Observations indicated that, although elimination of para-JEM BLUE from the broth increased false-positive instrument signals, its inclusion has an adverse effect on the growth of certain MAP, which suggests that its elimination from broth cultures may increase sensitivity.

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.