Development of SYBR GreenⅠReal-time RT-PCR for the Detection of Cucumber green mottle mosaic virus
TIAN Yi-Min1, WU Jian-Xiang2, CHEN Lei3, LUO Jin-Yan3, TENG Kai4, YE Jun1, DU Wei5, YU Cui1,*
1 Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs District, Shanghai 200135, China;
2 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China;
3 Shanghai Agricultural Technology Extension Center, Shanghai 201103, China;
4 Division of Animal and Plant Quarantine and Inspection of Shanghai Customs District, Shanghai 200082, China;
5 Agricultural Technology Extension Station of Ningxia, Yinchuan 750001, China
Abstract：Cucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus and is also transmitted by seeds which often causes seriously damages to productions of watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (Cucumis sativus), and other cucurbits. To guarantee the smooth proceeding of seed trade, it is necessary to establish more rapidly and precisely detection methods. This study developed a SYBR GreenⅠreal-time RT-PCR technique for detection of CGMMV in seeds. It is crucial to design and screen a pair of specific primers based on the conserved CGMMV gene sequences. The result showed that the development of SYBR GreenⅠreal time RT-PCR method in this study could detect CGMMV specially, and there was no cross-reactivity with other common cucurbits viruses. Sensitivity assay showed that the sensitivity of this method was 1 000 times that of RT-PCR and 100 times that of TaqMan RT-PCR. And the whole detection process could be completed within 2.5 h, indicating that the SYBR GreenⅠreal-time RT-PCR was a rapid, sensitive and simple detection method. Cucurbitaceae seeds, seedlings, and fruits imported and purchased from domestic market were employed to be detected by SYBR GreenⅠreal-time RT-PCR and TaqMan real-time RT-PCR simultaneously. The result showed that the value of kappa of the two method was above 0.82, indicating that the novel SYBR GreenⅠreal-time RT-PCR method could be effective and reliable. The present study provides new technical means for rapid clearance of seeds.
田沂民, 吴建祥, 陈磊, 罗金燕, 滕凯, 叶军, 杜伟, 于翠. 黄瓜绿斑驳花叶病毒SYBR GreenⅠ实时荧光RT-PCR检测方法的建立[J]. 农业生物技术学报, 2022, 30(8): 1640-1648.
TIAN Yi-Min, WU Jian-Xiang, CHEN Lei, LUO Jin-Yan, TENG Kai, YE Jun, DU Wei, YU Cui. Development of SYBR GreenⅠReal-time RT-PCR for the Detection of Cucumber green mottle mosaic virus. 农业生物技术学报, 2022, 30(8): 1640-1648.
[1] 车海彦, 曹学仁, 贺延恒, 等. 2020. 海南岛西瓜病毒病种类鉴定及发生分布[J]. 植物病理学报, 50(5): 632-636.
(Che H Y, CAO X R, HE Y H, et al.2020. Distribution and identification of watermelon viruses in Hainan island[J]. Acta Phytopathological Sinica, 50(5): 632-636.)
[2] 陈青, 陈红运, 廖富荣, 等. GB/T 28071-2011. 黄瓜绿斑驳花叶病毒检疫鉴定方法[S]. 北京: 中国标准出版社, 2011.
(Chen Q, Chen H Y, Liao F R, et al.GB/T 28071-2011. Detection and identification of Cucumber green mottle mosaic virus[S]. Beijing: National Standards Press, 2011.
[3] 陈京, 李明福. 2007. 新入侵的有害生物: 黄瓜绿斑驳花叶病毒[J]. 植物检疫, 21(2): 94-96.
(Chen J, Li M F.2007. New invasive pest: Cucumber green mottle mosaic virus[J]. Plant Quarantine, 21(2): 94-96.)
[4] 陈红运, 林石明, 陈青, 等. 2009. 黄瓜绿斑驳花叶病毒辽宁分离物全基因组序列测定[J]. 病毒学报, 25(1): 68-72.
(Chen H Y, Lin S M, Chen Q, et al.2009. Complete genomic sequence of a watermelon isolate of Cucumber green mottle mosaic virus in northern China[J]. Chinese Journal of Virology, 25(1): 68-72.)
[5] 程兆邦, 任春梅, 廖倩, 等. 2013. 江苏黄瓜绿斑驳花叶病毒的发生和防治[J]. 江苏农业学报, 41(2): 114-117.
(Cheng Z B, Ren C M, Liao Q, et al.2013. Occurrence and control of Cucumber green mottle mosaic virus in Jiangsu[J]. Jiangsu Journal of Agricultural Sciences,41(2): 114-117.)
[6] 邓丛良, 江明, 汪万春, 等. 2008. 应用MNP-RT-PCR方法检测黄瓜绿斑驳花叶病毒[J]. 植物病理学报, 38(4): 436-440.
(Deng C L, Jiang M, Wang W C, et al.2008. Detection of Cucumber green mottle mosaic virus using MNP-RT-PCR method[J]. Acta Phytopathologica Sinica, 38(4): 436-440.)
[7] 胡亚萍, 董萍, 赵驾浩, 等. 2013. 上海郊区黄瓜绿斑驳花叶病毒的鉴定和防治对策[J]. 上海农业学报, 29(1): 55-58.
(Hu Y P, Dong P, Zhao J H, et al.2013. Identification and control measure of Cucumber green mottle mosaic virus in Shanghai suburbs[J]. Acta Agriculturae, 29(1): 55-58.)
[8] 阚春月, 于翠, 杨翠云, 等. 2011. 黄瓜绿斑驳花叶病毒分子检测方法的建立及评价[J]. 上海交通大学学报(农业科学版), 28(5): 457-461, 466.(Kan C Y, Yu C, Yang C Y, et al. 2010. Establish and evaluate molecular detection methods of Cucumber green mottle mosaic virus[J]. Journal of Shanghai Jiaotong University (Agricultural Science), 28(5): 457-461, 466.)
[9] 李俊香, 古勤生. 2015. 黄瓜绿斑驳花叶病毒传播方式的研究进展[J]. 中国蔬菜, 1: 13-18.
(Li J X, Gu Q S.2015. Research progress in transmission of Cucumber mosaic virus[J]. China Vegetables, 1: 13-18.)
[10] 李小妮, 任小平, 王琳, 等. 2009. 广东省黄瓜绿斑驳花叶病毒分子检测及防疫[J]. 植物保护学报, 36(3): 283-284.
(Li X N, Ren X P, Wang L, et al.2009. Molecular detection and epidemic prevention of Cucumber green mottle mosaic virus in Guangdong, China[J]. Journal of Plant Protection, 36(3): 283-284.)
[11] 刘华威, 罗来鑫, 朱春雨, 等. 2016. 黄瓜绿斑驳花叶病毒防治研究进展[J]. 植物保护, 42(6): 29-37.
(Liu H W, Luo L X, Zhu C Y, et al.2016. Research progress in management of Cucumber green mottle mosaic virus[J]. Plant Protection, 42(6): 29-37.)
[12] 孙晓辉, 高利利, 刘锦, 等. 2018. 番茄褪绿病毒SYBR GreenⅠ实时荧光定量PCR方法[J]. 植物病理学报, 48(5): 700-706.
(Sun X H, Gao L L, Liu J, et al.2018. SYBR GreenⅠquantitative real-time PCR (qPCR) for Tomato chlorosis virus[J]. Acta Phytopathologica Sinica, 48(5): 700-706.)
[13] 孙鑫, 张健, 杨磊, 等. 2020. 基于SYBR GreenⅠ桑树皱叶病毒的qPCR检测方法的建立及应用[J]. 蚕业科学, 46(2): 140-145.
(Sun X, Zhang J, Yang L, et al.2020. Establishment and application of SYBR GreenⅠbased on qPCR assay for the detection of Mulberry crinkle leaf virus[J]. Science of Sericulture, 46(2): 140-145.)
[14] 肖彩丽, 郑红英, 吴晓花, 等. 2016. 黄瓜绿斑驳花叶病毒基因组全序列分析及病害的田间调查[J]. 植物保护学报, 43(4): 529-536.
(Xiao C L, Zhen H Y, Wu X H, et al.2016. The complete genomic sequence analysis of three isolates of Cucumber green mottle mosaic virus in Zhejiang and the application of prepared antiserum[J]. Journal of Plant Protection, 43(4): 529-536.)
[15] 张珣. 2008. 六种植物病毒Real-time PCR定量方法的建立及其应用[D]. 博士学位论文, 中国农业科学院, 导师: 王锡锋, pp. 79-80.
(Zhang X.2008. Establishment and application of real-time quantitative PCR methods for the detection of 6 plant viruses[D]. Thesis for Ph.D., Chinese Academy of Agricultural Sciences, Supervisor: Wang X F, pp. 79-80.)
[16] 张卫东, 权永兵, 廖力, 等. 2011. RT-PCR法特异性快速检测黄瓜绿斑驳花叶病毒[J]. 江西农业大学学报, 33(1): 43-46.
(Zhang W D, Quan Y B, Liao L, et al.2011. Rapid detection of Cucumber green mottle mosaic virus by RT-PCR[J]. Acta Agriculturae Universitatis Jiangxiensis, 33(1): 43-46.
[17] Ainsworth G C.1935. Mosaic disease of the cucumber[J]. Annals of Applied Biology, 22(1): 55-67.
[18] Chen H Y, Zhao W J, Gu Q S, et al.2008. Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus[J]. Journal of Virological Methods, 149(2): 326-329.
[19] Cho I S, Yoon J Y, Chung B N, et al.2021. First report of Cucumber green mottle mosaic virus infecting Perilla frutescens in Korea and the world[J]. Journal of Plant Pathololy, 103: 347.
[20] Dombrovsky A, Tran-Nguyen L T T, Jones R A C.2017. Cucumber green mottle mosaic virus: Rapidly increasing global distribution, etiology, epidemiology, and management[J]. Review in Advance, 55: 231-256.
[21] Jiao Y B, Jiang J J, Wu Y H.2019. Rapid detection of Cucumber green mottle mosaic virus in watermelon through a recombinase polymerase amplification assay[J]. Journal of Virology Methods, 270: 146-149.
[22] Liu H W, Luo L X, Li J Q, et al.2014. Pollen and seed transmission of Cucumber green mottle mosaic virus in cucumber[J]. Plant Pathology, 63(1): 72-77.
[23] Vani S, Varma A.1993. Properties of Cucumber green mottle mosaic virus isolated from water of river Yamuna[J]. India Phytopathology, 46(2): 118-122.
