title: High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.
abstract: BACKGROUND
  Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.
  
  
  RESULTS
  Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.
  
  
  CONCLUSIONS
  HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.
authors.affiliation: Physiology Department, Biosciences Institute, São Paulo University, São Paulo, São Paulo, Brazil.
authors.forename: Ricardo Andrade
authors.initials: RA
authors.lastname: Zampieri
authors.affiliation: Physiology Department, Biosciences Institute, São Paulo University, São Paulo, São Paulo, Brazil.
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Laranjeira-Silva
authors.affiliation: Physiology Department, Biosciences Institute, São Paulo University, São Paulo, São Paulo, Brazil.
authors.forename: Sandra Marcia
authors.initials: SM
authors.lastname: Muxel
authors.affiliation: Pathology Department, Medical Faculty, São Paulo University, São Paulo, São Paulo, Brazil.
authors.forename: Ana Carolina
authors.initials: AC
authors.lastname: Stocco de Lima
authors.affiliation: Parasitology Department, Biomedical Institute, São Paulo University, São Paulo, São Paulo, Brazil.
authors.forename: Jeffrey Jon
authors.initials: JJ
authors.lastname: Shaw
authors.affiliation: Physiology Department, Biosciences Institute, São Paulo University, São Paulo, São Paulo, Brazil.
authors.forename: Lucile Maria
authors.initials: LM
authors.lastname: Floeter-Winter
mesh_terms: D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D050884:HSP72 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007891:Leishmania / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007896:Leishmaniasis; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D016054:DNA, Protozoan; D050884:HSP72 Heat-Shock Proteins; D015800:Protozoan Proteins