Molecular Biology Methods for Detection and Identification of Cryptosporidium Species in Feces, Water, and Shellfish

Abstract

Techniques based on nucleic acid amplification have proven to be essential for the detection and epidemiological tracking of members of the genus Cryptosporidium. This gastrointestinal protozoan parasite cannot be routinely cultivated and it has an extremely low infectious dose, possibly below 100 oocysts. As Cryptosporidium is an important pathogen, particularly in immuno-compromised hosts, there is a pressing need to employ sensitive and discriminatory systems to monitor the organism. A number of fairly standard target genes have been assessed as detection targets, including 18S rRNA, microsatellites, and heat-shock (stress) proteins. As our knowledge of the biology of the organism increases, and as the full genome information becomes available, the choice of target may change. Genes encoding parasite-specific surface proteins (gp60, TRAP-C2, COWP) have already been examined. Much of the effort expended in molecular diagnostics of Cryptosporidium has been directed toward developing robust nucleic acid extraction methods. These are vital in order to recover amplifiable DNA from environments where small numbers of oocysts, often fewer than 100, may exist. Methodology based on adaptation of commercial kits has been developed and successfully employed to recover amplifiable DNA directly from water, food (particularly seafood), and fecal samples.