猪星状病毒4型SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立  

Establishment and Application of a SYBR Green Ⅰ Real-time PCR Technique for the Detection of Porcine Astrovirus 4

作　　者：陈少杰 刘立辉 吴志勇 肖娜 袁广富 王晶[1] 邢亚茹 刘如月 范京惠[1] 左玉柱[1] CHEN Shaojie;LIU Lihui;WU Zhiyong;XIAO Na;YUAN Guangfu;WANG Jing;XING Yaru;LIU Ruyue;FAN Jinghui;ZUO Yuzhu(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Dingzhou Bureau of Agricultural and Rural Affairs,Dingzhou 073000,China;Handan Animal Husbandry Technology Promotion Station,Handan 056000,China)

机构地区：[1]河北农业大学动物医学院,保定071001 [2]定州市农业农村局,定州073000 [3]邯郸市畜牧技术推广站,邯郸056000

出　　处：《畜牧兽医学报》2020年第2期404-408,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：河北省重点研发计划项目(19226622D);河北省农业产业技术体系生猪创新团队(HBCT2018110207)。

摘　　要：为建立一种快速检测猪星状病毒4型(PAstV4)的SYBR GreenⅠ荧光定量RT-PCR检测方法,根据已知的猪星状病毒4型序列的ORF1a基因设计了特异性引物,并将扩增片段克隆到pMD19-T载体上;将构建的重组质粒作为标准品建立SYBR GreenⅠ荧光定量RT-PCR检测方法,并对建立方法的灵敏性、特异性及重复性进行验证。结果显示,建立的SYBR GreenⅠ荧光定量PCR方法最低检测量为50.3拷贝·μL-1,其灵敏度是普通PCR的100倍;与CSFV、PRRSV、PRV、PEDV、PDCoV无交叉反应,特异性较好;建立的标准曲线呈良好的线性关系,相关系数R2=1.00。应用该方法检测了2018-2019年收集的43份临床样品,阳性率为18.6%。本研究为猪星状病毒4型的临床检测建立了一种快速、灵敏、特异性强的方法。In order to establish a SYBR Green Ⅰ fluorescence quantitative RT-PCR method for the rapid detection of Porcine astrovirus 4(PAstV4), a pair of specific primer was designed based on the conservative ORF1 a gene. The amplified fragment was cloned into pMD19-T vector. The acquared recombinant plasmid, as a standard, was detected by SYBR Green Ⅰ fluorescence quantitative RT-PCR assay. And the sensitivity, specificity and repeatability were verified. The results showed that the minimum detectable amount of the method was 50.3 copies·μL-1, which was 100 times more sensitive than the traditional PCR, and there was no cross reaction with CSFV, PRRSV, PRV, PEDV and PDCoV. The established standard curve showed a good linear relationship with R2=1.00. The established method was used to detect 43 clinical samples collected from 2018 to 2019, with a positive rate of 18.6%. These results indicated that a convenient, specific and sensitive qPCR assay was successfully established for the detection of PAstV4.

关 键 词：猪星状病毒 荧光定量 SYBR GreenⅠ 

分 类 号：S852.659.6[农业科学—基础兽医学]