Molecular Identification of Anaplasma spp. Using RFLP

A. phagocytophilum and A. bovis were identified by digesting the 16S rRNA nPCR products of 868–870 bp in length (the PCR amplicon of 924–926 bp without primer sequences) using two restriction enzymes for the RFLP assay [9 (link)]. The restriction enzymes AleI and BtgZI were used for the RFLP assay conducted using the CLC Main Workbench 6.7.2 (CLC Bio, Qiagen, Aarhus, Denmark). The solution subjected restriction digestion comprised 10 μL of PCR product, 5 μL of buffer (10×, 1 μL of AleI (10,000 U/mL; New England Biolabs, Hitchin, UK) or 2 μL of BtgZI (5000 U/mL; New England Biolabs), and distilled water to obtain a final volume of 50 μL. For BtgZI or AleI, the reactions were incubated for 1 h at 60 °C or 30 °C, respectively. The restricted fragments were separated through electrophoresis on a 3% agarose gel in TAE solution at 100 V for 60 min. The gel was then stained with ethidium bromide and subjected to UV visualization.