Kawaji Satoko, Nagata Reiko, Mori Yasuyuki
Bacterial and Parasitic Disease Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-0856, Japan.
J Vet Med Sci. 2014 Jan;76(1):65-72. doi: 10.1292/jvms.13-0366. Epub 2013 Sep 20.
An efficient protocol for the manual fluorescent MGIT culture system combined with rapid confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) growth in the broth culture was established and evaluated for the detection of viable MAP in direct quantitative PCR (QPCR) positive bovine feces. Manually detected fluorescence emissions from MGIT tubes were analyzed objectively using an open source software, ImageJ. For molecular confirmation of MAP growth, DNA samples harvested by simply boiling the broth, an inexpensive and time- and labor-saving DNA preparation method, yielded adequate results. The sheep strain of MAP required longer incubation time relative to the cattle strain, suggesting that the MGIT system may not support well the growth of ovine isolates as described previously. Of 61 direct QPCR positive bovine feces, the recovery rate of MAP in the MGIT system (62.3%) was significantly 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 was several weeks earlier compared to solid media. In MGIT culture positive samples, the time to detect fluorescence was correlated with the DNA quantity detected in fecal QPCR. As a positive 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. For this purpose, the manual MGIT system is a sensitive and rapid culture method at least for bovine samples.
建立了一种高效的手动荧光MGIT培养系统方案，并结合快速确认肉汤培养中副结核分枝杆菌（MAP）的生长情况，用于检测直接定量PCR（QPCR）阳性牛粪便中的活MAP。使用开源软件ImageJ客观分析手动检测的MGIT管荧光发射。为了从分子水平确认MAP的生长，通过简单煮沸肉汤收获DNA样本，这是一种廉价且省时省力的DNA制备方法，结果令人满意。与牛源菌株相比，MAP的羊源菌株需要更长的培养时间，这表明MGIT系统可能如先前所述不能很好地支持绵羊分离株的生长。在61份直接QPCR阳性的牛粪便中，MGIT系统中MAP的回收率（62.3%）显著高于（P<0.05）使用基于7H10琼脂的斜面培养基的回收率（44.3%）。与固体培养基相比，MGIT系统获得粪便培养最终结果的时间要早几周。在MGIT培养阳性样本中，检测到荧光的时间与粪便QPCR中检测到的DNA量相关。由于直接粪便QPCR检测呈阳性结果并不意味着粪便中排出活的MAP，因此可以通过粪便培养进行细菌分离以验证QPCR结果。为此，手动MGIT系统至少对于牛样本而言是一种灵敏且快速的培养方法。
