A TaqMan RT-PCR Method for Detecting Porcine Circovirus 3
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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。 -
关键词:
- 猪圆环病毒3型 /
- Rep基因 /
- TaqMan实时荧光定量PCR方法 /
- 检测
Abstract:Objective A TaqMan RT-PCR method was established for detecting porcine circovirus 3 （PCV3） infection in swine. Method Specific primers and probe were designed by Oligo 7 software targeting the Rep gene after genetic comparison. Result The established TaqMan RT-PCR method could detect 42.2 copies·uL−1 with no positive signal on common porcine infectious diseases indicating high specificity of the methodology. On the constructed positive plasmids, the coefficients of variation for the intra- and inter-assays were less than 1.48% showing a high detection reproducibility. From the 193 clinical specimens collected in Fujian from 2014 to 2018, the newly developed method showed a high prevalence of PCV3 at 65.80% and high co-infection with PCV2 at 52.85%. Conclusion The established TaqMan RT-PCR method was made available for studying the function of Rep in PCV3. -
Key words:
- Porcine circovirus 3 /
- Rep gene /
- TaqMan RT-PCR method /
- detection
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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 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 
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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 Number PCV3 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
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