Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp paratuberculosis Cells
Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp paratuberculosis Cells
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DOI：
10.1128/aem.01432-10
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发表时间：
2010-11-01
影响因子：
4.4
通讯作者：
Grant, Irene R.
中科院分区：
文献类型：
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作者：
Foddai, Antonio;Elliott, Christopher T.;Grant, Irene R.
为了引入鸟分枝杆菌亚种的特异性。在噬菌体扩增测定之前，根据M的捕获效率（以百分比表示）评估涉及抗体或肽的各种磁性分离方法。鸟亚种副结核分枝杆菌细胞和其他分枝杆菌属的非特异性结合的百分比。将涂覆有化学合成的M.鸟亚种副结核病特异性肽生物素化aMp 3和生物素化aMptD（即，肽介导的磁性分离[PMS]）被证明是实现85 - 100%捕获M的最佳磁性分离方法。鸟亚种副结核分枝杆菌和其他分枝杆菌的非特异性回收率极低（< 1%）。（特别是如果珠在使用前用1%脱脂乳封闭）从含有10（3）至10（4）CFU/ml的肉汤样品中分离。当PMS与最近优化的噬菌体扩增法耦合并用于检测M。鸟亚种对于50 ml体积的加标牛奶中的副结核菌素，平均50%检测限（LOD 50）为14.4 PFU/50 ml牛奶（相当于0.3 PFU/ml）。这种PMS-噬菌体检测代表了一种新的，快速的方法，用于检测和计数活M。鸟亚种牛奶中的副结核杆菌微生物，以及可能的其他样品基质，48小时内可获得结果。
In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (< 1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 10(3) to 10(4) CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD50) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h.