title: Comparison of PCR methods for diagnosis of canine visceral leishmaniasis in conjunctival swab samples.
abstract: Four PCR assays for detection of Leishmania DNA in conjunctival swab samples were compared. All methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or seminested). Two methods (kDNA PCR-hybridization and kDNA snPCR) used primers targeted to the minicircles of kinetoplast DNA (kDNA) and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of ribosomal rRNA genes. kDNA PCR-hybridization was positive for 22/23 dogs (95.6%) and for 40/46 samples (86.9%), considering the right and the left conjunctivas. kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The ITS-1 nPCR and LnPCR were both able to detect the parasites in 17/23 dogs (73.9%) and 29/46 (63%) and 30/46 (65.2%) samples, respectively. The positivities of the kDNA based methods were significantly higher; however the choice of the best method will depend on the kind of information required with the diagnosis.
authors.affiliation: Centro de Desenvolvimento da Tecnologia Nuclear, Cidade Universitária-Campus da UFMG, 31120-970 Belo Horizonte, MG, Brazil. marciapilatti@yahoo.com.br
authors.forename: Marcia Maria
authors.initials: MM
authors.lastname: Pilatti
authors.forename: Sidney de Almeida
authors.initials: Sde A
authors.lastname: Ferreira
authors.forename: Maria Norma
authors.initials: MN
authors.lastname: de Melo
authors.forename: Antero Silva Ribeiro
authors.initials: AS
authors.lastname: de Andrade
mesh_terms: D000818:Animals; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004283:Dog Diseases / Q000175:diagnosis / Q000469:parasitology*; D004285:Dogs; D005784:Gene Amplification; D017125:Genes, Protozoan / Q000235:genetics; D007891:Leishmania / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000235:genetics / Q000662:veterinary*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D016053:RNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D012335:RNA, Ribosomal