El-Osta Youssef G Abs, Chalmers Rachel M, Gasser Robin B
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria, Australia.
Mol Cell Probes. 2003 Aug;17(4):127-34. doi: 10.1016/s0890-8508(03)00030-6.
A mutation scanning-selective sequencing approach was employed for the genotypic identification and differentiation of Cryptosporidium parvum isolates. Genomic DNA samples (n=158) from Cryptosporidium oocysts from humans with clinical cryptosporidiosis (following recent foreign travel or during different outbreaks) in the UK were subjected to PCR-based single-strand conformation polymorphism (SSCP) analysis of a heat shock protein 70 gene region (p-hsp70; 448 bp). Samples representing different SSCP profiles were then subjected to sequencing. The analysis allowed the classification of 149 of the 158 samples as type-1 ( approximately 49%) or type-2 ( approximately 46%); amplicons from the remaining nine samples were consistent in size with p-hsp70 but represented non-specific, faecal contaminants. The percentages reflected those of a previous study in the UK for autochtonous, sporadic cases of cryptosporidiosis ( approximately 49% for type-1 and approximately 47% for type-2; n approximately 4000), but contrasted another survey of sporadic cases where type-2 dominated ( approximately 62%; n approximately 1000). The ability of the present SSCP-sequencing approach to accurately screen for C. parvum genotypes and to reliably discern erroneous amplicons has significant implications for the accurate diagnosis and monitoring of cryptosporidiosis and for population genetic studies.
