A RT-PCR Assay for Quantitative Detection of Bovine Viral Diarrhea Virus
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摘要:目的 设计一种能够快速检测出牛病毒性腹泻病毒（bovine viral diarrhea virus, BVDV）的方法。方法 依据GenBank公布的BVDV 5′UTR基因属于特异性保守区域的前提，构建具有特异性的探针和引物，以体外转录病毒RNA作为绝对定量标准品。对荧光定量RT-PCR方法的各反应条件进行优化，创建BVDV荧光定量PCR检测方法。结果 5.026 7 copies·μL−1是此检测方法的最低下限值，该方法拥有良好的重复性，变异系数在组内、组间均＜1%。特异性较高，对其他病毒核酸的扩增均为阴性，如猪瘟病毒、口蹄疫病毒等。结论 建立的BVDV荧光定量PCR方法敏感性高、特异性强、重复性好，为牛病毒性腹泻的早期诊断提供了重要的技术支撑。Abstract:Objective A rapid detection and quantification method for bovine viral diarrhea virus (BVDV) was established.Method For the methodology development, specific primers and probes were designed based on the target regions of the BVDV 5′UTR gene published by GenBank. The RNA of in vitro transcription viruses was used as the absolute quantitative standard. Reaction conditions of the fluorescent quantitative RT-PCR were optimized.Result The newly developed assay had a minimum detection limit of 5.0267copies/μL and an intragroup variation coefficient of less than 1% with high repeatability and specificity. Other than BVDV, it amplified no viral nucleic acids of viruses such as swine fever and foot-and-mouth diseases.Conclusion The established fluorescence quantitative RT-PCR method was highly sensitive, specific, and repeatable in detecting and quantifying BVDV. It appeared appropriate for early diagnosis of bovine viral diarrhea.
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Keywords:
- Bovine viral diarrhea virus /
- 5′UTR /
- RT-PCR /
- standard RNA
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图 1 标准品引物扩增产物
注：M为DNA marker；1为PCR产物；2为阴性对照。
Figure 1. Amplification products of RNA standard primers
Note: M: DNA marker; 1: PCR product; 2: negative control.
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图 2 荧光定量PCR方法特异性试验
注：A为阳性样品（BVDV NADL标准毒株）；B为FMDV；C为IBRV；D为水（阴性对照）；E为CSFV；F为PRRS。
Figure 2. Specificity of RT-PCR assay
Note: A: BVDV positive control; B: FMDV; C: IBRV; D: negative control; E: CSFV; F: PRRS.
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图 6 荧光定量PCR方法的应用
注：1为阳性样品（BVDV NADL标准毒株）；2～5为临床样品检出BVDV阳性。
Figure 6. Application of RT-PCR assay
Note: 1: BVDV positive control, 2-5: BVDV positive clinical samples were detected.
表 1 引物和探针
Table 1 Primers and probes applied
引物名称
Primers引物序列（5′→3′）
Primer sequences（5′-3′）目的片段大小/bp
Product size/bpA-F TAATACGACTCACTATAGGG-CATGCCCATAGTAGGAC 325 B-R TTTTTTTTTTTTTTTTTTTTTTT CCATGTGCCATGTACAG Q-F GAGGGTAGCAACAGTGGTGA 76 Q-R AGGCGTCGAACCACTGACG 探针 FAM-GTGGTGAGTTCGTTGGATGGCT-BHQ1 
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表 2 核酸浓度
Table 2 Nucleic acid concentration
扩增曲线
Amplification curve核酸浓度
Nucleic acid concentration（copies ·μL−1）1 5.026 7×108 2 5.0267×107 3 5.026 7×106 4 5.026 7×105 5 5.026 7×104 6 5.026 7×103 7 5.026 7×102 8 5.026 7×101 9 5.026 7 10 0.502 67
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表 3 实时荧光定量PCR方法的变异系数
Table 3 Coefficients of variation between inter- and intra-groups of RT-PCR assay
稀释度
Dilution rate组内重复性 Reproducibility intra-batch 组间重复性 Reproducibility inter-batch Ct平均值
Ct average values组内变异系数
Coefficient of variation/%Ct平均值
Ct average values组间变异系数
Coefficient of variation/%浓度1 21.82±0.12 0.55 21.79±0.08 0.37 浓度2 25.31±0.19 0.75 24.93±0.49 1.97 浓度3 28.63±0.27 0.94 28.45±0.16 0.56 浓度4 31.21±0.27 0.87 31.27±0.07 0.22 浓度5 35.65±0.27 0.76 35. 14±0.48 1.37
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