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通过对多种鸡球虫和松鼠球虫18SrRNA和28SrRNA进行序列比对分析,在18SrRNA 3′端和28SrRNA 5′端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8SrRNA-ITS2序列,其大小为1 178bp,其中ITS1序列长度为423bp,5.8SrRNA为155bp,ITS2为600bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列相似性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。
Through the sequence alignment of 18S rRNA and 28S rRNA of various chicken coccidia and squirrel coccidia, the universal primer of Eimeria was designed on the 3 ’end of 18S rRNA and the conserved region of 5’ end of 28S rRNA, The complete ITS1-5.8SrRNA-ITS2 sequence of Ehrlichia spp. Was successfully cloned from strain LY oocyst genomic DNA for the first time with a size of 1 178 bp, of which ITS1 sequence length was 423 bp, 5.8SrRNA was 155 bp and ITS2 was 600 bp . The ITS1 / 2 sequence of Listeria monocytogenes was highly variable, and its sequence similarity with that of coccidia and rodent was less than 60%. Then we designed specific primers for the ITS1 / 2 hypervariable region of Ehrlichia spp., And established a sensitive and specific PCR assay. The results of this study will provide an effective molecular tool for the clinical diagnosis of the causative species of rabbit coccidia and the genetic characteristics of the population of rabbit coccidia.