title: Development of a quantitative PCR for the detection of Rangelia vitalii.
abstract: The aim of this study was to develop and validate a SYBR Green qPCR assay to detect and quantify a fragment of the 18S rRNA gene of Rangelia vitalii in canine blood. Repeatability of the qPCR was determined by the intra- and inter-assay variations. The qPCR showed efficiency of E=101.30 (r(2)=0.996), detecting as few as one copy of plasmid containing the target DNA. Specificity of the assay was performed using DNA samples of Babesia canis, B. gibsoni, Ehrlichia canis, E. ewingii and Leishmania sp. No cross-reactivity was observed. Field samples consisting of blood from 265 dogs from Porto Alegre, Brazil were also tested. A total of 24 (9.05%) samples were positive for R. vitalii. Amplicons of 50% of positive samples were confirmed to be R. vitalii by Sanger sequencing. The positive samples had an average of 3.5×10(5) organisms/mL of blood (range: 1.27×10(3)-1.88×10(6)) based on the plasmid-generated standard curve. In conclusion, the SYBR Green qPCR assay developed herein is sensitive and specific and can be used as a diagnostic tool for detection and quantification of R. vitalii in canine blood samples.
authors.affiliation: Laboratory of Veterinary Clinical Analysis, Universidade Federal e Santa Maria, Santa Maria, RS, Brazil. Electronic address: franchimelo@gmail.com.
authors.forename: Francine Chimelo
authors.initials: FC
authors.lastname: Paim
authors.affiliation: Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.
authors.forename: Andrea Pires
authors.initials: AP
authors.lastname: dos Santos
authors.affiliation: Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.
authors.forename: Naíla Cannes
authors.initials: NC
authors.lastname: do Nascimento
authors.affiliation: Laboratory of Veterinary Clinical Analysis, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Camila Serina
authors.initials: CS
authors.lastname: Lasta
authors.affiliation: Department of Veterinary Medicine, Universidade Federal do Paraná, Curitiba, RS, Brazil.
authors.forename: Simone Tostes
authors.initials: ST
authors.lastname: Oliveira
authors.affiliation: Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.
authors.forename: Joanne Belle
authors.initials: JB
authors.lastname: Messick
authors.affiliation: Laboratory of Veterinary Clinical Analysis, Universidade Federal e Santa Maria, Santa Maria, RS, Brazil.
authors.forename: Sonia Terezinha dos Anjos
authors.initials: ST
authors.lastname: Lopes
mesh_terms: D000818:Animals; D001938:Brazil; D004283:Dog Diseases / Q000097:blood / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D016792:Piroplasmida / Q000235:genetics*; D011529:Protozoan Infections, Animal / Q000097:blood / Q000175:diagnosis* / Q000469:parasitology; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D012337:RNA, Ribosomal, 18S
keywords: 18S rRNA gene; Dog; Quantitative PCR; Rangeliosis