凡纳滨对虾野田村病毒RT-PCR检测方法的建立及应用
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摘要
【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.
【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.
引文
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[12]谢数涛,何建国,杨晓明,等.套式PCR检测斑节对虾白斑症病毒(WSSV)[J].青岛海洋大学学报,2001,31(2):220-224.
[13]徐丽美,吴成林,邱名毅,等.对虾桃拉病毒(TSV)RT-PCR快速诊断技术的研究[J].厦门大学学报(自然科学版),2005,44(2):264-267.
[14]任聪,龚艳清,陈信忠,等.双重PCR同时检测对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)[J].福建水产,2008(4):38-42.
[2]孙成波.南美白对虾“偷死综合征”的病因分析及调控措施探讨[J].中国水产,2008(3):65-67.
[3]刘宏刚,刘磊.南美白对虾“偷死症”的辨证防治[J].科学养鱼,2012(9):66.
[4]刘宏刚,刘建生,杨公社,等.南美白对虾冬棚偷死症的综合防治[J].科学养鱼,2014(1):61-62.
[5]卓然江,蔡小琴,卓莹,等.南美白对虾“偷死病”的病因分析及防治措施[J].科学养鱼,2015(8):58-59.
[6]徐承旭.黄海所发现对虾偷死病疑似病原[J].水产科技情报,2015,42(1):52.
[7]Zhang Q L,Liu Q,Liu S,et al.A new nodavirus is associated with covert mortality disease of shrimp[J].Journal of general virology,2014,95:2700-2709.
[8]张达云.对虾偷死野田村病毒(CMNV)RT-PCR检测方法的建立[J].畜禽防治,2016,3:43.
[9]潘晓艺,刘杜娟,沈锦玉,等.罗氏沼虾野田村病毒和双顺反子病毒双重RT-PCR检测方法与序列分析[J].上海海洋大学学报,2012,21(6):996-1002.
[10]雷燕,肖洋,张会军,等.凡纳滨对虾对虾杆状病毒PCR检测方法的建立及初步应用[J].广东海洋大学学报,2015,35(4):63-66.
[11]雷燕,肖洋,张会军,等.凡纳滨对虾肠道上皮细胞微胞子虫PCR检测方法的建立与应用[J].广东海洋大学学报,2016,36(4):50-54.
[12]谢数涛,何建国,杨晓明,等.套式PCR检测斑节对虾白斑症病毒(WSSV)[J].青岛海洋大学学报,2001,31(2):220-224.
[13]徐丽美,吴成林,邱名毅,等.对虾桃拉病毒(TSV)RT-PCR快速诊断技术的研究[J].厦门大学学报(自然科学版),2005,44(2):264-267.
[14]任聪,龚艳清,陈信忠,等.双重PCR同时检测对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)[J].福建水产,2008(4):38-42.