苹果茎痘病毒双重RT-PCR检测体系的建立及应用
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摘要
<正>病毒病是危害苹果(Malus domestica)的一类重要病害。已报道的苹果病毒种类很多,在我国发生普遍的主要有苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)、苹果茎沟病毒(Apple stem grooving virus,ASGV)、苹果茎痘病毒(Apple stem pitting virus,ASPV)、苹果花叶病毒(Apple mosaic virus,ApM V)以及苹果锈果类病毒(Apple scar skid viroid,ASSVd)~([1～3])。病毒或类病毒混合
Apple stem pitting virus( ASPV) has a worldwide distribution in pome fruit trees and leads to tremendous economic losses. A dual RT-PCR method was developed for ASPV detection from apple plants. The primer combination ASPV-F1/R1-F3R3 could simultaneously amplify two c DNA fragments covering two positions of viral genome,with the size 508 bp and 291 bp,respectively. The dual RT-PCR reaction was optimized at 60℃,using 0.6 uL ASPV-F1/R1 and 0.4 μL ASPV-F3R3( 10 mmol·L~(-1)),and 2 μL cDNA. Moreover,the reaction system could be used for new leaves,phloem of shoot and in vitro apple plants and the amplification efficiencies of the tree materials were almost the same. The dual RT-PCR was simple,rapid and sensitive for ASPV detection.
Apple stem pitting virus( ASPV) has a worldwide distribution in pome fruit trees and leads to tremendous economic losses. A dual RT-PCR method was developed for ASPV detection from apple plants. The primer combination ASPV-F1/R1-F3R3 could simultaneously amplify two c DNA fragments covering two positions of viral genome,with the size 508 bp and 291 bp,respectively. The dual RT-PCR reaction was optimized at 60℃,using 0.6 uL ASPV-F1/R1 and 0.4 μL ASPV-F3R3( 10 mmol·L~(-1)),and 2 μL cDNA. Moreover,the reaction system could be used for new leaves,phloem of shoot and in vitro apple plants and the amplification efficiencies of the tree materials were almost the same. The dual RT-PCR was simple,rapid and sensitive for ASPV detection.
引文
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[3]Guo R,Li S F,Dong Y F,et al.Occurrence of Apple scar skin disease and sequence analysis of Apple scar skin viroid in Liaoning(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2005,35(5):472-474.
[4]Hu G J,Zhang Z P,Fan X D,et al.Research progress of main apple viruses of China(in Chinese)[J].China Fruits(中国果树),2017,(3):71-74.
[5]Yanase H.Back transmission of Apple stem grooving virus to apple seedlings and induction of symptoms of apple topw orking disease in Mitsuba Kaido(Malus sieboldii)and Kobano Zumi(Malus sieboldii var.arborescens)rootstocks[J].Acta Horticuturae,1983,130:117-122.
[6]Yanase H,Koganezawa H,Fridlund P R.Correlation of pear necrotic spot with pear vein yellows and apple stem pitting,and a flexuous filamentous virus associated with them[J].Acta Horticulturae,1989,235:157-158.
[7]Liu H Z,Yang Z K,Zhou Y B,et al.Study on genom esequence and small RNA of Apple stem pitting virus infecting a pear plant(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2017,47(5):584-590.
[8]Yang J L,Dong Y F,Li S F,et al.Detection of Apple stem pitting virus(ASPV)by dot blot hybridization with digoxigenin-labelled viral c DNA probe(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2006,36(5):470-472.
[9]Cembali T,Folwell R J,Wandschneider P,et al.Economic implications of a virus prevention program in deciduous tree fruits in the US[J].Crop Protection,2003,22(10):1149-1156.
[10]Li L L,Dong Y F,Zhang Z P,et al.RT-PCR detection and molecular variability of Apple stem pitting virus(in Chinese)[J].Acta Horticulturae Sinica(园艺学报),2010,37(1):9-16.
[11]van Oosten H J,Meijnske C A R,Peerbooms H.Growth,yield and fruit quality of virus-infected and virus-free golden delicious apple trees,1968-1982[J].Acta Horticulturae,1982,130:213-220.
[12]Hu G J,Zhang Z P,Dong Y F,et al.Efficiency of virus elimination from potted apple plants by thermotherapy coupled with shoot-tip grafting[J].Australasian Plant Pathology,2015,44(2):167-173.
[13]Chamberian J S,Gibbs R A,Ranier J E,et al.Delection screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification[J].Nucleic Acids Research,1988,16(23):11141-11156.
[14]Fan X D,Dong Y F,Zhang Z P,et al.Multiplex RT-PCR assay for simultaneous detection of three latent apple viruses(in Chinese)[J].Acta Horticulturae Sinica(园艺学报),2009,36(12):1821-1826.
[15]Chen H X,Shao J Z,Sun J S,et al.Quick detection of apple latent viruses by two-step multiplex RT-PCRassay(in Chinese)[J].Journal of Fruit Science(果树学报),2012,29(4):695-701.
[16]Yang F,Wang G,Xu W,et al.A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses[J].Journal of Virological M ethods,2017,247:61-67.
[2]Hu G J,Dong Y F,Zhang Z P,et al.Occurrence and genetic diversity analysis of Apple stem pitting virus isolated from apples in China[J].Archives of virology,2017,162(8):2397-2402.
[3]Guo R,Li S F,Dong Y F,et al.Occurrence of Apple scar skin disease and sequence analysis of Apple scar skin viroid in Liaoning(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2005,35(5):472-474.
[4]Hu G J,Zhang Z P,Fan X D,et al.Research progress of main apple viruses of China(in Chinese)[J].China Fruits(中国果树),2017,(3):71-74.
[5]Yanase H.Back transmission of Apple stem grooving virus to apple seedlings and induction of symptoms of apple topw orking disease in Mitsuba Kaido(Malus sieboldii)and Kobano Zumi(Malus sieboldii var.arborescens)rootstocks[J].Acta Horticuturae,1983,130:117-122.
[6]Yanase H,Koganezawa H,Fridlund P R.Correlation of pear necrotic spot with pear vein yellows and apple stem pitting,and a flexuous filamentous virus associated with them[J].Acta Horticulturae,1989,235:157-158.
[7]Liu H Z,Yang Z K,Zhou Y B,et al.Study on genom esequence and small RNA of Apple stem pitting virus infecting a pear plant(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2017,47(5):584-590.
[8]Yang J L,Dong Y F,Li S F,et al.Detection of Apple stem pitting virus(ASPV)by dot blot hybridization with digoxigenin-labelled viral c DNA probe(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2006,36(5):470-472.
[9]Cembali T,Folwell R J,Wandschneider P,et al.Economic implications of a virus prevention program in deciduous tree fruits in the US[J].Crop Protection,2003,22(10):1149-1156.
[10]Li L L,Dong Y F,Zhang Z P,et al.RT-PCR detection and molecular variability of Apple stem pitting virus(in Chinese)[J].Acta Horticulturae Sinica(园艺学报),2010,37(1):9-16.
[11]van Oosten H J,Meijnske C A R,Peerbooms H.Growth,yield and fruit quality of virus-infected and virus-free golden delicious apple trees,1968-1982[J].Acta Horticulturae,1982,130:213-220.
[12]Hu G J,Zhang Z P,Dong Y F,et al.Efficiency of virus elimination from potted apple plants by thermotherapy coupled with shoot-tip grafting[J].Australasian Plant Pathology,2015,44(2):167-173.
[13]Chamberian J S,Gibbs R A,Ranier J E,et al.Delection screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification[J].Nucleic Acids Research,1988,16(23):11141-11156.
[14]Fan X D,Dong Y F,Zhang Z P,et al.Multiplex RT-PCR assay for simultaneous detection of three latent apple viruses(in Chinese)[J].Acta Horticulturae Sinica(园艺学报),2009,36(12):1821-1826.
[15]Chen H X,Shao J Z,Sun J S,et al.Quick detection of apple latent viruses by two-step multiplex RT-PCRassay(in Chinese)[J].Journal of Fruit Science(果树学报),2012,29(4):695-701.
[16]Yang F,Wang G,Xu W,et al.A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses[J].Journal of Virological M ethods,2017,247:61-67.