Department of Chemistry and Research Institute of Physics and Chemistry, Chonbuk National University, Jeonju, Republic of Korea.
J Sep Sci. 2010 Apr;33(8):1109-14. doi: 10.1002/jssc.200900766.
We report a diagnostic method for Anaplasma phagocytophilum (A. phagocytophilum) infection in cattle using a nested PCR and microchip electrophoresis (ME). A. phagocytophilum causes human granulocytic anaplasmosis and granulocytic ehrlichiosis, which are emerging tick-borne zoonotic diseases. Nested PCR was used to amplify genomic DNA samples extracted from cattle blood. The amplified PCR products were analyzed under a sieving gel matrix of 0.7% poly(ethyleneoxide) (M(r)=8,000,000) in a conventional glass microchip. In the ME assay, A. phagocytophilum was analyzed within 35 s with a relative standard deviation of 1.30% (n=5) using a programmed field strength gradient (PFSG) as follows: 615.3 V/cm for 0-24 s, 66.7 V/cm for 24-34 s, 615.3 V/cm for 34-100 s. The ME-PFSG assay was clinically validated by comparing the 16S rRNA gene levels obtained by this method with those measured using conventional slab gel electrophoresis performed with ten cattle blood samples suspected of A. phagocytophilum infection. In contrast to slab gel electrophoresis, the proposed ME-PFSG methodology had increased sensitivity (200-450 pg/microL), a faster analysis time (<35 s), and required a smaller sample volume (approximately 162 fL).
我们报告了一种使用巢式 PCR 和微芯片电泳 (ME) 诊断牛类无形体感染的方法。无形体引起人类粒细胞无形体病和粒细胞埃立克体病，这是两种新兴的蜱传动物源性传染病。巢式 PCR 用于扩增从牛血中提取的基因组 DNA 样本。扩增的 PCR 产物在常规玻璃微芯片中的 0.7%聚氧化乙烯（M(r)=8,000,000）筛分凝胶基质中进行分析。在 ME 分析中，使用编程场强梯度 (PFSG) 在 35 秒内分析无形体，相对标准偏差为 1.30%（n=5），PFSG 程序如下：0-24 秒为 615.3 V/cm，24-34 秒为 66.7 V/cm，34-100 秒为 615.3 V/cm。通过将该方法获得的 16S rRNA 基因水平与使用疑似无形体感染的十份牛血样本进行的常规平板凝胶电泳测量的水平进行比较，对 ME-PFSG 检测进行了临床验证。与平板凝胶电泳相比，建议的 ME-PFSG 方法具有更高的灵敏度（200-450 pg/microL）、更快的分析时间（<35 秒）和更小的样本量（约 162 fL）。
