High resolution genotyping of cryptosporidium by mutation scanning
El-Osta, Y.G.A., Zhu, X.Q.X., Chalmers, R., Caccio, S., Widmer, G., Morgan-Ryan, U.M., Thompson, R.C.A., Pozio, E. and Gasser, R.B. (2003) High resolution genotyping of cryptosporidium by mutation scanning. In: Thompson, R.C.A., Armson, A. and Morgan, U., (eds.) Cryptosporidium: From Molecules to Disease. Elsevier B.V., Amsterdam, The Netherlands, pp. 173-175.
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This chapter focuses on the high resolution genotyping of Cryptosporidium by mutation scanning. A range of different molecular approaches has been developed to characterize Cryptosporidium species and genotypes. Some of these approaches may not necessarily precisely resolve sequence variation because they depend on the size-separation of DNA molecules. Some methods are laborious and time-consuming to perform when the analysis of large numbers of samples is needed. Such limitations may be overcome by employing mutation detection methods, such as Single-Strand Conformation Polymorphism (SSCP). This chapter presents a study, the aim of that was to evaluate an SSCP approach for the genotyping of Cryptosporidium oocyst isolates. The present SSCP methods have advantages over some approaches to screen for genetic variation in Cryptosporidium. In contrast to arbitrarily primed-PCR, SSCP employs amplicons produced at higher stringency using specific primers, thus minimizing the co-amplification of extraneous DNA and maximizing reproducibility. SSCP can be used to screen relatively large numbers of samples for variation prior to selective DNA sequence analysis, which reduces considerably time, labor, and expense, and in contrast to PCR-coupled RFLP, which screens for sequence variation at a small number of endonuclease restriction sites, SSCP scans the entire length of an amplicon for variability. The results presented in this chapter indicate that the present approach can be used as a diagnostic tool to identify any of the currently recognized species and genotypes of Cryptosporidium. Combined with DNA sequencing, the approach also provides a powerful analytical tool.
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