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.

Fig 1

Derivative melting curve (A), normalized melting curve (B), and difference melting curve (C) for coccidian. Toxoplasma gondii (black), Neospora caninum (red), Cryptosporidium parvum (blue), Sarcocystis neurona (green) and multiplex reaction containing DNA from all four protozoa (orange).

Fig 2. Derivative melting curve (left) and normalized melting curve (right) of HRM duplex assays.

(A) Toxoplasma gondii and Neospora caninum; (B) T. gondii and Sarcocystis neurona; (C) T. gondii and Cryptosporidium parvum; (D) N. caninum and S. neurona; (E) N. caninum and C. parvum; and (F) S. neurona and C. parvum.