Tenter A M, Luton K, Johnson A M
Institut für Parasitologie der Tierärztlichen Hochschule Hannover, Germany.
Appl Parasitol. 1994 Sep;35(3):173-88.
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.
对感染绵羊的四种形成包囊的球虫（柔嫩肉孢子虫、绵羊肉孢子虫、巨型肉孢子虫、刚地弓形虫）的小亚基（SSU）核糖体RNA（rRNA）基因进行了分析，以寻找可作为物种特异性聚合酶链反应（PCR）引物位点的独特靶序列。总共11个推测为物种特异性的寡核苷酸与针对真核生物SSU rRNA基因保守区域设计的通用寡核苷酸组合进行了测试。对于每种寄生虫，发现其中两对寡核苷酸是用于SSU rRNA基因片段物种特异性扩增的理想引物。通过使用源自柔嫩肉孢子虫、绵羊肉孢子虫、巨型肉孢子虫、刚地弓形虫和绵羊的基因组DNA制剂测试不同的PCR参数（退火温度、镁离子浓度、扩增循环数），优化了PCR检测的特异性。新的PCR检测是对三种绵羊肉孢子虫属物种和刚地弓形虫进行物种特异性区分的有力工具。
