摘要
根据GenBank中登录的隐孢子虫小亚基rRNA基因序列设计1对引物,并以标准基因组DNA为模板,初步建立了检测乳牛微小隐孢子虫和安氏隐孢子虫的SYBR Green real time PCR方法,并对乳牛微小隐孢子虫和安氏隐孢子虫阳性样品和上海40份乳牛粪便进行了检测。结果表明,此次建立的real timePCR对微小隐孢子虫和安氏隐孢子虫均能扩增出曲线,且其他寄生虫(鸡贝氏隐孢子虫、刚地弓形虫、犬新孢子虫)和大肠杆菌均未检测到;标准基因组DNA的检测阈值达到5个拷贝,牛粪中卵囊的最低检测量为每克粪便5个卵囊,乳牛粪便阳性率为15%(6/40)。表明,建立的荧光定量PCR快速、特异、敏感,可用于乳牛隐孢子虫病的流行病学调查。
A SYBR Green real time PCR method for detecting Cryptosporidium parvum and Cryptosporidium andersoni was established with a pair of primers designed according to the SSU rRNA gene of Cryptosporidium available in GenBank and with the recombinant plasmid as DNA template. The PCR assay was validated on positive samples with C. parvurn or C. andersoni and used to detect fecal samples collected from 40 dairy cows in Shanghai. The products were amplified from C. parvum and C. andersoni, but not from C. bailey, Toxoplasma gondii, Neospora caninum and Escherichia coli, by the established SYBR Green real time PCR. The sensitivity of the assay was 5 copies of recombinant plasmid and 5 oocysts per gram in feces. The preliminary application indicated that 6 out of 40（15 %） were Cryptosporidium-posirive. The results showed that the established PCR was rapid, sensitive and specific, and could be served as a valuable tool for epidemiological investigation.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2009年第6期510-515,共6页
Chinese Veterinary Science
基金
上海市科技兴农重点攻关项目(沪农科攻字2005第3-4号)
