Detection of Cryptosporidium spp. from human faeces by PCR-RFLP, cloning and sequencing.

In this study, we compared and validated a nested ABC polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay to amplify Cryptosporidium parvum oocyst wall protein (COWP) gene fragment with a previous nested PCR-RFLP method. When RFLP of COWP gene did not provide clear results, we further analysed samples by cloning and sequencing for species and genotype identification. ABC-PCR was performed on DNA extracted from human faecal samples collected in England where Cryptosporidium was previously detected by conventional methods. COWP gene amplification was successful for all specimens; RFLP analyses using both RsaI and AluI enzymes recognised 54/55 restriction profiles. Among these, 40 (72.7%) characteristic patterns were generated for C. parvum and six (10.9%) for Cryptosporidium hominis; four (7.3%) showed species mixtures, while the remaining four samples (7.3%) showed atypical genotypes. Cloning and sequencing of mixtures and atypical genotypes identified one C. parvum, one C. hominis and three Cryptosporidium meleagridis. Indeed, molecular tools improved PCR-RFLP results. In conclusion, this study showed that ABC-PCR-RFLP technique was quick, simple and specific. Cloning and sequencing were helpful in characterising mixtures or previously uncharacterised RFLP patterns. All these epidemiological findings are useful information for any preventive intervention to control disease diffusion.