为建立鸭腺病毒3型(duck adenovirus 3,DAdV-3)一种基于SYBR Green Ⅰ染料的荧光定量PCR诊断方法,本试验根据DAdV-3基因组序列中相对保守的Fiber-2基因,通过普通PCR扩增,构建pMD19-Fiber2重组质粒为阳性标准品,在此基础上设计合成特异性引物进行实时荧光定量PCR方法的建立,对其灵敏性、特异性和重复性进行评价,并初步应用于临床样本检测.结果显示,针对DAdV-3的Fiber-2基因建立的SYBR Green Ⅰ荧光定量PCR检测方法线性关系良好,相关系数0.998 9;检测DAdV-3的灵敏度为3.6 × 102copies/μL,敏感性比普通PCR检测方法高100倍;该方法对鸭腺病毒1型、鸭疫里默杆菌等其他常见病原均无交叉扩增反应,批内、批间变异系数均小于5％;临床样本检测与普通PCR符合率100％.以上结果表明,所建方法特异性强、敏感性高且重复性好,适用于临床检测,该方法可为DAdV-3的临床诊断、流行病学调查以及防控提供技术支持.
Establishment and application of SYBR Green Ⅰ real-time fluorescence quantita-tive PCR detection method for duck adenovirus type 3
In order to establish a fluorescence quantitative PCR(qPCR)diagnostic method based on SYBR Green Ⅰ dye for detecting duck adenovirus 3(DAdV-3),the conserved Fiber-2 gene of DAdV-3 genome sequence was amplified by PCR method and ligated to pMD19-T vector.The re-combinant plasmid acted as a positive standard.After that,specific primers were designed and syn-thesized to establish a real-time fluorescence qPCR method.The sensitivity,specificity,and repeat-ability of the qPCR method were evaluated,and then preliminarily applied in the clinical detection.Results showed that the established qPCR method had a positive linear relationship with a correla-tion coefficient of 0.998 9.While the sensitivity was 3.6 ×102 copies/μL,which 100 times higher than that of ordinary PCR detection method.No cross-amplification reaction was found with other common duck pathogens such as duck adenovirus type 1 and Riemerella anatipestifer.The coeffi-cient of variation within and between batches were both less than 5％,indicating that the repeatability was good.When compared with ordinary PCR method,the coincidence rate was 100％.All these findings revealed that the established qPCR method had strong specificity,high sensitivi-ty and good repeatability.It could be applied in the clinical testing and provide technical support for the diagnosis,epidemiological investigation,and prevention and control of DAdV-3.
duck adenovirus type 3Fiber-2 genereal time fluorescence quantitative PCR
NETL
NSTL
万方数据
