title: Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis.
abstract: The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3% were co-negatives, but 27.6% were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.
authors.affiliation: Laboratório de Imunoparasitologia Veterinária, Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista - UNESP, Rod. Paulo Donato Castellane, km 5, CEP 14884-900 Jaboticabal - SP, Brazil. andreahn@gmail.com
authors.forename: Andrea Cristina Higa
authors.initials: AC
authors.lastname: Nakaghi
authors.forename: Rosangela Zacarias
authors.initials: RZ
authors.lastname: Machado
authors.forename: Jesus Aparecido
authors.initials: JA
authors.lastname: Ferro
authors.forename: Marcelo Bahia
authors.initials: MB
authors.lastname: Labruna
authors.forename: Andreas Lazaros
authors.initials: AL
authors.lastname: Chryssafidis
authors.forename: Marcos Rogério
authors.initials: MR
authors.lastname: André
authors.forename: Cristiane Divan
authors.initials: CD
authors.lastname: Baldani
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D004283:Dog Diseases / Q000175:diagnosis* / Q000382:microbiology*; D004285:Dogs; D041061:Ehrlichia canis / Q000235:genetics* / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D005798:Genes, Bacterial / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins