实时荧光定量PCR检测对虾白斑综合症病毒方法的建立 |
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引用本文: | 程晓艳,刘庆慧,黄倢. 实时荧光定量PCR检测对虾白斑综合症病毒方法的建立[J]. 安徽农业科学, 2010, 38(26): 14265-14267. DOI: 10.3969/j.issn.0517-6611.2010.26.028 |
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作者姓名: | 程晓艳 刘庆慧 黄倢 |
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作者单位: | 中国海洋大学,山东青岛,266003;农业部海洋渔业资源可持续利用重点开放实验室,中国水产科学院黄海水产研究所,山东青岛,266071;农业部海洋渔业资源可持续利用重点开放实验室,中国水产科学院黄海水产研究所,山东青岛,266071 |
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基金项目: | 国家自然科学基金项目,国家重点基础研究项目,对虾行业专项 |
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摘 要: | [目的]根据对虾白斑综合症病毒(White Spot Syndrome Virus,WSSV)的保守序列,利用Primer5.0软件设计引物,建立了WSSV的荧光实时定量PCR检测体系。[方法]构建含有WSSV目扩增片段的质粒为标准品,进行定量PCR扩增,探索反应条件。[结果]确定了最佳退火温度(61℃)和最佳引物浓度(0.2μmol/L),标准品浓度在8.8×1098.8×104个拷贝数之间有典型的"s"型扩增曲线,标准曲线中模板拷贝数(X)与Ct值的关系为Ct=-3.597lgX+42.547,相关系数R2=0.993。[结论]该检测方法特异性强,对传染性皮下和造血器官坏死病毒(IHHNV)、肝胰腺细小病毒(HPV)没有扩增反应。
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关 键 词: | 实时荧光定量PCR 白斑综合症病毒 检测 |
Establish of Real-time Fluorescent Quantitative PCR Assay for Detecting White Spot Syndrome Virus |
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Affiliation: | CHENG Xiao-yan et al(Ocean University of China,Qingdao,Shandong 266003) |
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Abstract: | [Objective] A real-time fluorescent quantitative PCR assay utilizing SYBR green Ⅰdye was developed for detection and quantization of white spot syndrome virus particles isolated from infected shrimp.[Method]A pair of specific primers which could amplify a 221 bp fragment were designed based on Primer Express 5.0 software.The standards were established with plasmid containing the conserved region of WSSV.Reaction conditions were explored.[Result] The result showed that 61 ℃ was the best annealing temperature and 0.2 μmol/L was the best primers concentration.Typical "s" amplification curves were showed between concentrations of 8.8×109-8.8×104 copies.The linear relationship between virus concentration(X)and Ct was Ct=-3.597 lgX+42.547 with the correlation coefficient R2 =0.993.[Conclusion]The primers were specific for WSSV and did not react with either Infectious Hypodermal and Hematopoietic Necrosis Virus(IHHNV)or Hepatopancreatic Parvovirus(HPV). |
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Keywords: | SYBR GreenⅠreal-time PCR WSSV Detection |
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