基因2型鹅星状病毒EvaGreen荧光定量一步法RT-PCR的建立与应用

Establishment and application of EvaGreen one-step qRT-PCR for goose astrovirus 2

目的:建立快速、灵敏、特异的基因2型鹅星状病毒(Goose astrovirus 2,GoAstV-2)检测方法。方法:以GoAstV-2 ORF2基因为靶标,设计特异性检测引物并构建含目标基因的重组质粒作为阳性质粒标准品,以其为模板优化建立一种EvaGreen饱和染料荧光定量一步法RT-PCR检测方法。结果:所建立方法采用dUTP/UDG酶防污染体系,其标准曲线在2.58×10^(1)~2.58×10^(7) copies/μL质粒模板量呈现良好的线性关系,斜率为-3.413,相关系数(R^(2))为0.995,熔解温度T_(M)为(85.5±0.5)℃;该方法对质粒标准品检测灵敏度下限为25.8 copies/μL;该方法特异性检测GoAstV-2,而新城疫病毒(NDV)、H5型禽流感病毒(AIV-H5)、禽呼肠孤病毒(ARV)等11种常见禽传染病病原检测结果均为阴性。重复性试验显示,组内和组间变异系数均小于2%,表明其具有良好的重复性。GoAstV-2感染病死鹅组织定量检测显示,心脏、肝脏、脾脏、肺脏、肾脏、腺胃、胰腺、小肠、大脑和肌肉组织均可检测出GoAstV-2,其中肾脏病毒含量最高(2.58×10^(11) copies/g),大脑和肌肉组织含毒量较低。32份临床送检病死鹅组织样品的检测显示,9份存在GoAstV-2感染,阳性率为28.1%。结论:EvaGreen荧光定量一步法RT-PCR能高效检测GoAstV-2,可应用于禽类GoAstV-2感染的诊断和净化检测。

Objective:To establish a rapid,sensitive and specific detection method for Goose astrovirus 2(GoAstV-2).Methods:Specific detection primers of ORF2 gene were designed at the conserved region.The target gene was amplified and cloned to plasmid vector.The standard plasmid was used as the positive plasmid standard to establish an EvaGreen saturated dye one-step qRT-PCR.Results:The established method adopted dUTP/UDG enzyme to prevent pollution of products,and a good linear relationship when the standard plasmid template was among in 2.58×10^(1)-2.58×10^(7) copies/μL,with a correla tion coefficient(R^(2))of 0.995,and a slope of-3.413.The melting temperature(T_(M))was(85.5±0.5)℃.The limited detection contend of standard plasmid of this method was 25.8 copies/μL.The detection of GoAstV-2 was specific,and that the detection results of eleven common avian infectious diseases such as Newcastle disease virus(NDV),H5 type Avian influenza virus(AIV-H5)and avian reovirus(ARV)were negative.The intra-group and inter-group coefficients of variation were less than 2%,indicating that it had good repeatability.The quantitative detection of tissues of dead geese infected with GoAstV-2 showed that it could be detected in heart,liver,spleen,lung,kidney,glandular stomach,pancreas,small intestine,brain and muscle tissues,and the content of virus was the highest(2.58×10^(11) copies/g)in kidney.The detection of 32 clinical dead goose tissue samples showed that nine samples were positive(28.1%).Conclusion:The EvaGreen one-step qRT-PCR method can effectively detect GoAstV-2,and can be applied to the diagnosis and purification detection of GoAstV-2 infection.