Department of Electrical and Computer Engineering, University of Alberta, Edmonton, AB, Canada.
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada.
J Microbiol Methods. 2022 Jun;197:106490. doi: 10.1016/j.mimet.2022.106490. Epub 2022 May 18.
Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 10 CFU/mL, 1.68 × 10 CFU/mL and 1.68 × 10 CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 10 CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 10 CFU/mL from feces and no less than 10 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 10 and 2 × 10 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease.
未涂层甲苯磺酰基活化磁珠用于从粪便、牛奶和尿液中捕获耻垢分枝杆菌和禽分枝杆菌副结核亚种（MAP）。离心和未涂层磁珠分别回收了 1.68×10 CFU/mL、1.68×10 CFU/mL 和 1.68×10 CFU/mL 悬浮在磷酸盐缓冲液中的耻垢分枝杆菌细胞的 99%以上和 93%以上。与离心相比，磁珠更有效地浓缩 1.68×10 CFU/mL 的耻垢分枝杆菌细胞。同样，使用 F57-qPCR 检测从磁珠或离心回收的 MAP 细胞时，检测到的细胞存在差异；用磁珠回收的 MAP 细胞的循环阈值（Ct）更低（p<0.05），表明回收率更高。通过检测 F57 和 IS900 序列，证明了从牛奶、尿液和粪便标本中分离 MAP 细胞的磁分离。从粪便中捕获的磁珠不少于 10 CFU/mL，从牛奶和尿液悬浮液中捕获的磁珠不少于 10 CFU/mL。在另一种检测策略中，分枝杆菌噬菌体 D29 感染偶联到磁珠上的耻垢分枝杆菌。在含有 2×10 和 2×10 CFU/mL 耻垢分枝杆菌的尿液样本中孵育 24 小时后观察到噬菌斑形成单位。这项研究的结果为结核病和约翰氏病的诊断提供了一种有前途的工具。
