Fehlberg Hllytchaikra Ferraz, Maciel Bianca Mendes, Albuquerque George Rêgo
Graduation program in Animal Science, Santa Cruz State University, Ilhéus (BA), Brazil.
Department of Biological Sciences, Santa Cruz State University, Ilhéus (BA), Brazil.
PLoS One. 2017 Mar 27;12(3):e0174168. doi: 10.1371/journal.pone.0174168. eCollection 2017.
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.
本研究的目的是通过使用单一引物对扩增18S核糖体DNA（rDNA），规范用于鉴定和区分刚地弓形虫、肉孢子虫属、新孢子虫属和隐孢子虫属的高分辨率熔解方法。分析在单个反应（每个反应包含来自一种原生动物的DNA）、双重反应（每个反应包含来自两种原生动物的DNA）和多重反应（单个反应包含四种寄生虫的DNA）上进行。所提出的方法使我们能够通过分析导数熔解曲线、标准化熔解曲线和差异熔解曲线来鉴定和区分这四种物种，实验之间和实验内部具有很高的重现性，变异系数较低（分别小于2.2%和2.0%）证明了这一点。这是首次将该方法用于在单个反应中区分这四种原生动物物种的研究。
