【摘要】 目的建立一种方便快捷的检测微小隐孢子虫的实时荧光定量PCR(Real-time PCR)方法。方法根据GenBank公布的微小隐孢子虫18s rRNA基因序列设计1对引物,通过质粒DNA和卵囊检测标准曲线的绘制及特异性和重复性的研究,建立检测微小隐孢子虫的基于SYBR Green的Real-time PCR方法,进而对奶牛粪便进行检测。结果建立的Real-time PCR法对检测微小隐孢子虫具有很高的特异性,质粒DNA和卵囊的检测阈值分别达到1个拷贝和10个卵囊,奶牛粪便阳性率为25.93%(21/81),高于普通PCR检测率20.99%(17/80)。结论建立的Real-timePCR方法简便、快速,特异性强,敏感度高,可用于微小隐孢子虫的快速定量检测。更多还原

【Abstract】 Objective To develop a real-time fluorescent quantitative PCR assay(real-time PCR) for detection of Cryptosporidium parvum. Methods A pair of primers was designed according to the small subunit ribosomal RNA of C.parvum in GenBank,and real-time PCR assay was established for detection of C.parvum by drawing standard curves of recombinant plasmid and oocysts,and the sensitivity,specificity,and repeatability of the assay were determined by detection in cattle feces.Results Real-time PCR was highly differential to C.parvum,and low amounts detected by real-time PCR were single copies of plasmid DNA and ten oocysts in a gram of feces.A total of 21(25.53%,21/81) cows were positive according to the assay,which was a higher detection rate than that of typical PCR(20.99%,17/81).Conclusion The real-time PCR assay is simple,quick,and suitable for the quantitative detection of C.parvum with exceptional sensitivity and specificity.更多还原