鹅圆环病毒实时荧光定量PCR检测方法的建立

Development of a real-time PCR assay for detection of goose circovirus

为建立鹅圆环病毒(GoCV)快速检测方法,本研究根据GenBank中登录的GoCV全长基因组序列,在其保守基因区设计并合成1对引物,采用荧光嵌合法(SYBR GreenⅠ)建立检测GoCV的实时荧光定量PCR(real-time PCR)方法。以本实验室构建的含有GoCV永康株(GoCV-yk01)全长基因组的重组质粒为标准模板,经退火温度、循环次数等反应条件的优化,绘制GoCV real-time PCR的标准曲线,并进行融解曲线分析。试验结果表明:建立的GoCV real-time PCR方法特异性强,检测范围广(Ct值范围12～32),最低检出病毒拷贝数为1.46×102。该方法能够对组织中病毒进行定量检测,为快速检测GoCV提供了有效的技术手段。

A SYBR GreenⅠreal-time PCR for detecting Goose circovirus（GoCV） was established using primers derived from the published sequences.A plasmid containing GoCV-yk01 genome was constructed and served as a standard template to generate the standard curve.The assay had high specificity with a wide detection range（Ct value： 12 to 32） and could detect as low as 1.46×102 copies of virus DNA.This method could be used for rapid detection of GoCV in clinical samples.