DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction

Abstract

The objective of this project was to construct specific and sensitive molecular probes and amplification primers for Cryptosporidium parvum that could be used in diagnosis, retrospective tissue studies, and in epidemiologic surveys. Whole genomic DNA was extracted from oocysts of C. parvum purified from human and bovine feces. A genomic library was constructed in plasmid pUC18 and propagated in Escherichia coli DH5 alpha. Transformants were screened by colony hybridization and autoradiography. The 2.3-kilobase segment in plasmid pHC1, a clone specific for C. parvum, was sequenced by the Sanger method. Computer analysis gave a G+C content of 35%. A 400-base region (bases 470-870) was selected as an amplification target because it contained a unique restriction endonuclease site that could serve as a useful marker. Primers of 26 nucleotides each were synthesized. Sensitive and specific amplification of the target sequence was demonstrated both by ethidium bromide staining of agarose and acrylamide gels, and by hybridization with chemiluminescence-labeled synthetic oligonucleotide probes.