title: SYBR Green-based real-time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases.
abstract: BACKGROUND
  Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk.
  
  
  RESULTS
  A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95 °C ± 0.01 and 77.36 °C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities.
  
  
  CONCLUSIONS
  For all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Leônidas Deane, sala 209, Avenida Brasil 4365, Manguinhos, 21040-360 Rio de Janeiro, RJ, Brazil.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pita-Pereira
authors.forename: Rachel
authors.initials: R
authors.lastname: Lins
authors.forename: Marcia P
authors.initials: MP
authors.lastname: Oliveira
authors.forename: Rosimar B
authors.initials: RB
authors.lastname: Lima
authors.forename: Bernardo A S
authors.initials: BA
authors.lastname: Pereira
authors.forename: Otacilio C
authors.initials: OC
authors.lastname: Moreira
authors.forename: Reginaldo P
authors.initials: RP
authors.lastname: Brazil
authors.forename: Constança
authors.initials: C
authors.lastname: Britto
mesh_terms: D000293:Adolescent; D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D001938:Brazil; D002648:Child; D002675:Child, Preschool; D017124:Conserved Sequence / Q000235:genetics; D018105:DNA, Kinetoplast / Q000737:chemistry / Q000235:genetics*; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology*; D007891:Leishmania / Q000235:genetics / Q000302:isolation & purification*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology; D007898:Leishmaniasis, Visceral / Q000175:diagnosis* / Q000469:parasitology; D008297:Male; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D010802:Phylogeny; D011576:Psychodidae / Q000469:parasitology*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D052160:Benzothiazoles; D018105:DNA, Kinetoplast; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I