斯氏艾美耳球虫ITS1-5.8S rRNA-ITS2序列的克隆及PCR检测方法的建立

摘要

通过对多种鸡球虫和松鼠球虫18S rRNA和28S rRNA进行序列比对分析,在18S rRNA 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ITS1-5.8S rRNA-ITS2序列,其大小为1 178 bp,其中ITS1序列长度为423 bp,5.8SrRNA为155 bp,ITS2为600 bp,斯氏艾美耳球虫LY株1TS1/2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.%18S rRNA and 28S rRNA sequences of several Eimeria species from chickens and squirrels were aligned to design common primers for Eimeria parasites from various hosts based on the conserved sequences of both 3' end of 18S rRNA and 5' end of 28S rRNA. 1 178 bp of the ITS1-5. 8S rRNA-ITS2 complete sequence of E. stiedai, including 423 bp of ITS1, 155 bp of 5. 8S rRNA, and 600 bp of ITS2, was firstly cloned with the common primers and genomic DNA of oocysts of LY isolate as templates. In contrast to 5. 8S rRNA fragment, ITS1 and ITS2 sequences of E. stiedai LY isolate was more variable, and less than 60% of ITS1 and ITS2 sequences of LY isolate was identical to those of Eimeria species in chickens and other rodent hosts. A sensitive and specific PCR diagnostic assay based on the ITS1-5. 8S rRNA-ITS2 sequence was developed to identify E. stiedai, one of high pathogenic species from rabbits by designing specific primers for E. stiedai at the mutative sites of ITS1 and ITS2. These findings will provide a powerful tool for clinical differentiation of high pathogenic Eimeria species in rabbits and revealing population genetic characteristics of rabbit coccidia.