Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces

Aims: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. Methods and Results: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. Conclusions: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. Significance and Impact of the study: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.