奶牛隐孢子虫Nested PCR—RFLP方法的建立及初步应用

为快速检测并准确鉴别奶牛隐孢子虫种，以隐孢子虫18S rRNA基因的特殊区域为基础，设计内、外引物，并根据软件分析确定相应的内切酶EcoT141，采用Nested PCR-RFLP方法进行虫种种型鉴别分析。在Nested PCR两次PCR反应中，以微小隐孢子虫（Cryptos poridium parvum，C．p）和安氏隐孢子虫（Cryptosporidium andersoni，C．an）卵囊提取的DNA为模板，均能扩增出长约800bp和500bp的明亮条带，且特异性强，其他虫种不能扩增出条带，该方法最低可检测到5个卵囊／g粪便；对于由内引物扩增出的500bp的条带，C.an的PCR产物能被内切酶EcoT141酶切，酶切后的片段分别为416bp和92bp，C.p的PCR产物不能被此酶酶切。用所建立的Nested PCR-RFLP法对上海奶牛389头和进口奶牛200头的共计589份粪样进行检测，Nested PCR的结果表明上海奶牛和进口奶牛的隐孢子虫阳性率分别为19．02％和3．5％，RFLP的结果表明上海奶牛感染的主要是Cp和C．an，进口奶牛感染的主要是C．p。研究结果表明，本研究建立的检测奶牛粪便中的隐孢子虫的NestedPCR-RFLP法，可用于奶牛隐孢子虫流行病学调查并有效鉴别奶牛隐孢子虫种。

DEVELOPMENT AND APPLICATION OF THE NESTED PCR-RFLP METHOD FOR DETECTION AND DIFFERENTIATION OF CRYPTOSPORIDIUM IN THE DAIRY COWS

A nested PCR-RFLP-based method was developed to detect Cryptosporidiosis and differentiate Cryptosporidium species. Two pairs of primers were designed based on published sequence of 18S rRNA gene of Cryptosporidiurn. The first pair of primers was derived from conserved areas within the 18S rRNA of Cryptosporidium and the amplified fragment was 800 bp. The second pair of primers was derived from various areas within conserved areas of the 18S rRNA and the amplified fragment was 500 bp. The PCR products were digested with EcoT14I. The PCR product of C. andersoni was digested with EcoT14I into two visible bands of 416 bp and 92 bp but the PCR product of C. parvum were not. This method could detect 5 oocysts in one gram of fecal sample from diary cattle. Preliminary application for testing 389 fecal samples from Shanghai domestic dairy cows revealed that Cryptosporidium infection rate was 19.02% and both C. andersoni and C. parvum were detected. The Cryptosporidium infection rate was 3. 5% in 220 fecal samples of imported dairy cows and only C. parvum was detected. This nested PCR-RFLP method could be used for investigation of Cryptosporidium infection and differentiation of Cryptosporidium species.