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

在这项研究中，我们开发并评估了荧光原位杂交和原位聚合酶链式反应技术在从水样中鉴定微小隐孢子虫卵囊的有效性。首先，利用荧光标记寡核苷酸探针Cryl(Vasey et al.1998)、CP18-1和CP18-2针对微小隐孢子虫小亚单位核糖体RNA(SSUrRNA)中的特定序列，进行了荧光原位杂交技术。按Deere at el的方法进行原位杂交。(1998)利用微小隐孢子虫、小隐孢子虫和百雷隐孢子虫的卵囊。在荧光免疫分析(IFA)的同时，在荧光显微镜下寻找特异的荧光信号。虽然在荧光显微镜下，特别是用CRYL探针在微小毛滴虫卵囊中检测到了强阳性信号，但在白雷毛囊和毛囊虫卵囊中也观察到了强阳性信号。这些结果表明，原位杂交技术不足以将微小隐孢子虫与隐孢子虫的其他种类区分开来。在此基础上，我们建立了原位聚合酶链式反应技术，可以扩增微小螺旋体的特异性基因。针对微小隐孢子虫SSUrRNA基因586bp的部分片段，设计引物进行原位聚合酶链式反应。原位聚合酶链式反应后，从卵囊中提取DNA，琼脂糖凝胶电泳法证实SSUrRNA基因的扩增。在完整的微小隐孢子虫卵囊中成功扩增出586bP基因，且卵囊通透性超过5min。经50%乙醇固定后用2 mg/ml胰酶处理。这些结果表明，原位聚合酶链式反应技术将成为鉴别微小螺旋体与其他物种的有用工具。

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.