Species-specific identification of Sarcocystis and Toxoplasma by PCR amplification of small subunit ribosomal RNA gene fragments.

Abstract

The small subunit (SSU) ribosomal RNA (rRNA) genes of four cyst-forming coccidia (Sarcocystis tenella, Sarcocystis arieticanis, Sarcocystis gigantea, Toxoplasma gondii) infecting sheep were analysed for unique target sequences which could be used as priming sites for species-specific polymerase chain reactions (PCR). A total of 11 putatively species-specific oligonucleotides were tested in combination with universal oligonucleotides designed for conserved regions of the SSU rRNA genes of eukaryotes. For each parasite species two of these oligonucleotide pairs were found to be ideal primers for species-specific amplification of SSU rRNA gene fragments. The specificity of the PCR assays was optimised by testing different PCR parameters (annealing temperature, magnesium ion concentration, number of amplification cycles) with genomic DNA preparations derived from S. tenella, S. arieticanis, S. gigantea, T. gondii and sheep. The new PCR assays are a powerful tool for species-specific differentiation of the three ovine Sarcocystis spp. and T. gondii.