| 三种对虾病毒多重实时荧光PCR检测方法的建立 |
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| 引用本文： | 谢芝勋,谢丽基,庞耀珊,卢兆发,谢志勤,孙建华,邓显文,刘加波,唐小飞.三种对虾病毒多重实时荧光PCR检测方法的建立[J].农业生物技术学报,2008,16(5). |
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| 作者姓名： | 谢芝勋 谢丽基 庞耀珊 卢兆发 谢志勤 孙建华 邓显文 刘加波 唐小飞 |
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| 作者单位： | 1. 广西兽医研究所,南宁,530001
2. 广西水产畜牧局,南宁,530022 |
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| 基金项目： | 国家百千万人才工程人选专项资金项目，广西科技攻关项目 |
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| 摘 要： | 根据基因库中白斑综合征病毒（WSSV）、传染性皮下及造血器官坏死病毒（IHHNV）和桃拉综合征病毒（TSV）的基因序列，设计了WSSV 、IHHNV和TSV的三对特异性引物和三条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化，建立了能够同时检测WSSV 、IHHNV和TSV的三重实时荧光PCR方法。该方法特异性好，对WSSV 、IHHNV和TSV的检测敏感性分别达到20000、20和20000个模板拷贝数；此外抗干扰能力强，对WSSV 、IHHNV和TSV不同模板浓度进行组合，仍可有效地同时检测这三个病毒。对保存的45份经常规PCR检测仅为WSSV 、IHHNV和TSV阳性的样品进行二重实时荧光PCR检测，结果都为阳性，其中2份为WSSV和IHHNV混合感染。本研究建立的三重实时荧光PCR方法用于WSSV、IHHNV和TSV的检测具有特异、敏感、快速、定量等优点。
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| 关 键 词： | 白斑综合征病毒:传染性皮下及造血器官坏死病毒 桃拉综合征病毒 多重荧光PCR |
| 收稿时间： | 2007-11-26 |
| 修稿时间： | 2008-1-31 |
Development of a Multiplex Real-time PCR Assay for Detection of Three Kinds of Viruses in Penaeid Shrimp |
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| XIE Zhi-xun,XIE Li-ji,PANG Yao-shan,LU Zhao-fa,XIE Zhi-qin,SUN Jian-hua,DENG Xian-wen,LIU Jia-bo,TANG Xiao-fei.Development of a Multiplex Real-time PCR Assay for Detection of Three Kinds of Viruses in Penaeid Shrimp[J].Journal of Agricultural Biotechnology,2008,16(5). |
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| Authors: | XIE Zhi-xun XIE Li-ji PANG Yao-shan LU Zhao-fa XIE Zhi-qin SUN Jian-hua DENG Xian-wen LIU Jia-bo TANG Xiao-fei |
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| Abstract: | White spot syndrome virus (WSSV) , Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and and Taura syndrome virus (TSV ) are responsible for significant economic loss in the shrimp industry. In order to simultaneously and massively identify WSSV , IHHNV and TSV, three pair of primers and three TaqMan probes were designed and synthesized according to the conserved gene sequences of WSSV (AF369029) , IHHNV (AF218226) and TSV(NC003005) in GenBank. The reaction parameters such as the concentration of three pair of primers, three TaqMan probes and the reaction buffer were optimized to develop a multiplex real-time PCR assay for the rapid detection of WSSV , IHHNV and TSV. The multiplex real-time PCR assay was found to be specific and to be able to detect and differentiate WSSV , IHHNV and TSV, and no positive results were observed when nucleic acid from Vibrio and Streptococcus were used as multiplex real-time PCR templates. The developed multiplex real-time PCR assay was compared with that of routine PCR. The sensitivity of multiplex real-time PCR assay was 20000, 20 and 20000 template copies for WSSV , IHHNV and TSV respectively, and its sensitivity was 10, 1000 and 10 times higher than that of the routine PCR. The samples were examined using the multiplex real-time PCR repeatedly and the results indicated that the multiplex real-time PCR was reproducible. Different concentrations of WSSV , IHHNV and TSV when mixed together still could be identified by this assay, which implied the assay could be applied to clinical confirmation for simultaneous infection of WSSV , IHHNV and TSV. The multiplex real-time PCR results of 45 routine PCR positive samples showed that one specific amplified curve was displayed when shrimp was infected by only one of these three viral pathogens, whereas two or three specific amplified curves were displayed when shrimp was infected by two or three viral pathogens. The result indicated that multiplex real-time PCR was able to detect and differentiate the presence of each pathogen in infected clinical shrimp. This multiplex real-time PCR assay was a quick, sensitive, specific and quantitative tool for detection of WSSV , IHHNV and TSV, and will be useful for the control of WSS, IHHN and TS in shrimp. |
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