Detection and Confirmation of Mycobacteriumavium subsp. paratuberculosis in DirectQuantitative PCR Positive Fecal Samples by the Manual Fluorescent MGIT CultureSystem

[摘要] References(23)Cited-By(1)An efficient protocol for the manual fluorescent MGIT culture system combinedwith rapid confirmation of Mycobacterium avium subsp.paratuberculosis (MAP) growth in the broth culture was established andevaluated for the detection of viable MAP in direct quantitative PCR (QPCR) positivebovine feces. Manually detected fluorescence emissions from MGIT tubes were analyzedobjectively using an open source software, ImageJ. For molecular confirmation of MAPgrowth, DNA samples harvested by simply boiling the broth, an inexpensive and time- andlabor-saving DNA preparation method, yielded adequate results. The sheep strain of MAPrequired longer incubation time relative to the cattle strain, suggesting that the MGITsystem may not support well the growth of ovine isolates as described previously. Of 61direct QPCR positive bovine feces, the recovery rate of MAP in the MGIT system (62.3%) wassignificantly higher (P<0.05) than that using 7H10 agar-based slants(44.3%). The time to obtain a final result for fecal culture by the MGIT system wasseveral weeks earlier compared to solid media. In MGIT culture positive samples, the timeto detect fluorescence was correlated with the DNA quantity detected in fecal QPCR. As apositive result in the direct fecal QPCR test does not mean fecal excretion of viable MAP,bacterial isolation by fecal culture could be conducted to verify the QPCR result. Forthis purpose, the manual MGIT system is a sensitive and rapid culture method at least forbovine samples.

[学科分类] 兽医学

[关键词] fecal culture;ImageJ;MGIT;paratuberculosis;real-time quantitative PCR