Molecular Detection of Coxiella burnetii in Blood

Detection of C. burnetii in blood by end-point PCR was performed using primers and procedures that were modified from previous reports.^{[9 (link)]} The gene target was derived from the transposase gene insertion element IS1111a of C. burnetii isolate LBCE 13265 (NCBI Nr. KT 965031.1). The forward (5’-CGG GTT AAG CGT GCT CAG TAT GTA-3’) and reverse (5’-TGC CAC CGC TTT TAA TTC CTC CTC-3’) primers were synthesized at around 24 bp. The end-point PCR process consisted of an initial denaturation step at 95°C for 15 minutes; 45 cycles of 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 30 seconds; and a final elongation step at 72°C for 7 minutes. Amplification of 5 μL of DNA was performed in a total volume of 25 μL containing 10X PCR buffer (Qiagen), 2.5 mM MgCl₂, 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase (Qiagen). Gel electrophoresis was used to separate PCR products on a 2% agarose gel containing ethidium bromide, and visualized using a GelDoc System (Clinx Science Instruments, Shanghai, China).