畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (2): 404-408.doi: 10.11843/j.issn.0366-6964.2020.02.023

• 研究简报 • 上一篇    

猪星状病毒4型SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

陈少杰1, 刘立辉2, 吴志勇3, 肖娜2, 袁广富1, 王晶1, 邢亚茹1, 刘如月1, 范京惠1*, 左玉柱1*   

  1. 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
  • 收稿日期:2019-09-19 出版日期:2020-02-23 发布日期:2020-02-22
  • 通讯作者: 左玉柱,主要从事动物传染病学的教学、科研和社会服务工作,E-mail:zuoyuzhu@163.com;范京惠,主要从事兽医微生物与免疫学的教学、科研工作,E-mail:jinghui76@163.com
  • 作者简介:陈少杰(1993-),男,河北邢台人,硕士生,主要从事动物传染病防控研究,E-mail:274559723@qq.com
  • 基金资助:
    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)

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

CHEN Shaojie1, LIU Lihui2, WU Zhiyong3, XIAO Na2, YUAN Guangfu1, WANG Jing1, XING Yaru1, LIU Ruyue1, FAN Jinghui1*, ZUO Yuzhu1*   

  1. 1. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China;
    2. Dingzhou Bureau of Agricultural and Rural Affairs, Dingzhou 073000, China;
    3. Handan Animal Husbandry Technology Promotion Station, Handan 056000, China
  • Received:2019-09-19 Online:2020-02-23 Published:2020-02-22

摘要: 为建立一种快速检测猪星状病毒4型(PAstV4)的SYBR Green Ⅰ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 Green Ⅰ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 Green Ⅰ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。

关键词: 猪星状病毒, 荧光定量, SYBR Green Ⅰ

Abstract: 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 ORF1a 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.

Key words: Porcine astrovirus, RT-PCR, SYBR Green Ⅰ

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