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| Real-time PCR技术用于中国大菱鲆虹彩病毒的组织敏感性检测及病毒流行情况调查 | |
| 引用本文： | 吴成龙,史成银,黄倢,孔晓瑜.Real-time PCR技术用于中国大菱鲆虹彩病毒的组织敏感性检测及病毒流行情况调查[J].农业生物技术学报,2008,16(5). |
| 作者姓名： | 吴成龙 史成银 黄倢 孔晓瑜 |
| 作者单位： | 1. 中国水产科学研究院黄海水产研究所,农业部海洋渔业资源可持续利用重点开放实验室,青岛,266071;中国海洋大学水产学院,教育部海水养殖重点实验室,青岛,266003 2. 中国水产科学研究院黄海水产研究所,农业部海洋渔业资源可持续利用重点开放实验室,青岛,266071 3. 中国海洋大学水产学院,教育部海水养殖重点实验室,青岛,266003 |
| 基金项目： | 国家重点基础研究发展计划(973计划)，国家自然科学基金，中国水产科学研究院黄海水产研究所基本科研业务费专项，国家高技术研究发展计划(863计划) |
| 摘 要： | 中国大菱鲆虹彩病毒(turbot reddish body iridovirus, TRBIV)是一种感染养殖大菱鲆的鱼类虹彩病毒，它可以引起大菱鲆病毒性红体病并导致养殖大菱鲆大量死亡。本研究利用TRBIV主要衣壳蛋白基因序列设计的一对引物，结合内嵌式核酸染料SYBR Green ?，建立了TRBIV特异的Real-time PCR检测方法。实验结果表明，该对引物具有较高的灵敏度和较强的特异性，能够检测相当于102数量级的TRBIV基因组拷贝，而不与健康大菱鲆组织DNA、淋巴囊肿病毒DNA发生交叉反应。最后，应用建立的Real-time PCR检测方法，开展了TRBIV的组织敏感性检测和病毒流行情况调查。结果发现，大菱鲆的脾脏、肾脏、脑、鳃、心脏、肝脏、消化道、血液等组织中均可检测到TRBIV的存在，其中脾和肾是TRBIV的最主要的靶器官，每毫克组织的病毒含量分别高达5.23?106个和2.18?106个。分子流行病学调查结果显示，在山东半岛的多个大菱鲆养殖场中均存在TRBIV的感染和流行。 |
| 关 键 词： | 大菱鲆虹彩病毒 实时定量PCR SYBR Green ? 主要衣壳蛋白 检测 |
| 收稿时间： | 2008-1-22 |
| 修稿时间： | 2008-4-11 |
Real-time PCR Assay for Sensitive Organ Detection and Epidemic Investigation of Turbot reddish body iridovirus |
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| WU Cheng-long,SHI Cheng-yin,HUANG Jie,KONG Xiao-yu.Real-time PCR Assay for Sensitive Organ Detection and Epidemic Investigation of Turbot reddish body iridovirus[J].Journal of Agricultural Biotechnology,2008,16(5). | |
| Authors: | WU Cheng-long SHI Cheng-yin HUANG Jie KONG Xiao-yu |
| Abstract: | A rapid and sensitive real-time PCR assay using the Roter Gene 3000 sequence detection system coupled with SYBR Green І chemistry was developed for the quantitative detection of turbot reddish body iridovirus (TRBIV) isolated from farmed turbot, Scophthalmus maximus. The assay involved the amplification of a 152 bp DNA fragment from a conserved region of TRBIV MCP gene. The PCR mixture contains a fluorescence dye, SYBR Green І, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid pUCm-T/TRBIV MCP containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (Ct ) value and logarithmic positive plasmid concentration were close to one (r2=0.99806) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23106 and 2.18106 viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed large number of cultured turbots were infected and TRBIV has been epidemic widely in turbot farms locating at Shandong peninsula. These results suggested that the real-time PCR assay reported here could be used as a rapid, sensitive and quantification method for TRBIV. |
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