罗非鱼罗湖病毒RT-PCR检测方法的建立及初步应用
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摘要
【目的】建立快速检测罗非鱼罗湖病毒(Tilapia Lake Virus,Ti LV)的RT-PCR方法。【方法】参照GenBank中罗非鱼罗湖病毒全基因序列,依据其假定蛋白基因片段3设计合成1对特异性扩增引物,提取罗湖病毒RNA,逆转录成cDNA,进行PCR扩增,通过优化扩增条件和扩增体系,建立快速检测罗非鱼罗湖病毒的RT-PCR方法。用该方法对自然感染罗湖病毒发病的罗非鱼样品进行RT-PCR扩增。【结果】RT-PCR扩增得到与试验设计相符的499 bp的特异性目的条带,而对健康罗非鱼、野生罗非鱼及其他病毒对照组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10fg/mL的质粒DNA,具有较好的特异性和较高的敏感性。用该方法对332份临床样品进行RT-PCR检测,其中36份样品扩增出特异性的目的条带,经测序比对,检测结果正确。【结论】建立的检测方法可用于TiLV的检测。
【Objective】To establish a method for rapidly detecting tilapia lake virus(TiLV) by RT-PCR.【Method】A pair of specific primers were designed according to the complete genome hypothetical protein segment 3 of a novel RNA virus, called TiLV, and RNA of TiLV was extracted and reversely transcribed to cDNA as template for PCR. A rapid RT-PCR method was established for detection of TiLV by optimization of the reaction parameters. And the Tilapia which naturally infected with TiLV was detected by the RT-PCR method. 【Result】The specific band of 499 bp was amplified from the positive sample, but no specific band was found from healthy tilapia, wild tilapia and other control.Sequencing analysis indicated that this method was accurate, and 10 fg/mL DNA plasmid of the TiLV could be detected. 332 clinical samples were collected and 36 of those were positive, and the detection result was verified by sequencing and sequence alignment. 【Conclusion】 The findings indicated that the RT-PCR assay could be used for the detection of TiLV. The established RT-PCR assay could lay the foundation for the detection and epidemiological investigation of TiLV infection.
【Objective】To establish a method for rapidly detecting tilapia lake virus(TiLV) by RT-PCR.【Method】A pair of specific primers were designed according to the complete genome hypothetical protein segment 3 of a novel RNA virus, called TiLV, and RNA of TiLV was extracted and reversely transcribed to cDNA as template for PCR. A rapid RT-PCR method was established for detection of TiLV by optimization of the reaction parameters. And the Tilapia which naturally infected with TiLV was detected by the RT-PCR method. 【Result】The specific band of 499 bp was amplified from the positive sample, but no specific band was found from healthy tilapia, wild tilapia and other control.Sequencing analysis indicated that this method was accurate, and 10 fg/mL DNA plasmid of the TiLV could be detected. 332 clinical samples were collected and 36 of those were positive, and the detection result was verified by sequencing and sequence alignment. 【Conclusion】 The findings indicated that the RT-PCR assay could be used for the detection of TiLV. The established RT-PCR assay could lay the foundation for the detection and epidemiological investigation of TiLV infection.
引文
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[7]雷燕,赵振峰,李海涛.池塘养殖发病罗非鱼检出罗湖病毒.http://bbs.liyang-tech.com/forum.php?mod=view thread&tid=14932&highlight=%C2%DE%BA%FE%B2%A1%B6%BE&_dsign=01c74355.
[8]DONG H T,SIRIROOB S,MEEMETTA W,et al.Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection[J].Aquaculture,2017,476:111-118.
[9]王玮,丁建乐,房金岑.罗非鱼产业标准化现状及分析[J].上海海洋大学学报,2012,21(6):976-981.
[10]杨弘.中国罗非鱼产业发展趋势与建议[J].海洋与渔业,2010(5):33-34.
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[12]王民权,葛朋彪.罗非鱼主要病害控制技术[J].中国水产,2006(7):57.
[13]郭玉娟,张德锋,樊海平,等.中国南方地区罗非鱼无乳链球菌的分子流行病学研究[J].水产学报,2012,36(3):399-405.
[14]杨宁,黄海,张希,等.尼罗罗非鱼嗜水气单胞菌病的病原分离鉴定和药敏试验[J].水产科学,2014,33(5):306-310.
[15]陈善真,潘忠超,段元慧,等.一株尼罗罗非鱼致病性嗜水气单胞菌的分离鉴定及药敏试验[J].广东农业科学,2014(7):158-161.
[16]李聪,蔡岩,周永灿,等.海南罗非鱼致病性维氏气单胞菌分离鉴定及药敏特性研究[J].水产科学,2015,34(10):640-646.
[17]韦阳道,杨军,文衍红,等.罗非鱼竖鳞病病原维氏气单胞菌的分离鉴定与致病性研究[J].中国畜牧兽医,2016,43(11):3047-3052.
[18]ERAN B,NISCHAY M,THOMAS B,et al.Characterization of a novel orthomyxo-like virus causing mass die-offs of Tilapia[J].American Society for Microbiology,2016,7(2):1-7.
[19]KEMBOU TSOFACK J E,ZAMOSTIANO R,WATTEDS,et al.Detection of Tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR[J].Journal of Clinical Microbiology,2017,55(3):759.
[2]EYNGOR M,ZAMOSTIANO R,KEMBOU TSOFACKJ E,et al.Identification of a novel RNA virus lethal to tilapia[J].J Clin Microbiol,2014,52:4137-4146.
[3]BACHARACH E,MISHRA N,BRIESE T,et al.Characterization of a novel rthomyxo-like virus causing mass die-offs of tilapia[J].MBio,2016,7:e00431-00416.
[4]DEL-POZO J,MISHRA N,KABUUSU R,et al.Syncytial hepatitis of tilapia(Oreochromis niloticus L.)is associated with Orthomyxovirus-like virions in hepatocytes[J].Vet Pathol,2017,54:164-170.
[5]FATHI M,DICKSON C,DICKSON M,et al.Identification of tilapia lake virus in Egypt in Nile tilapia affected by“summer mortality”syndrome[J].Aquaculture,2017,473:430-432.
[6]DONG H T,SIRIROOB S,MEEMETTA W,et al.Awarning and an improved PCR detection method for tilapia lake virus(TiLV)disease in Thai tilapia farms.https://www.researchgate.net/publication/316283907.
[7]雷燕,赵振峰,李海涛.池塘养殖发病罗非鱼检出罗湖病毒.http://bbs.liyang-tech.com/forum.php?mod=view thread&tid=14932&highlight=%C2%DE%BA%FE%B2%A1%B6%BE&_dsign=01c74355.
[8]DONG H T,SIRIROOB S,MEEMETTA W,et al.Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection[J].Aquaculture,2017,476:111-118.
[9]王玮,丁建乐,房金岑.罗非鱼产业标准化现状及分析[J].上海海洋大学学报,2012,21(6):976-981.
[10]杨弘.中国罗非鱼产业发展趋势与建议[J].海洋与渔业,2010(5):33-34.
[11]卢迈新.罗非鱼链球菌病研究进展[J].南方水产,2010,6(1):75-79.
[12]王民权,葛朋彪.罗非鱼主要病害控制技术[J].中国水产,2006(7):57.
[13]郭玉娟,张德锋,樊海平,等.中国南方地区罗非鱼无乳链球菌的分子流行病学研究[J].水产学报,2012,36(3):399-405.
[14]杨宁,黄海,张希,等.尼罗罗非鱼嗜水气单胞菌病的病原分离鉴定和药敏试验[J].水产科学,2014,33(5):306-310.
[15]陈善真,潘忠超,段元慧,等.一株尼罗罗非鱼致病性嗜水气单胞菌的分离鉴定及药敏试验[J].广东农业科学,2014(7):158-161.
[16]李聪,蔡岩,周永灿,等.海南罗非鱼致病性维氏气单胞菌分离鉴定及药敏特性研究[J].水产科学,2015,34(10):640-646.
[17]韦阳道,杨军,文衍红,等.罗非鱼竖鳞病病原维氏气单胞菌的分离鉴定与致病性研究[J].中国畜牧兽医,2016,43(11):3047-3052.
[18]ERAN B,NISCHAY M,THOMAS B,et al.Characterization of a novel orthomyxo-like virus causing mass die-offs of Tilapia[J].American Society for Microbiology,2016,7(2):1-7.
[19]KEMBOU TSOFACK J E,ZAMOSTIANO R,WATTEDS,et al.Detection of Tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR[J].Journal of Clinical Microbiology,2017,55(3):759.
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