Specific amplification of Sarcocystis cruzi DNA using a randomly primed polymerase chain reaction assay

The polymerase chain reaction (PCR) method to randomly amplify polymorphic DNA (RAPD) was used to differentiate between Sarcocystis cruzi DNA and bovine DNA. This assay was also exploited to identify a S. cruzi DNA fragment which may be useful as a probe. Five primers ranging in length from 16 to 20 nucleotides were analyzed for their ability to direct the amplification of either bovine or parasite DNA fragments. Two primers, TGA and TGD, preferentially amplified bovine DNA in a mixture of S. cruzi and bovine DNA. The primers TGB and TGF each directed the amplification of S. cruzi DNA instead of bovine DNA. Assays using TGF and S. cruzi DNA resulted in the production of a unique 0.8 kilobase (kb) DNA fragment. This fragment was not amplified from two other closely related coccidian species, Toxoplasma gondii and Sarcocystis campestris. When the 0.8 kb DNA fragment was purified and used as a DNA probe, it only hybridized with DNA from S. cruzi. The results of this study indicate that this DNA fragment may be developed into a useful DNA probe for S. cruzi, and that the RAPD-PCR method may be successfully exploited for the rapid development of DNA probes for parasites and other organisms.