凡纳滨对虾野田村病毒RT-PCR检测方法的建立及应用

Development and Application of a RT-PCR Assay of Litopenaeus vannamei Nodavirus

【目的】建立快速检测凡纳滨对虾野田村病毒的RT-PCR方法。【方法】根据GenBank中获得的凡纳滨对虾野田村病毒的基因序列,应用Oligo6.0软件设计合成1对特异性扩增引物,对反应条件和反应体系进行优化,建立快速检测凡纳滨对虾野田村病毒的RT-PCR方法。用该方法对感染野田村病毒的凡纳滨对虾进行RT-PCR检测。【结果】RT-PCR可扩增出与设计相符的413 bp的特异性目的条带,而对白斑症病毒(WSSV)阳性虾、对虾杆状病毒(BP)阳性虾、传染性皮下及造血器官坏死病毒(IHHNV)阳性虾、肝胰腺细小病毒(HPV)阳性虾、黄头病毒(YHV)阳性虾、传染性肌肉坏死病毒(IMNV)阳性虾、桃拉病毒(TSV)阳性虾和健康虾的扩增结果均为阴性。测序比对结果表明,该方法检测结果准确,最低可检测出约100 fg/mL的野田村病毒重组质粒DNA;用该方法对从福建、广东、海南、广西、江苏、浙江、山东等地的386份临床样品进行RT-PCR检测,共检出野田村病毒阳性样品64份。【结论】该方法可用于凡纳滨对虾野田村病毒的快速检测。

【Objective】To establish a rapid RT-PCR method to detect Litopenaeus vannamei Nodavirus.【Method】A pair of specific primers was designed by Oligo6.0 according to the published gene sequence in GenBank, and a rapid RT-PCR method was established for detection of Litopenaeus vannamei nodavirus by optimization of the reaction parameters. 【Result】The specific band of 413 bp were amplified by RT-PCR from the positive shrimps, but no specific band was found from shrimps with White spot syndrome virus(WSSV), Baculovirus penaei(BP), Infectious hypodermal and hematopoietic necrosis virus(IHHNV), Hepatopancereatic parvovirus(HPV), Yellow head virus(YHV),Infectious Myonecrosis Virus(IMNV), Taura syndrome virus(TSV), and healthy shrimps. Sequencing analysis indicated that this method was accurate, and 100 fg/mL DNA plasmid of the Litopenaeus vannamei nodavirus was detected. A total of 386 clinical shrimp samples were collected from provinces of Fujian, Guangdong, Hainan, Guangxi, Jiangsu, Zhejiang and Shandong, and 64 positive samples were detected by the new method. 【Conclusion】The new RT-PCR assay could be used in the rapid detection of Litopenaeus vannamei samples.