Microchip electrophoretic separation for the fast diagnosis of Anaplasma phagocytophilum infection in cattle

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 24-34s, 615.3 V/cm for 34-100s. 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/mu L), a faster analysis time (<35 s), and required a smaller sample volume (similar to 162 fL).