| 许建明,张念之,蒋一男,张利峰,夏春.Taqman MGB探针快速定量检测VHSV方法的研究[J].高技术通讯(中文),2010,20(2):208~213 |
| Taqman MGB探针快速定量检测VHSV方法的研究 |
| Absolute quantitative real time RT PCR assay for rapid detection of viral hemorrhagic septicemia virus (VHSV) with Taqman MGB prob |
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| DOI： |
| 中文关键词: 病毒性出血性败血症病毒(VHSV)，Taqman MGB探针，荧光RT PCR法，定量检测 |
| 英文关键词: viral hemorrhagic septicemia virus (VHSV), Taqman MGB probe, real time RT PCR, quantitative detection |
| 基金项目:国家质检总局（2007IK031）资助项目 |
| 作者 | 单位 | | 许建明 | 中国农业大学动物医学院农业部预防兽医学重点实验室 | | 张念之 | 中国农业大学动物医学院农业部预防兽医学重点实验室 | | 蒋一男 | 中国农业大学动物医学院农业部预防兽医学重点实验室 | | 张利峰 | 北京出入境检验检疫局 | | 夏春 | 中国农业大学动物医学院农业部预防兽医学重点实验室 |
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| 中文摘要: |
| 为建立准确实时地定量检测病毒性出血性败血症病毒（VHSV），在VHSV N基因保守区设计了Taqman MGB探针与引物对，随后，采用体外转录技术获得了VHSV N基因RNA，并以此为绝对定量标准品，建立了绝对定量(AQ)检测VHSV的实时荧光RT PCR法(AQ RT PCR方法)，并与世界动物卫生组织(OIE)推荐的普通RT PCR法进行了比较。此荧光RT PCR法特异性好，与其他鱼类弹状病毒无交叉反应。检测线性范围为1010～102拷贝/反应，灵法度达102 拷贝/反应。此检测灵敏度比OIE推举的RT PCR法高出5个数量级，比嵌套RT PCR高出1个数量级。此法是出入境检疫VHSV的有效方法。 |
| 英文摘要: |
| An absolute quantitative (AQ) real time RT PCR (AQ RT PCR) method is established and developed for viral hemorrhagic septicemia virus (VHSV) detection. Firstly, the Taqman MGB probe and the primers are designed from highly conserved regions of nucleoprotein (N) gene of VHSV. Secondly, the AQ RT PCR method is established using the quantitative standard samples obtained from the in vitro transcripted VHSV N gene. The comparison of the AQ RT PCR with the conventional RT PCR shows that the AQ RT PCR is more specific and there are no cross reactions with other fish Rhabdoviridae viruses. The linear range of the AQ RT PCR assay is from 1010 copies/reaction to 100 copies/reaction. The low quantitative detection limit is 100 copies/reaction. The sensitivity of the AQ RT PCR is higher than the conventional RT PCR by five orders of magnitude and also by one order of magnitude than the nested PCR. The AQ RT PCR method will be efficiently used in entr exit detection of VHSV. |
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