Cere N, Humbert J F, Licois D, Corvione M, Afanassieff M, Chanteloup N
INRA, Centre de Tours, Nouzilly, France.
Exp Parasitol. 1996 Mar;82(2):132-8. doi: 10.1006/expr.1996.0017.
In this work, we described a new approach for the isolation of a species-specific probe for the Eimeria media parasite of the rabbit based on the use of the random amplified polymorphic DNA (RAPD) technique. A specific fragment of 800 bp of the studied species was isolated after RAPD and then cloned and DIG-radiolabeled. After dot-blotting, we observed that this probe was specific for E. media. Sequencing of the 3' and 5' ends of this probe enabled the determination of two primers that could be used in a PCR reaction. The amplified product of 750 bp was specific E. media. The use of these primers and of our probe allowed the detection of a very small number of oocysts. With a new protocol of DNA purification, 10 purified oocysts were detected by PCR. The efficiency of the amplification was not changed when two species were mixed. The threshold of detection of oocysts in fecal matter was equal to 30.
在本研究中，我们描述了一种基于随机扩增多态性DNA（RAPD）技术分离兔艾美耳球虫特异性探针的新方法。经RAPD后分离出所研究物种的一个800 bp的特异性片段，然后进行克隆并用地高辛（DIG）放射性标记。斑点杂交后，我们观察到该探针对中型艾美耳球虫具有特异性。对该探针的3'端和5'端进行测序，确定了可用于PCR反应的两种引物。扩增得到的750 bp产物是中型艾美耳球虫特异性的。使用这些引物和我们的探针能够检测到极少量的卵囊。采用新的DNA纯化方案，通过PCR检测到了10个纯化的卵囊。当两种物种混合时，扩增效率没有改变。粪便中卵囊的检测阈值为30。
