Small mammal and chigger DNA extraction and PCR

DNA was extracted from individual chiggers, pools of chiggers (~20-50 unidentified individuals of possible multispecies from the same host animal) and rodent tissues using the Qiagen Blood and Tissue Kit (Qiagen, USA). Chiggers were rinsed with distilled water and individuals cut through the mid-gut using a sterile 30G needle under a dissecting microscope. Pools were crushed using a sterile polypropylene motorized pestle (motorized pellet pestle {"type":"entrez-nucleotide","attrs":{"text":"Z35991","term_id":"536403","term_text":"Z35991"}}Z35991, Sigma Aldrich, St Louis, MO). Rodent tissues were cut into a small piece (≤10mg of spleen or ≤25mg of liver or lung). Samples were incubated with proteinase K at 56°C for 3 hours. The rest of the steps followed the manufacturer’s protocol. Chigger samples were eluted in 45μl and rodent samples in 100μl of buffer AE (Qiagen, Hilden, Germany). Samples were stored at -20°C before PCR. Real-time PCR targeting the 47kDa O. tsutsugamushi outer-membrane protein was performed on all samples as previously described[24], except for chigger samples where 5 μl of DNA template was added. PCR was run on a Bio Rad CFX96 (Bio Rad, USA).