title: Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis.
abstract: The objective of this study was to standardize the high-resolution melting method for identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by amplification of 18S ribosomal DNA (rDNA) using a single primer pair. The analyses were performed on individual reactions (containing DNA from a single species of a protozoan), on duplex reactions (containing DNA from two species of protozoa in each reaction), and on a multiplex reaction (containing DNA of four parasites in a single reaction). The proposed method allowed us to identify and discriminate the four species by analyzing the derivative, normalized, and difference melting curves, with high reproducibility among and within the experiments, as demonstrated by low coefficients of variation (less than 2.2% and 2.0%, respectively). This is the first study where this method is used for discrimination of these four species of protozoa in a single reaction.
authors.affiliation: Graduation program in Animal Science, Santa Cruz State University, Ilhéus (BA), Brazil.
authors.forename: Hllytchaikra Ferraz
authors.initials: HF
authors.lastname: Fehlberg
authors.affiliation: Department of Biological Sciences, Santa Cruz State University, Ilhéus (BA), Brazil.
authors.forename: Bianca Mendes
authors.initials: BM
authors.lastname: Maciel
authors.affiliation: Department of Agricultural and Environmental Sciences, Santa Cruz State University, Ilhéus (BA), Brazil.
authors.forename: George Rêgo
authors.identifier: http://orcid.org/0000-0002-0556-8856
authors.initials: GR
authors.lastname: Albuquerque
mesh_terms: D000818:Animals; D003048:Coccidiosis / Q000175:diagnosis / Q000469:parasitology; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000469:parasitology; D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D018688:Neospora / Q000235:genetics / Q000302:isolation & purification*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011528:Protozoan Infections / Q000175:diagnosis* / Q000469:parasitology; D012522:Sarcocystis / Q000235:genetics / Q000302:isolation & purification*; D012523:Sarcocystosis / Q000175:diagnosis / Q000469:parasitology; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis / Q000469:parasitology
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal