Molecular Detection of Anaplasma spp. in Equine Samples

Genomic DNA was extracted from the whole blood and lung tissue samples using a DNeasy Blood & Tissue Kit (Qiagen, Melbourne, Australia) following the manufacturer’s instructions. The extracted DNA was kept at −20 °C before use. PCR amplification was carried out using an AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Republic of Korea). First, infection with Anaplasma spp. was screened via the amplification of 16S rRNA fragments using nPCR with the primer pairs EE1/EE2 and EE3/EE4 to obtain an expected amplicon of 924–926 bp in length [8 (link),9 (link)]. For species identification, the 16S rRNA genes of A. phagocytophilum and A. bovis were identified by re-amplifying the PCR-positive samples using the primer pairs EE1/EE2 and SSAP2f/SSAP2r and EE1/EE2 and AB1f/AB1r, respectively [9 (link)], to obtain the expected amplicons of 641 and 551 bp in length, respectively. The positive control for each PCR reaction comprised the A. phagocytophilum [11 (link)] and A. bovis [12 (link)] strains that were previously identified in horses from mainland Korea. For each PCR reaction, a sample with distilled water and PCR reagents but no DNA was used as the negative control.
We addressed concerns regarding the amplicon contamination of nPCR using a distinct positive control sequence to ensure the differentiation of true positive results from those caused by possible contamination. The HKG of the 18S rRNA expressed with high stability in horse tissue and cultured cells [19 (link)] was used as an internal positive control. To identify the 18S rRNA HKG, horse DNA samples were amplified using previously published primer sequences to obtain the expected 204 bp long amplicons [19 (link)]. A horse blood sample infected with C. burnetii [43 (link)] was used as the internal negative control using the primer sets EE1/EE2 and SSAP2f/SSAP2r and EE1/EE2 and AB1f/AB1r.
All primers and amplification conditions used in the present study are presented in Supplementary Table S1.