快速荧光定量RT-PCR检测对虾Taura综合征病毒(TSV)的方法建立与应用  

Development and application of fast real-time quantitative RT-PCR assay for detection of taura syndrome virus in penaeid shrimp

作　　者：顾松叶[1] 王忠发[1] 施慧[2] 沈飚 

机构地区：[1]浙江省舟山市疾病预防控制中心,浙江舟山316021 [2]浙江省海洋水产研究所,浙江舟山316100 [3]舟山出入境检验检疫局,浙江舟山316000

出　　处：《中国卫生检验杂志》2011年第9期2213-2216,共4页Chinese Journal of Health Laboratory Technology

基　　金：浙江省科技厅项目(2008C32035);浙江省卫生厅项目(2007A199)

摘　　要：目的:建立桃拉综合征病毒快速荧光定量RT-PCR用于提高我市对虾出口检验检疫工作效率和疫病防控。方法:选取TSV基因组的保守序列,利用Primer Express 2.0软件设计引物和探针,扩增产物长度为73 bp。构建TSV目的扩增片段的质粒为标准品,对TSV的核酸提取、RT-PCR进行优化。结果:TSV核酸提取20 min、RT-PCR34 min,检测灵敏度为0.03 fg/μl,组内的实验变异系数为0.25%～1.17%、组间实验变异系数为1.51%～2.60%,与10种病原微生物无非特异反应,定量标准曲线相关系数=-1。结论:本方法用于TSV核酸定量检测速度极快,灵敏度与特异度高、重复性好、定量准确,各项指标均优于标准方法。Objective： A fast real-time quantitative RT-PCR method for Taura Syndrome Virus（TSV） detection was developed and would be implemented for improving the efficiency of penaeid shrimp export inspection and epidemic disease control and prevention.Methods： By using the Primer express 2.0,primers and probe which give an amplicon of 73 bp were designed based on a conserved region from TSV genome.And a plasmid containing target TSV sequence was constructed and served as the standard.In this study,Virus RNA extraction and RT-PCR reaction time were also optimized.Results： The time for RNA extraction and RT-PCR was reduced to 20 minutes and 34 minutes respectively.Detection limit of our method could reach 0.03 fg/μl.The inter coefficient variations（CV） ranged from 0.25% to 1.17%,while the intra CV were from 1.51% to 2.60%.And the correlation coefficient（r） equaled-1.No non-specific reactions were found with ten common pathogenic microorganism.Conclusion： This method for quantification of TSV copy is extremely quick and highly sensitive and specific.The reproducibility and accuracy are excellent as well.It is a way better than standard method in all aspects.

关 键 词：TAURA综合征病毒 定量检测 快速荧光定量RT-PCR RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]