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

在这项研究中，我们开发和评估的有用性荧光原位杂交和原位PCR技术，以确定隐孢子虫卵囊的水样。首先，利用三种荧光标记的寡核苷酸探针Cryl（Vasey et al.1998）、CP 18 -1和CP 18 -2针对微小隐球菌小亚基核糖体RNA（SSUrRNA）中的特定序列进行荧光原位杂交技术。根据Deere at el.（1998），利用C.小隐孢子虫、鼠隐孢子虫和贝氏隐孢子虫卵囊。在荧光免疫分析（IFA）的同时，在荧光显微镜下寻找特异性荧光信号。虽然在荧光显微镜下，特别是使用Cryl探针，在微小隐孢子虫卵囊中检测到强阳性信号，但在贝雷隐孢子虫卵囊和鼠隐孢子虫卵囊中也观察到该信号。这些结果表明，原位杂交技术在鉴别微小隐孢子虫和其他隐孢子虫时不够敏感。在此基础上，我们发展了原位PCR技术，可以扩增小隐孢子虫的特异性基因。以微小隐孢子虫SSUrRNA基因586 bp片段为靶序列，设计引物进行原位PCR。原位PCR后，从卵囊中提取DNA，通过琼脂糖凝胶电泳证实SSUrRNA基因扩增。用2 mg/ml胰蛋白酶处理卵囊5 min以上，经50%乙醇固定后，成功扩增出586 bp的基因。这些结果表明，原位PCR技术将是一种有用的工具，以鉴定小隐孢子虫与其他物种。

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.