全预混冻干PCR试剂检测草鱼呼肠孤病毒Ⅱ型的研究
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摘要
草鱼呼肠孤病毒Ⅱ型是导致草鱼出血病的主要病原体之一,目前在我国广泛流行。本研究根据GenBank中草鱼呼肠孤病毒Ⅱ型基因序列,设计了草鱼呼肠孤病毒Ⅱ型特异性探针和PCR引物,通过优化反应条件,建立了草鱼呼肠孤病毒Ⅱ型一步法TaqMan荧光定量RT-PCR检测方法。试验对反转录和PCR反应体系进行摸索,通过优化的冻干工艺制备出草鱼呼肠孤病毒Ⅱ型全预混RT-PCR试剂,在荧光定量PCR中,冻干试剂的检测灵敏度可达10个模板,是普通PCR的100倍。批次间变异系数小于4%。特异性试验结果表明,该方法可特异性地检出草鱼呼肠孤病毒Ⅱ型,而对鲤春病毒血症病毒、传染性造血器官坏死症病毒无检测信号。该冻干试剂4℃保存12个月,室温25℃保存3个月,37℃保存15d,敏感性仍为10个,与第1d相同;利用手持单通道荧光PCR仪,可在42min内完成核酸诊断。本试验基于荧光定量PCR检测方法,结合核酸扩增体系冻干工艺制备的全预混草鱼呼肠孤病毒Ⅱ型RT-PCR试剂具有耐保存便于运输、准确性高、仪器拓展性好的特点,对草鱼呼肠孤病毒Ⅱ型快速诊断有重要意义。
Grass carp reovirus Ⅱ(GCRV Ⅱ)as one of the major causative pathogens involved in grass cap hemorrhagic disease is currently widespread in China,with an urgent need for development of diagnostic products with sensitivity and convenience.In this study,GCRV Ⅱ-specific probes and PCR primers were designed according to the GCRV Ⅱ gene sequences in GenBank.A TaqMan-based real-time quantitative RT-PCR assay was established by optimizing the reaction conditions.Premixed RT-PCR kits were prepared for optimizing the conditions of RT-PCR reactions as well as the freeze-dried process.This technique is shown to be highly sensitive with detection limit of nucleic acids(10copies),100 times as normal PCR.The intra-assay trials demonstrated that the variations were less than 4%for the lyophilized reagent,indicating that it is good reproducibility.The assay was highly specific for GCRVⅡ,without cross-signals to Spring viremia of Carp virus(SVCV)and Infectious hematopoietic necrosis virus(IHNV),and the lyophilized reagent was still effective when it was stored for 12 months at 4℃,for 3months at 25℃and for15 days at 37℃.However,the sensitivity was still 10 copies,the same as that at the first day.The nucleic acid diagnosis can be carried using handheld single-channel fluorescence PCR instrument within 42 min.The premixed GCRV Ⅱ RT-PCR method,combined fluorescence quantitative PCR detection,is characterized by resistance to storage and easy to transport,and will be of great significance in rapid clinical diagnosis on GCRVⅡ.
Grass carp reovirus Ⅱ(GCRV Ⅱ)as one of the major causative pathogens involved in grass cap hemorrhagic disease is currently widespread in China,with an urgent need for development of diagnostic products with sensitivity and convenience.In this study,GCRV Ⅱ-specific probes and PCR primers were designed according to the GCRV Ⅱ gene sequences in GenBank.A TaqMan-based real-time quantitative RT-PCR assay was established by optimizing the reaction conditions.Premixed RT-PCR kits were prepared for optimizing the conditions of RT-PCR reactions as well as the freeze-dried process.This technique is shown to be highly sensitive with detection limit of nucleic acids(10copies),100 times as normal PCR.The intra-assay trials demonstrated that the variations were less than 4%for the lyophilized reagent,indicating that it is good reproducibility.The assay was highly specific for GCRVⅡ,without cross-signals to Spring viremia of Carp virus(SVCV)and Infectious hematopoietic necrosis virus(IHNV),and the lyophilized reagent was still effective when it was stored for 12 months at 4℃,for 3months at 25℃and for15 days at 37℃.However,the sensitivity was still 10 copies,the same as that at the first day.The nucleic acid diagnosis can be carried using handheld single-channel fluorescence PCR instrument within 42 min.The premixed GCRV Ⅱ RT-PCR method,combined fluorescence quantitative PCR detection,is characterized by resistance to storage and easy to transport,and will be of great significance in rapid clinical diagnosis on GCRVⅡ.
引文
[1]柯丽华,方勤,蔡宜权.一株新的草鱼出血病病毒分离物的特性[J].水生生物学报,1990,14(2):153-159.
[2]曾伟伟,王庆,王英英,等.草鱼呼肠孤病毒三重PCR检测方法的建立及其应用[J].中国水产科学,2013,39(2):419-426.
[3]李贤.草鱼呼肠孤病毒GZ1208株的分离、鉴定及全基因组序列的分析[D].上海:上海海洋大学,2016.
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[5]刘永奎,王庆,曾伟伟,等.草鱼呼肠孤病毒JX-0902株的分离和鉴定[J].中国水产科学,2011,18(5):1077-1083.
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[8]郝贵杰,沈锦玉,潘晓艺,等.草鱼呼肠孤病毒湖州分离株的分离及鉴定[J].渔业科学进展,2011,32(1):47-52.
[9]徐洋,郝贵杰,沈锦玉,等.两株草鱼呼肠孤病毒江西株的分离与鉴定[J].淡水渔业,2010,40(3):44-49.
[10]Yan X Y,Wang Y,Xiong L F,et al.Phylogenetic analysis of newly isolated grass carp reovirus[J].Springer Plus,2014(3):190.
[11]杨映.南方草鱼呼肠孤病毒流行病学调查及双重荧光定量RT-PCR检测方法的建立[D].雅安:四川农业大学,2013.
[12]杨广志,罗毅志,叶雪平.葡萄球菌A蛋白协同凝集试验快速检测草鱼出血病病毒研究[J].水产学报,1991,15(1):27-33.
[13]江育林,李正秋.病毒感染的草鱼细胞产生类干扰素物质的研究[J].病毒学报,1991,7(1):30-35.
[14]邵建忠,项黎新,李亚南,等.应用Dot-ELISA技术检测草鱼出血病病毒研究[J].水产学报,1996,20(1):6-11.
[15]王铁辉,李军,易永兰,等.用逆转录酶链式反应监测草鱼出血病病毒的研究[J].海洋与湖沼,1997,28(1):1-6.
[16]草鱼出血病研究协作组.草鱼出血病病毒的电子显微镜观察初报[J].淡水渔业,1983(3):39-46.
[17]侯月娥,冯娟,宁章勇,等.军曹鱼Igκ轻链cDNA克隆及组织表达分析[J].中国水产科学,2011,18(1):48-58.
[18]侯月娥,伍建敏,李中圣,等.PEDV和TGEV双重TaqMan荧光定量一步法RT-PCR检测方法的建立及应用[J].中国动物传染病学报,2017,25(1):19-25.
[19]周勇,曾令兵,范玉顶,等.草鱼呼肠孤病毒TaqMan real-time PCR方法的建立[J].水产学报,2011,35(5):774-779.
[2]曾伟伟,王庆,王英英,等.草鱼呼肠孤病毒三重PCR检测方法的建立及其应用[J].中国水产科学,2013,39(2):419-426.
[3]李贤.草鱼呼肠孤病毒GZ1208株的分离、鉴定及全基因组序列的分析[D].上海:上海海洋大学,2016.
[4]曾伟伟,王庆,刘永奎,等.一株草鱼呼肠孤病毒弱毒株的分离、鉴定及免疫原性初步分析[J].水生生物学报,2011,35(5):790-795.
[5]刘永奎,王庆,曾伟伟,等.草鱼呼肠孤病毒JX-0902株的分离和鉴定[J].中国水产科学,2011,18(5):1077-1083.
[6] Fan Y D,Rao S J,Zeng L B,et al.Identification and genomic characterization of a novel fish reovirus,Hubei grass carp disease reovirus,isolated in 2009in China[J].Journal of General Virology,2013,94(Pt 10):2266-2277.
[7]张超,王庆,石存斌,等.草鱼呼肠孤病毒HZ08株的分离与鉴定[J].中国水产科学,2010,17(6):1257-1263.
[8]郝贵杰,沈锦玉,潘晓艺,等.草鱼呼肠孤病毒湖州分离株的分离及鉴定[J].渔业科学进展,2011,32(1):47-52.
[9]徐洋,郝贵杰,沈锦玉,等.两株草鱼呼肠孤病毒江西株的分离与鉴定[J].淡水渔业,2010,40(3):44-49.
[10]Yan X Y,Wang Y,Xiong L F,et al.Phylogenetic analysis of newly isolated grass carp reovirus[J].Springer Plus,2014(3):190.
[11]杨映.南方草鱼呼肠孤病毒流行病学调查及双重荧光定量RT-PCR检测方法的建立[D].雅安:四川农业大学,2013.
[12]杨广志,罗毅志,叶雪平.葡萄球菌A蛋白协同凝集试验快速检测草鱼出血病病毒研究[J].水产学报,1991,15(1):27-33.
[13]江育林,李正秋.病毒感染的草鱼细胞产生类干扰素物质的研究[J].病毒学报,1991,7(1):30-35.
[14]邵建忠,项黎新,李亚南,等.应用Dot-ELISA技术检测草鱼出血病病毒研究[J].水产学报,1996,20(1):6-11.
[15]王铁辉,李军,易永兰,等.用逆转录酶链式反应监测草鱼出血病病毒的研究[J].海洋与湖沼,1997,28(1):1-6.
[16]草鱼出血病研究协作组.草鱼出血病病毒的电子显微镜观察初报[J].淡水渔业,1983(3):39-46.
[17]侯月娥,冯娟,宁章勇,等.军曹鱼Igκ轻链cDNA克隆及组织表达分析[J].中国水产科学,2011,18(1):48-58.
[18]侯月娥,伍建敏,李中圣,等.PEDV和TGEV双重TaqMan荧光定量一步法RT-PCR检测方法的建立及应用[J].中国动物传染病学报,2017,25(1):19-25.
[19]周勇,曾令兵,范玉顶,等.草鱼呼肠孤病毒TaqMan real-time PCR方法的建立[J].水产学报,2011,35(5):774-779.