竹花叶病毒SYBR GreenⅡ反转录实时荧光定量PCR检测方法的建立及应用
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摘要
竹花叶病毒(Bamboo mosaic virus, BaMV)是目前为止被发现感染竹类植物唯一的RNA病毒,严重影响竹类植物经济价值。因此,快速准确检测BaMV,对其预防和控制具有重要意义。本研究参考BaMV外壳蛋白基因(coat protein, CP)保守区设计2对特异性引物,运用反转录实时荧光定量PCR (reverse transcription real-time fluorescence quantitative PCR, RT-qPCR)法对该病毒进行定性和定量分析,建立了一套基于SYBR GreenⅡ的BaMV的RT-qPCR检测体系。该检测体系的标准曲线循环阈值(Ct)与模板浓度的对数呈现良好的线性关系,扩增效率和相关系数分别为100%和0.999。重复性实验表明组内及组间变异系数均在1.5%以内。同时,利用此方法首次检测到8个不同竹种中亦存在BaMV。结果表明,SYBR GreenⅡRT-qPCR检测竹花叶病毒的方法灵敏、特异、重复性好,可用于竹花叶病毒的快速检测。
Bamboo mosaic virus(BaMV) is the only RNA virus that has been found to infect bamboo so far,seriously affects the economic value of bamboo. Therefore, development of a rapid and accurate detection method for the BaMV is of great significance for the prevention and control of BaMV. In this study, two pairs of specific primers according to the coat protein gene(CP) sequence of BaMV were designed. One sequence of BaMV CP gene were obtained from Phyllostachys bambusoides with mosaic symptoms by RT-qPCR. And the full-length sequence(GenBank accession number: KP256071) was 528 bp encoding 175 putative amino acid residues. The sequences shared more than 98% similarity at nucleotides level with those of Phyllostachys nigra BaMV strains.The recombinant plasmid was used as template for SYBR Green Ⅱ real-time PCR to generate standard and melting curves. The standard curve cycle threshold(Ct) had a good linear relationship with the logarithm of template concentration. Melting curve analysis indicated no primer-dimers and nonspecific products in the assay. The amplification efficiency and correlation coefficient were 100% and 0.99,respectively. Repeat ability tests indicated that inter-assay variability of the Ct values was 1.5%. The purpose strips could be successfully amplified with the primers BaMV-F/BaMV-R only in the bamboo infected mosaic sample and the healthy young leaves without purpose strips, which indicated that the primers is highly specific. The sensitivity of RT-qPCR was 100 times higher than that of regular RT-PCR. The minimum detectable concentration of BaMV plasmid standard in RT-qPCR assay was 8.5×101 copies/uL.The presence of BaMV in 8 bamboo species were firstly detected by this method. The results show that the SYBR Green Ⅱreal-time fluorescent quantitative PCR method for detecting BaMV is sensitive, specific, and reproducible, and could be used for rapid detection of BaMV.
Bamboo mosaic virus(BaMV) is the only RNA virus that has been found to infect bamboo so far,seriously affects the economic value of bamboo. Therefore, development of a rapid and accurate detection method for the BaMV is of great significance for the prevention and control of BaMV. In this study, two pairs of specific primers according to the coat protein gene(CP) sequence of BaMV were designed. One sequence of BaMV CP gene were obtained from Phyllostachys bambusoides with mosaic symptoms by RT-qPCR. And the full-length sequence(GenBank accession number: KP256071) was 528 bp encoding 175 putative amino acid residues. The sequences shared more than 98% similarity at nucleotides level with those of Phyllostachys nigra BaMV strains.The recombinant plasmid was used as template for SYBR Green Ⅱ real-time PCR to generate standard and melting curves. The standard curve cycle threshold(Ct) had a good linear relationship with the logarithm of template concentration. Melting curve analysis indicated no primer-dimers and nonspecific products in the assay. The amplification efficiency and correlation coefficient were 100% and 0.99,respectively. Repeat ability tests indicated that inter-assay variability of the Ct values was 1.5%. The purpose strips could be successfully amplified with the primers BaMV-F/BaMV-R only in the bamboo infected mosaic sample and the healthy young leaves without purpose strips, which indicated that the primers is highly specific. The sensitivity of RT-qPCR was 100 times higher than that of regular RT-PCR. The minimum detectable concentration of BaMV plasmid standard in RT-qPCR assay was 8.5×101 copies/uL.The presence of BaMV in 8 bamboo species were firstly detected by this method. The results show that the SYBR Green Ⅱreal-time fluorescent quantitative PCR method for detecting BaMV is sensitive, specific, and reproducible, and could be used for rapid detection of BaMV.
引文
程晓甜,阿地力·沙塔尔,张伟,等.2014.SYBR Green实时荧光PCR快速鉴定枣实蝇技术[J].林业科学,50(4):60-65.(Cheng X T,Adil S,Zhang W,et al.2014.Rapid identification of Carpomya vesuviana by real-time PCRwith SYBR Green chemical[J].Scientia Silvae Sinicae,50(4):60-65.)
李玲娣,周常勇,李中安,等.2013.褐色橘蚜中柑橘衰退病毒实时荧光定量RT-PCR检测方法的建立与应用[J].中国农业科学,46(3):525-533.(Li L D,Zhou C Y,Li ZA,et al.2013.Development and application of a realtime RT-PCR approach for quantification of CTV in Toxoptera citricida[J].Scientia Agricultura Sinica,46(3):525-533.)
梁芳,张燕,王若斓,等.2017.建兰花叶病毒SYBR Green实时荧光定PCR检测方法的建立[J].江西农业大学学报,39(03):572-580.(Liang F,Zhang Y,Wang R L,et al.2017.Development of a SYBR Green I Real-time Fluorescence quantitative PCR detection method for Cymbid?ium mosaic virus.[J].Acta Agriculturae Universitatis Jiangxiensis,39(03):572-580.)
林纳生,陈脉纪,江涛,等.1979.台湾BaMV之初步研究[J].台湾省林业试验所试验报告,317:4-10.(Lin N S,Cheng M J,Jiang T,et al.1979.Preliminary study of Taiwan BaMV[J].Taiwan Provincial Forestry Laboratory Test Report,317:4-10.)
林文武,杨文婷,张洁,等.2014.竹花叶病毒的研究进展及其作为表达载体的应用[J].武夷科学,17(1):175-186.(Lin W W,Yang W T,Zhang J,et al.2014.Advance in research on Bamboo mosaic virus and its application to gene expression vector[J].Wu Yi Science Journal,17(1):175-186.)
林文武,杨文婷,张洁,等.2016.福州和成都竹花叶病毒的RT-PCR法检测[J].植物病理学报,46(4):469-473.(Lin W W,Yang W T,Zhang J,et al.2016.Detection of Bam?boo mosaic virus by RT-PCR amplification in Fuzhou and Chengdu.[J].Acta Phytopathologica Sinica,46(4):469-473.)
柳爱春,刘超,李峰.2011.荧光RT-PCR法快速检测微量建兰花叶病毒研究[J].现代农业技术,21:176-179.(Liu A C,Liu C,Liu F.2011.Rapid detection of trace amounts of Cymbidium mosaic virus by real-time RT-PCR[J].Modern Agricultural Science and Technology,21:176-179.)
潘明森,王震烁,方敦煌,等.2015.土壤中黑胫病菌荧光定量PCR快速检测体系的建立及初步应用[J].江西农业大学学报,37(4):712-718.(Pan M S,Wang Z S,Fang D H,et al.2015.Development and preliminary application of fluorescence quantitative PCR system for rapid detection of Phytophthora nicotianae in soil.[J].Acta Agric Lturae Universitatis Jiangxiensis,37(4):712-718.)
王丽,王振东,乔奇,等.2014.甘薯褪绿矮化病毒西非株系实时荧光定量检测方法的建立及应用[J].植物病毒学报,44(5):461-468.(Wang L,Wang Z D,Qiao Q,et al.2014.Development and application of a real-time PCRmethod for detection of west African strain of Sweet po?tato chlorotic stunt virus.[J].Acta Phytopathologica Sinica,44(5):461-468.)
王艳娇,崔甜甜,黄爱军,等.2016.柑橘脉突病毒实时荧光定量RT-PCR检测体系的建立与应用[J].园艺学报,43(8):1613-1620.(Wang Y J,Cui T T,Huang A J,et al.2016.Development and application of a quantitative RT-PCR approach for quantification of Citrus vein enation virus[J].Acta Horticulturae Sinica,43(8):1613-1620.)
吴然,李君英,邵建柱,等.2015.苹果锈果类病毒实时荧光PCR检测方法的建立[J].果树学报,32(1):150-155.(Wu R,Li J Y,Shao J Z,et al.2015.Establishment of detection method for apple scar skin viroid(ASSVd)by real-time PCR.[J].Journal of Fruit Science,32(1):150-155.)
赵焕英,包金.2007.实时荧光定量PCR技术的原理及其应用研究进展[J].中国组织化学与细胞化学杂志,16(4).(Zhao H Y,Bao J.2007.Progress in the principle and application of a real-time fluorescent quantitative RT-PCR[J].Chinese Journal of Histochemistry and Cytochemisstry,16(4).)
张伟,徐硕,陈德鑫.2013.常用植物病毒病检测技术比较[J].南方农业,(12):24-28.(Zhang W,Xu S,Cheng D X.2013.Comparison of common plant viruses detection measures[J].South China Agriculture,(12):24-28.)
Bohm J,Hahn A,Schubert R,et al.2010.Real-time quantitative PCR:DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants[J].Journal of Phytopathology,147(7-8):409-416.
Chen X J,Xu X G,Li Y Z,et al.2013.Development of a realtime fluorescentquantitative PCR assay for detection of Impatiens necrotic spot virus[J].Journal of Virological Methods,189:299-304.
Hsu Y H,Annamalai P,Lin C S,et al.2010.A sensitive method for detecting bamboo mosaic virus(BaMV)and establishment of BaMV-free meristem-tip cultures.[J].Plant Pathology,49(1):101-107.
Hsu Y H,Tsai C H,Lin N S 2018.Editorial:Molecular Biology of Bamboo mosaic virus-A Type Member of the Potexvirus Genus[J].Frontiers in Microbiology,9:6-8.
Hull R,Milne R G,Van Regenmortel M H V.1991.List of proposed standard acronyms for plant viruses and viroids[J].Archives of Virology,120(1-2):151-164.
Kuo S Y,Lin Y C,Lai Y C,et al.2018.Production of fluorescent antibody-labeling proteins in plants using a viral vector and the application in the detection of Acidovorax citrulli and Bamboo mosaic virus[J].PloS One,13(2):e0192455.
Lin N S,Hsu Y H.1994.A satellite RNA associated with Bamboo mosaic potexvirus[J].Virology,202(2):707-714.
Lin N S,Lin F Z,Huang T Y,et al.1992.Genome properties of Bamboo mosaic virus[J].Phytopathology,82(7):731-734.
Lin W,Gao F,Yang W,et al.2016.Molecular characterization and detection of a recombinant isolate of bamboo mosaic virus from China[J].Archives of Virology,161(4):1091-1094.
Wang I N,Yeh W B,Lin N S 2017.Phylogeography and Coevolution of Bamboo Mosaic Virus and Its Associated Satellite RNA[J].Frontiers in Microbiology,8:886-898.
李玲娣,周常勇,李中安,等.2013.褐色橘蚜中柑橘衰退病毒实时荧光定量RT-PCR检测方法的建立与应用[J].中国农业科学,46(3):525-533.(Li L D,Zhou C Y,Li ZA,et al.2013.Development and application of a realtime RT-PCR approach for quantification of CTV in Toxoptera citricida[J].Scientia Agricultura Sinica,46(3):525-533.)
梁芳,张燕,王若斓,等.2017.建兰花叶病毒SYBR Green实时荧光定PCR检测方法的建立[J].江西农业大学学报,39(03):572-580.(Liang F,Zhang Y,Wang R L,et al.2017.Development of a SYBR Green I Real-time Fluorescence quantitative PCR detection method for Cymbid?ium mosaic virus.[J].Acta Agriculturae Universitatis Jiangxiensis,39(03):572-580.)
林纳生,陈脉纪,江涛,等.1979.台湾BaMV之初步研究[J].台湾省林业试验所试验报告,317:4-10.(Lin N S,Cheng M J,Jiang T,et al.1979.Preliminary study of Taiwan BaMV[J].Taiwan Provincial Forestry Laboratory Test Report,317:4-10.)
林文武,杨文婷,张洁,等.2014.竹花叶病毒的研究进展及其作为表达载体的应用[J].武夷科学,17(1):175-186.(Lin W W,Yang W T,Zhang J,et al.2014.Advance in research on Bamboo mosaic virus and its application to gene expression vector[J].Wu Yi Science Journal,17(1):175-186.)
林文武,杨文婷,张洁,等.2016.福州和成都竹花叶病毒的RT-PCR法检测[J].植物病理学报,46(4):469-473.(Lin W W,Yang W T,Zhang J,et al.2016.Detection of Bam?boo mosaic virus by RT-PCR amplification in Fuzhou and Chengdu.[J].Acta Phytopathologica Sinica,46(4):469-473.)
柳爱春,刘超,李峰.2011.荧光RT-PCR法快速检测微量建兰花叶病毒研究[J].现代农业技术,21:176-179.(Liu A C,Liu C,Liu F.2011.Rapid detection of trace amounts of Cymbidium mosaic virus by real-time RT-PCR[J].Modern Agricultural Science and Technology,21:176-179.)
潘明森,王震烁,方敦煌,等.2015.土壤中黑胫病菌荧光定量PCR快速检测体系的建立及初步应用[J].江西农业大学学报,37(4):712-718.(Pan M S,Wang Z S,Fang D H,et al.2015.Development and preliminary application of fluorescence quantitative PCR system for rapid detection of Phytophthora nicotianae in soil.[J].Acta Agric Lturae Universitatis Jiangxiensis,37(4):712-718.)
王丽,王振东,乔奇,等.2014.甘薯褪绿矮化病毒西非株系实时荧光定量检测方法的建立及应用[J].植物病毒学报,44(5):461-468.(Wang L,Wang Z D,Qiao Q,et al.2014.Development and application of a real-time PCRmethod for detection of west African strain of Sweet po?tato chlorotic stunt virus.[J].Acta Phytopathologica Sinica,44(5):461-468.)
王艳娇,崔甜甜,黄爱军,等.2016.柑橘脉突病毒实时荧光定量RT-PCR检测体系的建立与应用[J].园艺学报,43(8):1613-1620.(Wang Y J,Cui T T,Huang A J,et al.2016.Development and application of a quantitative RT-PCR approach for quantification of Citrus vein enation virus[J].Acta Horticulturae Sinica,43(8):1613-1620.)
吴然,李君英,邵建柱,等.2015.苹果锈果类病毒实时荧光PCR检测方法的建立[J].果树学报,32(1):150-155.(Wu R,Li J Y,Shao J Z,et al.2015.Establishment of detection method for apple scar skin viroid(ASSVd)by real-time PCR.[J].Journal of Fruit Science,32(1):150-155.)
赵焕英,包金.2007.实时荧光定量PCR技术的原理及其应用研究进展[J].中国组织化学与细胞化学杂志,16(4).(Zhao H Y,Bao J.2007.Progress in the principle and application of a real-time fluorescent quantitative RT-PCR[J].Chinese Journal of Histochemistry and Cytochemisstry,16(4).)
张伟,徐硕,陈德鑫.2013.常用植物病毒病检测技术比较[J].南方农业,(12):24-28.(Zhang W,Xu S,Cheng D X.2013.Comparison of common plant viruses detection measures[J].South China Agriculture,(12):24-28.)
Bohm J,Hahn A,Schubert R,et al.2010.Real-time quantitative PCR:DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants[J].Journal of Phytopathology,147(7-8):409-416.
Chen X J,Xu X G,Li Y Z,et al.2013.Development of a realtime fluorescentquantitative PCR assay for detection of Impatiens necrotic spot virus[J].Journal of Virological Methods,189:299-304.
Hsu Y H,Annamalai P,Lin C S,et al.2010.A sensitive method for detecting bamboo mosaic virus(BaMV)and establishment of BaMV-free meristem-tip cultures.[J].Plant Pathology,49(1):101-107.
Hsu Y H,Tsai C H,Lin N S 2018.Editorial:Molecular Biology of Bamboo mosaic virus-A Type Member of the Potexvirus Genus[J].Frontiers in Microbiology,9:6-8.
Hull R,Milne R G,Van Regenmortel M H V.1991.List of proposed standard acronyms for plant viruses and viroids[J].Archives of Virology,120(1-2):151-164.
Kuo S Y,Lin Y C,Lai Y C,et al.2018.Production of fluorescent antibody-labeling proteins in plants using a viral vector and the application in the detection of Acidovorax citrulli and Bamboo mosaic virus[J].PloS One,13(2):e0192455.
Lin N S,Hsu Y H.1994.A satellite RNA associated with Bamboo mosaic potexvirus[J].Virology,202(2):707-714.
Lin N S,Lin F Z,Huang T Y,et al.1992.Genome properties of Bamboo mosaic virus[J].Phytopathology,82(7):731-734.
Lin W,Gao F,Yang W,et al.2016.Molecular characterization and detection of a recombinant isolate of bamboo mosaic virus from China[J].Archives of Virology,161(4):1091-1094.
Wang I N,Yeh W B,Lin N S 2017.Phylogeography and Coevolution of Bamboo Mosaic Virus and Its Associated Satellite RNA[J].Frontiers in Microbiology,8:886-898.