Table 1 Primers, target genes and PCR conditions used in this study
Pathogen | Primer (5’-3’) | Target gene | Product size (bp) | PCR protocol | Reference |
|---|---|---|---|---|---|
Babesia spp./Hepatozoon spp. | Piroplasmid-F: CCAGCAGCCGCGGTAATTC | 18S rRNA | 350–400 | 95 °C for 10 min initial denaturation, followed by 35 cycles of 95 °C for 30 s, 64 °C for 20 s, 72 °C for 20 s, then 72 °C for 7 min for the final elongation | [38] |
Piroplasmid-R: CTTTCGCAGTAGTTYGTCTTTAACAAATCT | |||||
Ehrlichia spp./ Anaplasma spp. | EHR16SD: GGTACCYACAGAAGAAGTCC | 16S rRNA | 345 | 95 °C for 10 min initial denaturation, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, then 72 °C for 10 min for the final elongation | [39] |
EHR16SR: TAGCACTCATCGTTTACA GC | |||||
Coxiella burnetii | Trans-1: TATGTATCCACCGTAGCCAGT | IS1111a | 687 | 95 °C for 10 min initial denaturation, followed by 35 cycles of 95 °C for 30 s, 64 °C for 60 s, 72 °C for 60 s, then 72 °C for 7 min for the final elongation | [40] |
Trans-2: CCCAACAACACCTCCTTATTC | |||||
Bartonella spp. | ssrA-F: GCTATGGTAATAAATGGACAATGAAATAA | ssrA | 301 | 95 °C for 2 min initial denaturation, followed by 45 cycles of 95 °C for 15 s, 60 °C for 60 s | [41] |
ssrA-R: GCTTCTGTTGCCAGGTG | |||||
Probe: FAM-ACCCCGCTTAAACCTGCGACG | |||||
Rickettsia spp. | CS-78F: GCAAGTATCGGTGAGGATGTAAT | gltA | 401 | 95 °C for 10 min initial denaturation, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 40 s, then 72 °C for 7 min for the final elongation | [42] |
CS-323R: GCTTCCTTAAAATTCAATAAATCAGGAT | |||||
Spotted fever group rickettsiae | Rr190.70F: ATGGCGAATATTTCTCCAAAA | ompA | 632 | 95 °C for 10 min initial denaturation, followed by 35 cycles of 94 °C for 40 s, 58 °C for 30 s, 72 °C for 45 s, then 72 °C for 10 min for the final elongation | [43] |
Rr190.701R: GTTCCGTTAATGGCAGCATCT |