A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avian Eimeria species

Abstract

Coccidiosis is a significant disease of poultry caused by different species of Eimeria. Differentiation of Eimeria species is important for the quality control of the live attenuated Eimeria vaccines derived from monospecific lines of Eimeria spp. In this study, high-resolution melting (HRM) curve analysis of the amplicons generated from the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2) was used to distinguish between seven pathogenic Eimeria species of chickens, and the results were compared with those obtained from the previously described technique, capillary electrophoresis. Using a series of known monospecific lines of Eimeria species, HRM curve analysis was shown to distinguish between Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella. Computerized analysis of the HRM curves and capillary electrophoresis profiles could detect the dominant species in several specimens containing different ratios of E. necatrix and E. maxima and of E. tenella and E. acervulina. The HRM curve analysis identified all of the mixtures as “variation” to the reference species, and also identified the minor species in some mixtures. Computerized HRM curve analysis also detected impurities in 21 possible different combinations of the seven Eimeria species. The PCR-based HRM curve analysis of the ITS-2 provides a powerful tool for the detection and identification of pure Eimeria species. The HRM curve analysis could also be used as a rapid tool in the quality assurance of Eimeria vaccine production to confirm the purity of the monospecific cell lines. The HRM curve analysis is rapid and reliable and can be performed in a single test tube in less than 3 h.