Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction.
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 in Cryptosporidium parvum and C. muris. This sequence was found to contain 3 Mae I endonuclease restriction sites, 1 of which was present only in C. parvum. Mae I restriction of PCR products from 2 C. parvum isolates (one of human origin and the other of bovine origin), 1 C. muris isolate, and 1 C. baileyi isolate, showed a specific and reproducible profile for C. parvum that was different from the one obtained for both C. muris and C. baileyi. From these data, new Mae I restriction maps were proposed for the three species. The system was then used to screen 6 C. parvum isolates (from human and bovine hosts), and the C. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers of C. parvum oocysts in environmental samples (e.g. in water supplies).
Awad-el-Kariem
F M
FM
Department of Clinical Sciences, London School of Hygiene and Tropical Medicine.
Warhurst
D C
DC
McDonald
V
V
D000818:Animals; D001483:Base Sequence; D003458:Cryptosporidium / Q000145:classification* / Q000235:genetics; D016785:Cryptosporidium parvum / Q000145:classification* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D017125:Genes, Protozoan; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000737:chemistry* / Q000235:genetics; D015183:Restriction Mapping
D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S; C040836:CTAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific