猕猴桃植株中柑橘叶斑驳病毒实时荧光定量PCR检测技术的建立及应用
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摘要
柑橘叶斑驳病毒(Citrus leaf blotch virus,CLBV)在陕西省栽培猕猴桃中发生普遍。为监测CLBV发生情况,本研究建立了CLBV的实时荧光定量PCR(Real-time fluorescent quantitative polymerase chain reaction,RT-qPCR)检测方法。该方法特异性强,可准确检测目的病毒,标准曲线斜率为-3.378,决定系数R~2=0.997 9,扩增效率为97.7%,比普通RT-PCR灵敏度高100倍,可用于猕猴桃植株CLBV的批量检测或低丰度病毒样本(如猕猴桃休眠枝条)的检测。为苗木携带CLBV病毒的早期诊断、果园病毒病预测预报和防控奠定了基础。
During the virus detection of kiwifruit cultivars in Shaanxi province,Citrus leaf blotch virus was found to occur at high frequencies. In order to detect CLBV accurately,a specific RT-qPCR assay based on SYBR Green I fluorescent dye was established. The results showed that the RT-q PCR assay had good specificity and sensitivity for CLBV detection. The slope of the standard curve and determination coefficient R~2 were-3.378 and 0.997 9,and the amplification efficiency reached 97.7%,which was 100 times more sensitive than conventional RT-PCR. This method was suited to batch inspection of field samples and low-abundance virus samples( such as dormant shoots of kiwifruit). The RT-qPCR assay laid the foundation for the early diagnosis of CLBV-infected seedlings,forecast and control of CLBV in orchard.
During the virus detection of kiwifruit cultivars in Shaanxi province,Citrus leaf blotch virus was found to occur at high frequencies. In order to detect CLBV accurately,a specific RT-qPCR assay based on SYBR Green I fluorescent dye was established. The results showed that the RT-q PCR assay had good specificity and sensitivity for CLBV detection. The slope of the standard curve and determination coefficient R~2 were-3.378 and 0.997 9,and the amplification efficiency reached 97.7%,which was 100 times more sensitive than conventional RT-PCR. This method was suited to batch inspection of field samples and low-abundance virus samples( such as dormant shoots of kiwifruit). The RT-qPCR assay laid the foundation for the early diagnosis of CLBV-infected seedlings,forecast and control of CLBV in orchard.
引文
[1]Vives M C,Galipienso L,Navarro L,et al.Characterization of tw o kinds of subgenomic RNAs produced by Citrus leaf blotch virus[J].Virology,2002,295(2):328-336.
[2]Galipienso L,Vives M C,Moreno P.Partial characterization of Citrus leaf blotch virus,a new virus from Nagami kumquat[J].Archives of Virology,2001,146(2):357-368.
[3]Vives M C,Galipienso L,Navarro L,et al.The nucleotide sequence and genomic organization of Citrus leaf blotch virus:candidate type species for a new virus genus[J].Virology,2001,287(1):225-233.
[4]Guardo M,Sorrentino G,Marietta T,et al.First report of Citrus leaf blotch virus on kumquat in Italy[J].Plant Disease,2007,91(8):1054.
[5]Cao M J,Yu Y Q,Tian X,et al.First report of Citrus leaf blotch virus in lemon in China[J].Plant Disease,2017,101(8):1561.
[6]Blouin A G,Pearson M N,Chavan R R,et al.Viruses of kiw ifruit(Actinidia species)[J].Journal of Plant Pathology,2013,95(2):221-235.
[7]Wang J,Zhu D,Tan Y,et al.First report of Citrus leaf blotch virus in sw eet cherry[J].Plant Disease,2016,100(5):1027.
[8]Gress J C,Smith S,Tzanetakis I E.First report of Citrus leaf blotch virus in peony in the USA[J].Plant Disease,2017,101(4):637.
[9]Hernández-Rodríguez L,Pérez-Castro J M,GarcíaGarcía G,et al.Citrus leaf blotch virus in Cuba:first report and partial molecular characterization[J].Tropical Plant Pathology,2016,41(3):147-154.
[10]Agüero J,Vives M C,Velázquez K,et al.Citrus leaf blotch virus invades meristematic regions in nicotiana benthamiana and citrus[J].M olecular Plant Pathology,2013,14(6):610-616.
[11]Guardo M,Potere O,Castellano M A,et al.A new herbaceous host of Citrus leaf blotch virus[J].Journal of Plant Pathology,2009,91(2):485-488.
[12]Li R,Wu YF,Liu H.Kiwi virus disease symptoms and prevention methods(in Chinese)[J].Northw est Horticulture(西北园艺)2016,(5):35.
[13]Zhu C X,Wang G P,Zheng Y Z,et al.RT-PCR detection and sequence analysis of coat protein gene of Citrus leaf blotch virus infecting kiw ifruit trees(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2016,46(1):11-16.
[14]Guardo M,Sorrentino G,Caruso A.Characterization and incidence of Citrus leaf blotch virus(CLBV)in Southern Italy[J].Acta Horticulturae,2015(1065):825-830.
[15]Vives M C,Rubio L,Galipienso L,et al.Low genetic variation betw een isolates of Citrus leaf blotch virus from different host species and of different geographical origins[J].Journal of General Virology,2002,83(10):2587-2591.
[16]Galipienso L,Vives M C,Navarro L,et al.Detection of citrus leaf blotch virus using digoxigenin-labeled c DNA probes and RT-PCR[J].European Journal of Plant Pathology,2004,110(2):175-181.
[17]Dai J,Peng H,Chen W,et al.Development of multiplex real-time PCR for simultaneous detection of three Potyviruses in tobacco plants[J].Journal of Applied M icrobiology,2013,114(2):502-508.
[18]Ruiz-Ruiz S,Ambrós S,del Carmen Vives M,et al.Detection and quantitation of Citrus leaf blotch virus by TaqM an real-time RT-PCR[J].Journal of Virological M ethods,2009,160(1-2):57-62.
[19]Chavan R R,Blouin A G,Cohen D,et al.Characterization of the complete genome of a novel citrivirus infecting Actinidia chinensis[J].Archives of Virology,2013,158(8):1679-1686.
[2]Galipienso L,Vives M C,Moreno P.Partial characterization of Citrus leaf blotch virus,a new virus from Nagami kumquat[J].Archives of Virology,2001,146(2):357-368.
[3]Vives M C,Galipienso L,Navarro L,et al.The nucleotide sequence and genomic organization of Citrus leaf blotch virus:candidate type species for a new virus genus[J].Virology,2001,287(1):225-233.
[4]Guardo M,Sorrentino G,Marietta T,et al.First report of Citrus leaf blotch virus on kumquat in Italy[J].Plant Disease,2007,91(8):1054.
[5]Cao M J,Yu Y Q,Tian X,et al.First report of Citrus leaf blotch virus in lemon in China[J].Plant Disease,2017,101(8):1561.
[6]Blouin A G,Pearson M N,Chavan R R,et al.Viruses of kiw ifruit(Actinidia species)[J].Journal of Plant Pathology,2013,95(2):221-235.
[7]Wang J,Zhu D,Tan Y,et al.First report of Citrus leaf blotch virus in sw eet cherry[J].Plant Disease,2016,100(5):1027.
[8]Gress J C,Smith S,Tzanetakis I E.First report of Citrus leaf blotch virus in peony in the USA[J].Plant Disease,2017,101(4):637.
[9]Hernández-Rodríguez L,Pérez-Castro J M,GarcíaGarcía G,et al.Citrus leaf blotch virus in Cuba:first report and partial molecular characterization[J].Tropical Plant Pathology,2016,41(3):147-154.
[10]Agüero J,Vives M C,Velázquez K,et al.Citrus leaf blotch virus invades meristematic regions in nicotiana benthamiana and citrus[J].M olecular Plant Pathology,2013,14(6):610-616.
[11]Guardo M,Potere O,Castellano M A,et al.A new herbaceous host of Citrus leaf blotch virus[J].Journal of Plant Pathology,2009,91(2):485-488.
[12]Li R,Wu YF,Liu H.Kiwi virus disease symptoms and prevention methods(in Chinese)[J].Northw est Horticulture(西北园艺)2016,(5):35.
[13]Zhu C X,Wang G P,Zheng Y Z,et al.RT-PCR detection and sequence analysis of coat protein gene of Citrus leaf blotch virus infecting kiw ifruit trees(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2016,46(1):11-16.
[14]Guardo M,Sorrentino G,Caruso A.Characterization and incidence of Citrus leaf blotch virus(CLBV)in Southern Italy[J].Acta Horticulturae,2015(1065):825-830.
[15]Vives M C,Rubio L,Galipienso L,et al.Low genetic variation betw een isolates of Citrus leaf blotch virus from different host species and of different geographical origins[J].Journal of General Virology,2002,83(10):2587-2591.
[16]Galipienso L,Vives M C,Navarro L,et al.Detection of citrus leaf blotch virus using digoxigenin-labeled c DNA probes and RT-PCR[J].European Journal of Plant Pathology,2004,110(2):175-181.
[17]Dai J,Peng H,Chen W,et al.Development of multiplex real-time PCR for simultaneous detection of three Potyviruses in tobacco plants[J].Journal of Applied M icrobiology,2013,114(2):502-508.
[18]Ruiz-Ruiz S,Ambrós S,del Carmen Vives M,et al.Detection and quantitation of Citrus leaf blotch virus by TaqM an real-time RT-PCR[J].Journal of Virological M ethods,2009,160(1-2):57-62.
[19]Chavan R R,Blouin A G,Cohen D,et al.Characterization of the complete genome of a novel citrivirus infecting Actinidia chinensis[J].Archives of Virology,2013,158(8):1679-1686.