Establishment and Monitoring Analysis of Fluorescence RT-PCR for Detection of Bovine Viral Diarrhea Virus in Swine
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摘要: 根据牛病毒性腹泻病毒（BVDV）5'-UTR保守序列建立的特异性荧光RT-PCR，对采自福州市周边屠宰场的359份屠宰猪血清进行BVDV感染的病原学检测。结果待检的359份血清样品BVDV阳性52份，阳性率为14.5%（52/359）。对其中13份BVDV阳性样品5'端UTR片段进行序列测定分析，发现所测13份BVDV阳性样品5'端UTR片段均与VEDEVAC进化谱系较为密切，均为基因Ⅰ型。以上结果表明以牛源BVDV建立的特异性荧光RT-PCR方法适用于猪源BVDV感染的监测，并揭示福州市屠宰猪存在BVDV感染。
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关键词:
- 猪源牛病毒性腹泻病毒 /
- 荧光RT-PCR /
- 屠宰猪 /
- 病原学检测
Abstract: The study was aimed at developing a quantitative real-time fluorescence reverse transcriptase PCR(qRT-PCR) assay according to the conserved gene sequence of BVDV 5'-UTR for detection of bovine viral diarrhea virus(BVDV) obtained from 359 slaughtered swine serum samples of slaughterhouses in Fuzhou Prefecture. The results showed that the BVDV positive rate of slaughtered swines was 14.5%(52 positive samples out of 359 ones). Sequence analysis on 5'-UTR fragments of 13 BVDV positive samples showed that 5'-UTR fragments of all tested samples were closely related to VEDEVAC evolution lineages, all of which were genotype I. It was indicated that the specific qRT-PCR method established based on BVDV was suitable for detection of BVDV in swines. And the BVDV infection was common in slaughtered swines of Fuzhou Prefecture.-
Key words:
- BVDV in swine /
- fluorescence RT-PCR /
- slaughtered swine /
- pathogenic detection
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图 1 BVDV实时荧光定量RT-PCR扩增结果
注：P为阳性对照；N为阴性对照。
Figure 1. Real-time quantitative RT-PCR amplification of BVDV
图 2 BVDV敏感性实时荧光定量RT-PCR扩增结果
注：a~e为1×102.2~1×10-3.8TCID50/50 μL样品扩增曲线；g为阴性对照。
Figure 2. Susceptibility of real-time quantitative RT-PCR amplification of BVDV
图 3 BVDV组间重复性实时荧光定量RT-PCR扩增结果
注：a~e为组间重复样品扩增曲线；f为阴性对照。
Figure 3. Inter-group repeatability of real-time quantitative RT-PCR amplification of BVDV
图 4 BVDV实时荧光定量RT-PCR扩增结果
注：S1、S2为阳性样品扩增曲线；P为阳性对照；N为阴性对照。
Figure 4. Real-time quantitative RT-PCR amplification of BVDV
表 1 荧光RT-PCR反应体系
Table 1. The reaction system of Fluorescence RT-PCR
成分 用量/μL 10×RT-PCR buffer solution 2.5 MgCl2(25 mmol·L-1) 5.0 dNTP(10 mmol·L-1) 2.5 RNasin(40 U·μL-1) 0.5 AMV reverse transcriptase (5 U·μL-1) 0.5 Taq DNA polymerase(5 U·μL-1) 0.5 upstream primers(10 μmol·L-1) 0.5 downstream primers(10 μmol·L-1) 0.5 TaqMan probe(5 μmol·L-1) 0.5 RNA 5.0 RNase-Free water 7.0 
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