猪腺病毒3型SYBR GreenⅡ实时荧光定量PCR检测方法的建立与应用

Development and application of a SYBR GreenⅡreal-time PCR for detection of porcine adenovirus type 3

为建立一种针对猪腺病毒3型（PADV3）的检测方法,根据PADV3E1B基因保守序列设计1对引物,建立了检测PADV3的SYBR GreenⅡ实时荧光定量PCR。结果显示,该方法不能对猪瘟病毒、猪流行性腹泻病毒、猪博卡病毒、猪传染性胃肠炎、伪狂犬病病毒、猪圆环病毒2型、大肠杆菌、猪链球菌2型检测到荧光信号,特异性好;检测PADV3时在3.7×10^2-3.7×10^9 copies/μL范围内具有良好的线性关系,其扩增相关系数为0.999,扩增产物的熔解曲线仅出现单一特异峰,无引物二聚体,熔解温度为90.5℃;该方法组内变异系数为0.17%-1.23%,组间变异系数为0.73%-2.23%,重复性较好。应用该方法对临床56份腹泻粪便检测,检出11份阳性,且与常规PCR检测方法的阳性符合率为100%。上述结果表明,建立的实时荧光定量PCR为PADV3感染的早期诊断、流行病学调查奠定了技术基础。

In order to establish a real-time PCR based on SYBR GreenⅡfor detection of porcine adenovirus type 3（PADV3）,apair of special primers was designed according to the conserved sequences of E1 Bgene in GenBank.In result,the standard curve of established SYBR GreenⅡreal-time PCR had a wide dynamic range from 3.7×10^2 to 3.7×10^9 copies/μL with a linear correlation（r2）of 0.999.The melting curve analysis using SYBR GreenⅡdye showed one specific peak with a melting temperature（Tm）of 90.5℃.No amplification was detected from the DNA samples of classical swine fever virus,porcine epidemic diarrhea virus,porcine bocavirus,transmissible gastroenteritis virus of swine,pseudorabies virus,porcine circovirus type 2,Escherichia coli and Streptococcus suis type 2by this PCR,respectively.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.17% to1.23% and inter-assay of 0.73% to 2.23%.Further detection of 56 specimens showed that 11 of them were PADV3 positive by this method,and the results of the quantitative PCR were the same as that of the conventional PCR.In conclusion,the real-time PCR established in this study will be useful for earlier rapid laboratory diagnosis and epidemiological investigation of the infection of PADV3.