Real-time qPCR检测鸡胚中贝氏隐孢子虫方法研究

Development of a SYBR green real-time qPCR assay for detection of Cryptosporidium baileyi cultured in chicken embryo

为检测鸡胚中贝氏隐孢子虫,本研究根据GenBank公布的贝氏隐孢子虫和原鸡18SrRNA基因序列设计针对贝氏隐孢子虫的特异性引物,并以贝氏隐孢子虫卵囊基因组DNA为模板,建立检测贝氏隐孢子虫SYBR Green实时荧光定量PCR(Real-time qPCR)方法,同时对贝氏隐孢子虫在鸡胚中培养7d后尿囊膜和尿囊液样品中虫体DNA进行检测。结果表明,建立的SYBR Green real-time qPCR方法可特异性检测到贝氏隐孢子虫,建立的标准曲线线性关系良好(R2=0.998),卵囊基因组DNA检测阈值为10个卵囊,重复检测结果显示试验重现性较好。该方法成功检测到尿囊膜和尿囊液中虫体DNA。本研究建立了快速、特异的定量检测鸡胚中贝氏隐孢子虫real-time qPCR方法,可用于对贝氏隐孢子虫在鸡胚中繁殖规律分析和抗隐孢子虫药物筛选等研究。

In order to detect Cryptosporidium baileyi in chicken embryo,real-time qPCR method was established with primers designed according C. baileyi and chicken 18S rRNA gene from Gen- Bank. This method was validated on chorioallantoic membrane and allantoic fluid samples which were collected at 7 d after C. baileyi oocysts were innoculated. The results showed that amplifica- tion of DNA from C. baileyi oocyst were detected and cells of chorioallantoic membrane were not. Standard curves of real-time qPCR was precise （R2 =0. 998）. The sensitivity of the assay was 10 oocysts in PBS. In summary, a rapid and specific real-time qPCR method for the detection of C. baileyi in chicken embryo samples has been developed,which could be served as a valuable tool for evaluating reproduction and assessing potential drug against C. baileyi cultured in chicken embryo.