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猪圆环病毒3型TaqMan实时荧光定量PCR检测方法的建立

陈如敬 吴学敏 陈秋勇 车勇良 王隆柏 严山 周伦江

陈如敬,吴学敏,陈秋勇,等. 猪圆环病毒3型TaqMan实时荧光定量PCR检测方法的建立 [J]. 福建农业学报,2020,35(7):739−745 doi: 10.19303/j.issn.1008-0384.2020.07.007
引用本文: 陈如敬,吴学敏,陈秋勇,等. 猪圆环病毒3型TaqMan实时荧光定量PCR检测方法的建立 [J]. 福建农业学报,2020,35(7):739−745 doi: 10.19303/j.issn.1008-0384.2020.07.007
CHEN R J, WU X M, CHEN Q Y, et al. A TaqMan RT-PCR Method for Detecting Porcine Circovirus 3 [J]. Fujian Journal of Agricultural Sciences,2020,35(7):739−745 doi: 10.19303/j.issn.1008-0384.2020.07.007
Citation: CHEN R J, WU X M, CHEN Q Y, et al. A TaqMan RT-PCR Method for Detecting Porcine Circovirus 3 [J]. Fujian Journal of Agricultural Sciences,2020,35(7):739−745 doi: 10.19303/j.issn.1008-0384.2020.07.007

猪圆环病毒3型TaqMan实时荧光定量PCR检测方法的建立

doi: 10.19303/j.issn.1008-0384.2020.07.007
基金项目: 福建省科技计划公益类专项(2018R1023-8);福建省自然科学基金项目(2018J01042);福建省农业科学院自由探索科技创新项目(ZYTS2019021)
详细信息
    作者简介:

    陈如敬(1984−),男,硕士,助理研究员,研究方向:动物传染病(E-mail:fjchenrujing@163.com

    通讯作者:

    周伦江(1973−),男,博士,研究员,研究方向:动物病毒分子生物学(E-mail:lunjiang@163.com

  • 中图分类号: S 855.3

A TaqMan RT-PCR Method for Detecting Porcine Circovirus 3

  • 摘要:   目的  建立检测猪圆环病毒3型(Porcine circovirus 3, PCV3)感染的TaqMan实时荧光定量PCR方法。  方法  通过分析明确PCV3复制相关蛋白Rep基因特征,设计针对Rep基因的特异性引物和探针,经条件优化后建立检测PCV3感染的TaqMan实时荧光定量PCR方法。  结果  建立的TaqMan实时荧光定量PCR方法敏感性好,最低检测限为42.2拷贝·μL−1;特异性强,对常见猪群传染病均无交叉反应;重复性好,组内变异系数和组间变异系数均在1.48%以内。对福建省2014年至2018年保存的193份组织样品进行检测发现,PCV3在福建省猪群中存在较高的阳性感染率(65.80%),且和PCV2混合感染率较高(52.85%)。  结论  本方法的建立为开展Rep基因在PCV3复制和感染过程中的作用机制提供检测方法。
  • 图  1  PCV3不同代表株Rep基因遗传进化分析

    Figure  1.  Phylogenetic analysis based on Rep of PCV 3

    图  2  实时荧光定量PCR检测方法的扩增曲线

    注:0−6:质粒拷贝数分别为4.22×100~4.22×106 拷贝·μL−1

    Figure  2.  Amplification curve of RT-PCR

    Note: 0–6: numbers of plasmid copies from 4.22×100 to 4.22×106 copy·μL−1.

    图  3  实时荧光定量PCR检测方法的标准曲线

    Figure  3.  Standard curve of RT-PCR

    图  4  实时荧光定量PCR检测方法的特异性

    注:1:PCV3阳性;对照:PCV1、PCV2、PPV、PRV、v-PRV、PCMV、PBoV、PTTSuV 1a和PTTSuV 1b,样品均为阴性,无荧光扩增信号。

    Figure  4.  Specificity of TaqMan RT-PCR method

    Note: 1: PCV3 positive control; experimental control groups: PCV1, PCV2, PPV, PRV, v-PRV, PCMV, PBoV, PTTSuV 1a, and PTTSuV 1b (all negative with no fluorescent signal).

    表  1  实时荧光定量PCR方法的变异系数

    Table  1.   Coefficients of variation (CV) of TaqMan RT-PCR between inter- and intra-groups

    序号 Number拷贝数 Copies组内 (Intra-group) 组间 (Inter-group)
    Ct值±标准差 Ct value ± SD变异系数 CV/% Ct值±标准差 Ct value ± SD变异系数 CV/%
    1 4.22×106 14.82±0.09 0.58 14.83±0.13 0.85
    2 4.22×104 22.24±0.16 0.72 22.28±0.18 0.82
    3 4.22×102 29.64±0.33 1.10 29.78±0.44 1.48
    下载: 导出CSV

    表  2  临床样品的检测

    Table  2.   Detection on clinical samples by TaqMan RT-PCR method

    项目 Item样品数 NumberPCV3 PCV2 PCV2&PCV3
    阳性样品
    Positive samples
    阳性率
    Positive ratio/%
    阳性样品
    Positive samples
    阳性率
    Positive ratio/%
    阳性样品
    Positive samples
    阳性率
    Positive ratio/%
    2014年 25 7 28.00 21 84.00 5 20.00
    2015年 28 13 46.43 20 71.43 11 39.29
    2016年 41 28 68.29 34 82.93 18 43.90
    2017年 63 51 80.95 55 87.30 43 68.25
    2018年 36 28 77.78 31 86.11 25 69.44
    合计 Total 193 127 65.80 161 83.42 102 52.85
    下载: 导出CSV
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  • 收稿日期:  2019-09-23
  • 修回日期:  2020-05-05
  • 刊出日期:  2020-07-31

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