title: A SimpleProbe(®) real-time PCR assay for differentiating the cytochrome b M121I mutation in clinical specimens from dogs infected with Babesia gibsoni.
abstract: Babesia gibsoni (B. gibsoni) causes a canine tick-borne disease worldwide. The substitution of methionine with isoleucine (M121I) in the cytochrome b (CYTb) gene of B. gibsoni was identified as being associated with atovaquone resistance. Rapid identification of the drug-resistant strain is required to select a more effective combination of drugs, e.g., from atovaquone and azithromycin (AA) to clindamycin, diminazene, and imidocarb (CDI) combination. A SimpleProbe(®) real-time PCR assay was designed to detect the single nucleotide polymorphism at nucleotide 363 in CYTb gene of B. gibsoni and the sensitivity and specificity were evaluated by comparing the results from the conventional DNA sequencing method. Eighty-nine clinical blood samples were collected and analyzed in parallel with the SimpleProbe(®) assay and DNA sequencing. The assay identified 50 of 54 nt363G samples and had a sensitivity of 92.6% and a specificity of 100%. Thirty nt363T samples were correctly identified, as well, with a sensitivity of 100% and a specificity of 73.2%. However, this assay identified only one of 17 nt363A samples; the other 16 samples were misidentified as nt363T. The sensitivity of the nt363A identification was only 5.9%, and the specificity was 100%. When detecting the M121I mutation, 42 of 42 mutant samples were identified, with a sensitivity of 100%, and 45 of 47 wild type samples were identified, with a specificity of 95.7%. In conclusion, the SimpleProbe(®) assay could be used to detect the M121I mutation of the B. gibsoni CYTb from clinical specimens. This assay provides a reliable and sensitive tool for differentiating between the atovaquone-resistant strain and the non-resistant strain.
authors.affiliation: Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Pin-Chen
authors.initials: PC
authors.lastname: Liu
authors.affiliation: Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
authors.forename: Ya-Ling
authors.initials: YL
authors.lastname: Lin
authors.affiliation: Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan; Animal Disease Diagnostic Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
authors.forename: Chao-Nan
authors.initials: CN
authors.lastname: Lin
authors.affiliation: Institute of Veterinary Clinical Sciences, National Taiwan University, Taipei, Taiwan; National Taiwan University Veterinary Hospital, National Taiwan University, Taipei, Taiwan. Electronic address: bilingsu@ntu.edu.tw.
authors.forename: Bi-Ling
authors.initials: BL
authors.lastname: Su
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D053626:Atovaquone / Q000494:pharmacology; D001403:Babesia / Q000187:drug effects / Q000201:enzymology*; D001404:Babesiosis / Q000469:parasitology*; D045303:Cytochromes b / Q000235:genetics*; D004283:Dog Diseases / Q000469:parasitology*; D004285:Dogs; D004351:Drug Resistance; D020125:Mutation, Missense; D021261:Parasitic Sensitivity Tests / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D000890:Anti-Infective Agents; D045303:Cytochromes b; D053626:Atovaquone
keywords: Babesia gibsoni; Cytochrome b; M121I; SimpleProbe(®) real-time PCR