Identification of Cryptosporidium Sp. Oocysts in Water by Modified Immunomagnetic Separation and Rflp-pcr

Current methodologies for detection of Cryptosporidium sp. in water, despite some efforts for standardization (e.g. EPA Methods 1622/1623), has several drawback that limit the applicability or interpretation of the results of Cryptosporidium monitoring. Presence of some chemical dissolved in water, ageing of oocysts, reduced affinity of antibodies used in immunomagnetic separation (IMS) and immunofluorescent assay (IFA) results in reduced sensitivity and specificity. Against this background of molecular biology techniques used for detection of Cryptosporidium, are characterized by good sensitivity, reproducibility and acceptable cost analysis. In this paper, the authors showed the possibility of modifying the protocol of immunomagnetic isolation of oocysts and adjust its scale to the needs of a PCR reaction combined with an analysis of restriction fragments length polymorphism (RFLP-PCR). The method allows relatively rapid implementation of qualitative analysis in the direction of Cryptosporidium, combined with the identification of species. It is also connected to the determination of several pathogens (including Giardia and Toxoplasma) through a combination of antibodies used in the IMS and multiplexPCR.