大麦条纹花叶病毒实时荧光RT-PCR检测方法的建立及应用
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摘要
大麦条纹花叶病毒为禾本科作物上一种重要的病原,可造成严重的经济损失。该病毒的主要传播方式为种传,因此对种子进行检疫监管是控制病害传播的一种有效途径。本文依据病毒保守序列RNA2beta C蛋白设计引物,研发了一种实时荧光RT-PCR检测方法。经特异性与灵敏度评价,仅对大麦条纹花叶病毒有效,无法对小麦线条花叶病毒、甘蔗花叶病毒及玉米褪绿斑驳病毒的阳性样品进行鉴定;且扩增灵敏度与普通RT-PCR相当,处于0.02 ng/μL水平。该方法相较于已有的检测方法,操作更简便快速,不易污染,灵敏度高,特异性优。此外,本研究结合实时荧光RT-PCR与普通RT-PCR两种检测方法,对未知谷物粉进行大麦条纹花叶病毒的检测鉴定,证实了新方法的可行性。
Barley stripe mosaic virus is an important pathogen on gramineous crops,which can cause considerable economic loss. BSMV spreads via seeds mainly. Accordingly,seed quarantine is an effective way to control virus spread. In this paper,one set of primers was designed based on the conservative sequence in RNA2 beta C protein and a real-time RT-PCR method was established for BSMV quarantine.Specificity and sensitivity showed that this method could be only used for BSMV,but not Wheat streak mosaic virus,Sugarcane mosaic virus or Maize chlorotic mottle virus. Sensitivity test confirmed that this method detected samples at the level of 0.02 ng/μL,similar to normal RT-PCR. The real-time RT-PCR established in this study is simpler,faster,less contaminative,more sensitive and more specific compared to other existing methods. What's more,this method was combined with normal RT-PCR in BSMV detection and identification of an unknown crop powder sample,which turned out to be highly feasible.
Barley stripe mosaic virus is an important pathogen on gramineous crops,which can cause considerable economic loss. BSMV spreads via seeds mainly. Accordingly,seed quarantine is an effective way to control virus spread. In this paper,one set of primers was designed based on the conservative sequence in RNA2 beta C protein and a real-time RT-PCR method was established for BSMV quarantine.Specificity and sensitivity showed that this method could be only used for BSMV,but not Wheat streak mosaic virus,Sugarcane mosaic virus or Maize chlorotic mottle virus. Sensitivity test confirmed that this method detected samples at the level of 0.02 ng/μL,similar to normal RT-PCR. The real-time RT-PCR established in this study is simpler,faster,less contaminative,more sensitive and more specific compared to other existing methods. What's more,this method was combined with normal RT-PCR in BSMV detection and identification of an unknown crop powder sample,which turned out to be highly feasible.
引文
[1]张月季.大、小麦条纹花叶病毒病的研究.浙江农业科学,1984(2):63-65,60,107.
[2]周锡康,曹明生,孙光荣,等.我国引进大麦品种资源中的大麦条纹花叶病毒.上海农业学报,1988(3):35-40.
[3]彭学贤,曲士绵.大麦条纹花叶病毒研究进展.病毒学杂志,1989(4):329-335.
[4]于善谦,王鸣岐,陈仲宜,等.种传大小麦病毒病的研究(Ⅰ)---大麦条纹花叶病毒在我国的证实.复旦学报(自然科学版),1980(4):372-379.
[5]王志民,李维琪,赵民安,等.大麦条纹花叶病毒的研究.微生物学报,1981,21(4):446-448,522.
[6]李国英,汪国发.新疆春麦种子带大麦条纹花叶病毒调查.新疆农业科学,1981,11(6):31-32.
[7]И.И.Митрофанова,李国英.大麦条纹花叶病毒的传播和危害.麦类作物学报,1987(6):45-46.
[8]邱并生,裴美云,谢浩.我国大麦条纹花叶病毒的研究Ⅰ.用ELISA方法检测麦种中的大麦条纹花叶病毒.植物病理学报,1982(3):29-32.
[9]孙现超,安德荣,青玲,等.大麦条纹花叶病毒北京分离物的鉴定.西南大学学报(自然科学版),2007,29(10):51-54.
[10]周锡康,曹明生.我国大麦品种资源中条纹花叶病的初步调查研究.植物保护,1986,12(2):25-26.
[11]Carroll T W.Seed Transmissibility of Two Strains of Barley Stripe Mosaic Virus.Virology,1972,48(2):323-336.
[12]谢浩,王志民,李维琪,等.大麦条纹花叶病毒(BSMV)在新疆的发生.植物病理学报,1981(1):11-14.
[13]Hunter B G,Smith J,Fattouh F,et al.Relationship of Lychnis Ringspot Virus to Barley Stripe Mosaic Virus and Poa Semilatent Virus.Intervirology,1989,30(1):18-26.
[14]Jiang Z,Li Z,Yue N,et al.Construction of infectious clones of lychnis ringspot virus and evaluation of its relationship with barley stripe mosaic virus by reassortment of genomic RNA segments.Virus Research,2018,243:106-109.
[15]Polák Z,Slykhuis J T.Comparisons of poa semilatent and barley stripe mosaic viruses.Canadian Journal of Botany,1972,50(2):263-267.
[16]Petty I T,French R,Jones R W,et al.Identification of barley stripe mosaic virus genes involved in viral RNAreplication and systemic movement.EMBO J,1990,9(11):3453-3457.
[17]Petty I T D,Jackson A O.Mutational analysis of barley stripe mosaic virus RNAβ.Virology,1990,179(2):712-718.
[18]Zhang X,Dong K,Xu K,et al.Barley stripe mosaic virus infection requires PKA‐mediated phosphorylation ofγb for suppression of both RNA silencing and the host cell death response.New Phytol,2018,218(4):1570-1585.
[19]Yang M,Zhang Y,Xie X,et al.Barley Stripe Mosaic Virusγb Protein Subverts Autophagy to Promote Viral Infection by Disrupting the ATG7-ATG8 Interaction.Plant Cell,2018,30(7):1582-1595.
[20]Holzberg S,Brosio P,Gross C,et al.Barley stripe mosaic virus-induced gene silencing in a monocot plant.Plant J,2002,30(3):315-327.
[2]周锡康,曹明生,孙光荣,等.我国引进大麦品种资源中的大麦条纹花叶病毒.上海农业学报,1988(3):35-40.
[3]彭学贤,曲士绵.大麦条纹花叶病毒研究进展.病毒学杂志,1989(4):329-335.
[4]于善谦,王鸣岐,陈仲宜,等.种传大小麦病毒病的研究(Ⅰ)---大麦条纹花叶病毒在我国的证实.复旦学报(自然科学版),1980(4):372-379.
[5]王志民,李维琪,赵民安,等.大麦条纹花叶病毒的研究.微生物学报,1981,21(4):446-448,522.
[6]李国英,汪国发.新疆春麦种子带大麦条纹花叶病毒调查.新疆农业科学,1981,11(6):31-32.
[7]И.И.Митрофанова,李国英.大麦条纹花叶病毒的传播和危害.麦类作物学报,1987(6):45-46.
[8]邱并生,裴美云,谢浩.我国大麦条纹花叶病毒的研究Ⅰ.用ELISA方法检测麦种中的大麦条纹花叶病毒.植物病理学报,1982(3):29-32.
[9]孙现超,安德荣,青玲,等.大麦条纹花叶病毒北京分离物的鉴定.西南大学学报(自然科学版),2007,29(10):51-54.
[10]周锡康,曹明生.我国大麦品种资源中条纹花叶病的初步调查研究.植物保护,1986,12(2):25-26.
[11]Carroll T W.Seed Transmissibility of Two Strains of Barley Stripe Mosaic Virus.Virology,1972,48(2):323-336.
[12]谢浩,王志民,李维琪,等.大麦条纹花叶病毒(BSMV)在新疆的发生.植物病理学报,1981(1):11-14.
[13]Hunter B G,Smith J,Fattouh F,et al.Relationship of Lychnis Ringspot Virus to Barley Stripe Mosaic Virus and Poa Semilatent Virus.Intervirology,1989,30(1):18-26.
[14]Jiang Z,Li Z,Yue N,et al.Construction of infectious clones of lychnis ringspot virus and evaluation of its relationship with barley stripe mosaic virus by reassortment of genomic RNA segments.Virus Research,2018,243:106-109.
[15]Polák Z,Slykhuis J T.Comparisons of poa semilatent and barley stripe mosaic viruses.Canadian Journal of Botany,1972,50(2):263-267.
[16]Petty I T,French R,Jones R W,et al.Identification of barley stripe mosaic virus genes involved in viral RNAreplication and systemic movement.EMBO J,1990,9(11):3453-3457.
[17]Petty I T D,Jackson A O.Mutational analysis of barley stripe mosaic virus RNAβ.Virology,1990,179(2):712-718.
[18]Zhang X,Dong K,Xu K,et al.Barley stripe mosaic virus infection requires PKA‐mediated phosphorylation ofγb for suppression of both RNA silencing and the host cell death response.New Phytol,2018,218(4):1570-1585.
[19]Yang M,Zhang Y,Xie X,et al.Barley Stripe Mosaic Virusγb Protein Subverts Autophagy to Promote Viral Infection by Disrupting the ATG7-ATG8 Interaction.Plant Cell,2018,30(7):1582-1595.
[20]Holzberg S,Brosio P,Gross C,et al.Barley stripe mosaic virus-induced gene silencing in a monocot plant.Plant J,2002,30(3):315-327.