Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis

• Published in: PloS one 2017-03, Vol.12 (3), p.e0174168-e0174168
• Main Authors: Fehlberg, Hllytchaikra Ferraz, Maciel, Bianca Mendes, Albuquerque, George Rêgo
• Format: Article
• Language: English
• Subjects: Abortion; Acetal resins; Agronomy; Amine oxidase (flavin-containing); Amplification; Animal health; Animals; Antibiotic resistance; Antibiotics; Antigens; Apical membrane antigen 1; Assaying; B1 gene; Biochemistry; Biology and Life Sciences; Bradyzoites; Brain; Cadmium; Coccidiosis - diagnosis; Coccidiosis - parasitology; Complementarity; Computer programs; Concentration (composition); Cryptosporidiosis; Cryptosporidiosis - diagnosis; Cryptosporidiosis - parasitology; Cryptosporidium; Cryptosporidium - genetics; Cryptosporidium - isolation & purification; Denaturation; Deoxyribonucleic acid; Diagnosis; Dilution; Disease; Diseases; DNA; DNA methylation; DNA, Protozoan - genetics; DNA, Protozoan - isolation & purification; DNA, Ribosomal - genetics; DNA, Ribosomal - isolation & purification; Dyes; Epidemiology; Feces; Fluorescence; Free water; Genetic analysis; Genetic testing; Genomes; Genotypes; High resolution; Humans; Identification; Identification and classification; Inhibition; Intervention; Intestine; Life cycles; Meadows; Melting; Mice; Multiplexing; Neospora - genetics; Neospora - isolation & purification; Nucleic Acid Denaturation; Nucleotide sequence; Oocysts; Parasites; Peritoneum; pH effects; Physical Sciences; Polymerase Chain Reaction; Protozoa; Protozoan Infections - diagnosis; Protozoan Infections - parasitology; Research and Analysis Methods; Ribosomal DNA; Sarcocystis; Sarcocystis - genetics; Sarcocystis - isolation & purification; Sarcocystosis - diagnosis; Sarcocystosis - parasitology; Saturation; Sexually transmitted diseases; STD; Systems analysis; Temperature effects; Toxoplasma; Toxoplasma - genetics; Toxoplasma - isolation & purification; Toxoplasmosis; Toxoplasmosis - diagnosis; Toxoplasmosis - parasitology; Ultraviolet radiation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0174168

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.