ANALYSIS OF SPECIES AND TYPES OF CRYPTOSPORIDIUM FROM WATER USING MOLECULER BIOLOGICAL TECHNIQUES

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In this study we developed and evaluated the usefulness of fluorescence in situ hybridization and in situ PCR techniques to identify Cryptosporidium parvum oocysts from water samples. Firstly, fluorescence in situ hybridization technique was carried out by three fluorescently labelled oligonucleotide probes, Cryl( Vasey et al.1998) , CP18-1 and CP18-2, targeting specific sequences in small subunit ribosomal RNA (SSUrRNA) of C.parvum. In situ hybridization was performed according to the protocol of Deere at el. (1998) by using C. parvum, C.muris and C.bailei oocysts. The specific fluorescent signals were searched under fluorescence microscopy at the same time of fluorescence immuno assay (IFA). Although strong positive signals were detected under fluorescence microscopy in C.parvum oocysts especially using Cryl probe, the signals were also observed in C.bailei and C.muris oocysts. These results suggested that in situ hybridization technique is not sensitive enough for identify C.parvum from other species of Cryptosporidium. Then we developed in situ PCR technique which could amplify the specific gene of C.parvum. Primers which targeted the part of SSUrRNA gene, 586bp, of C.parvum were used for in situ PCR. After in situ PCR, DNA was isolated from oocysts to confirm the amplification of SSUrRNA gene by agarose gel electrophoresis. Amplification of 586bp gene were successfully identified in intact C.parvum oocysts which permeabilized for more than 5 min. by 2mg/ml trypsin after 50% ethanol fixation. These results suggested that in situ PCR technique will be the useful tool for identifying C.parvum from other species.