In total, 5,644 larval, nymphal, and adult ticks from 408 pools were collected from livestock (cattle and horse) and wild animals (elk, roe deer, raccoon, badger, wild boar, and wild rabbit) from two provinces, Gangwon and Jeju, in Korea between August and November 2019. The 235 pools collected from Gangwon Province were designated as 19M1–19M235, and the 173 samples collected from Jeju Province as 19T1–19T173. Samples were then stored at -20°C for the detection of C. burnetii.
Identification of tick species
The tick species were identified using a stereo microscope, Discovery.V8 (ZEISS, Oberkochen, Germany). The morphological identification was based on a previously established method .
Extraction of total nucleic acid
Total nucleic acids were extracted from tick samples using the Maxwell® RSC Viral Total Nucleic Acid Purification Kit using Maxwell® Instruments (Promega, Madison, WI, USA). One adult tick, 10 nymphs, or 50 larvae were homogenised in a tissue homogeniser using steel beads of diameter 2.381 mm (SNC, Hanam, Korea). The sample was lysed with 330 µl of lysis buffer in a Precellys 24 Tissue Homogeniser (Bertin Instruments, Montigny-le-Bretonneux, France). The homogenate was incubated at 56°C for 10 min and the following steps were performed using Maxwell® RSC Instrument, according to the manufacturer’s instructions. Finally, 50 µl of total nucleic acid was obtained from each sample.
Primers and standard DNA of C. burnetii
C. burnetii was detected by targeting a 295-bp long DNA fragment in the transposase of the insertion sequence (IS) element IS1111a, using the primers Cox-F (5′- GTCTTAAGGTGGGCTGCGTG-3′), and Cox-R (5′-CCCCGAATCTCATTGATCAGC-3′); and probe Cox-TM (FAM-AGCGAACCATTGGTATCGGACGTT-TAMRA) . DNA from the C. burnetii strain 493 (Nine Mile Phase I), preserved in our laboratory, was used as the positive control. The PCR product was cloned in the pGEM®-T vector system (Promega, Madison, WI, USA).
UR-qPCR was performed using a GENECHECKER® UF-300 PCR (Genesystem Co., Ltd., Daejeon, Korea) and 2× Rapi: Spec™ Probe Master mix (Cat. No. 9799100500; Genesystem Co., Ltd.). The reaction mix (10 µl) consisted of 0.4 µl (20 pmol) of each primer, 0.4 µl (2 pmol) of probe, 0.8 µl ddH2O, 5 µl PCR premix, and 3 µl of sample total nucleic acid. PCR conditions were set as follows: 95 °C for 30 s, 50 cycles of 95 °C for 5 s, and 60 °C for 10 s. Detection of C. burnetii was carried out in two steps; screening and detection. C. burnetii was screened from a pool of five samples, prepared by combining 10 µl of total nucleic acid from each sample. The individual samples from the PCR-positive pooled samples were then reanalysed to identify the exact sample that carried the pathogen.
The performance of the UR-qPCR system was compared to that of CFX96 Touch Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), by evaluating the amplification for each of the 408 tick pool nucleic acids using the same primers and probe. The 20 µl reaction mixture consisted of 1 µl (10 pmol) of each primer, 1 µl (5 pmol) of probe, 10 µL of iQTM Supermix as PCR premix (Bio-Rad), 3 µl of DNA template, and 4 µl of ddH2O. PCR conditions were set as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s.
Sensitivity and specificity of C. burnetii UR-qPCR
To check the sensitivity of UR-qPCR, serial dilutions of C. burnetii recombinant DNA template, from 2.8×108 to 2.8×100 copies, were used for PCR in triplicate to identify the minimum copy number at which the amplification was stable.
To evaluate the specificity of UR-qPCR for C. burnetii, DNA from five other tick-borne pathogens (Anaplasma phagocytophilum, Ehrlichia chaffeensis, Ehrlichia canis, Toxoplasma gondii, Borrelia burgdorferi) were tested with C. burnetii-specific primers and probe under the same PCR conditions.
Sequencing and phylogenetic analysis
The accuracy of UR-qPCR for C. burnetii detection was confirmed through sequencing of the PCR products using Cox-F/R primers. Phylogenetic analysis of the C. burnetii sequence was performed using a 687 bp DNA fragment of the IS1111 transposon gene, which was amplified using the primer pair Trans 1 (5′-TATGTATCCACCGTAGCCAGTC-3′)/Trans 2 (5′-CCCAACAACACCTCCTTATTC-3′) [27, 28]. The sequences were compared to the available C. burnetii sequences in NCBI using the Basic Local Alignment Search Tool (BLAST). Consensus sequences were aligned using the Clustal X2 program , overhanging ends were trimmed using BioEdit 7.2 , and a maximum likelihood phylogenetic tree was created using MEGA7 , bootstrapping 1000 times.