Sensitive genotyping of Cryptosporidium parvum by PCR-RFLP analysis of the 70-kilodalton heat shock protein (HSP70) gene.
A polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of a 587-bp region of the Cryptosporidium parvum 70-kDa heat shock protein (HSP70) gene was developed for the detection and discrimination of the two major genotypes of C. parvum, genotype 1 and genotype 2. Ten Cryptosporidium isolates from non-immunocompromised people were identified as genotypes 1 and 2 (five each) by DNA sequencing of the 587-bp PCR product. This distinction was also achieved with the combination of two endonucleases, HinfI and ScaI, which generated a specific pattern for each genotype. A thorough screening of published sequences showed that this combination of enzymes could also be used for the discrimination of other species/genotypes of Cryptosporidium, especially Cryptosporidium meleagridis and the 'dog' genotype of C. parvum, both of which are infectious in humans. The PCR, conducted on genotypes 1 and 2 of C. parvum, could detect one oocyst per reaction. This new and sensitive genotyping procedure should be of particular interest when applied to the monitoring of water resources in which low concentrations of parasites usually occur.
Gobet
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CSIRO Land and Water, Private Bag No. 5, Wembley, WA 6913, Australia. philippe.gobet@per.clw.csiro.au
Toze
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D000818:Animals; D003457:Cryptosporidiosis / Q000469:parasitology; D016785:Cryptosporidium parvum / Q000145:classification* / Q000235:genetics*; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific; D017125:Genes, Protozoan / Q000235:genetics*; D005838:Genotype; D018840:HSP70 Heat-Shock Proteins / Q000145:classification / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping
D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; C047020:endodeoxyribonuclease ScaI; D015252:Deoxyribonucleases, Type II Site-Specific