Detection and species identification ofCryptosporidiumoocysts using a system based on PCR and endonuclease restriction

Abstract

SUMMARY: The polymerase chain reaction (PCR) was used to produce a 556 bp nucleotide stretch, employing primers based on the published sequence of the 18S rRNA genes inCryptosporidium parvumandC. muris. This sequence was found to contain 3MaeI endonuclease restriction sites, 1 of which was present only inC. parvum. Mae I restriction of PCR products from 2C.parvumisolates (one of human origin and the other of bovine origin), 1C. murisisolate, and 1C. baileyiisolate, showed a specific and reproducible profile forC. parvumthat was different from the one obtained for bothC. murisandC. baileyi. From these data, newMaeI restriction maps were proposed for the three species. The system was then used to screen 6C. parvumisolates (from human and bovine hosts), and theC. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers ofC. parvumoocysts in environmental samples (e.g. in water supplies).