Open AccessIdentification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis
Author(s) -
Hllytchaikra Ferraz Fehlberg,
Bianca Mendes Maciel,
George Rêgo Albuquerque
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0174168
Subject(s) - sarcocystis , toxoplasma gondii , biology , neospora caninum , protozoa , cryptosporidium , polymerase chain reaction , ribosomal dna , apicomplexa , multiplex polymerase chain reaction , 18s ribosomal rna , ribosomal rna , microbiology and biotechnology , parasite hosting , genetics , phylogenetics , protozoal disease , feces , immunology , world wide web , computer science , gene , antibody , malaria
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.