A simple method of DNA extraction for Eimeria species

Abstract

A new, simple method is described for extracting DNA from coccidia (Eimeriidae) oocysts. In our hands this method works well for all Eimeria oocysts and, presumably, will work equally well for oocysts of other coccidia genera. This method combines the two steps of breaking oocyst and sporocyst walls, and dissolving the sporozoite membrane in one step. This greatly simplifies the currently used DNA extraction procedures for Eimeria species and overcomes the disadvantages of existing DNA extraction methods based on glass-bead grinding and sporozoite excystation procedures. Because all the procedures are done in a 1.5-ml microfuge tube, which minimizes the loss of DNA in the extraction procedures, this method is especially suitable for samples with small number of oocysts. In addition, this method directly lyses the oocyst and sporocyst walls as well as the sporozoite membrane in a continuous incubation; therefore, it does not require the sporozoites to be alive. The results of PCR experiments indicate that this method generates better quality of DNA than what the existing glass-bead grinding method does for molecular analysis, and is suitable for both large or small number (<10 2 oocysts) of living or dead oocyst samples.