WEI Shuang,YUAN Jun-jie,YANG Zhuo-yu,et al.Dual Priming Oligonucleotide System for Duplex Detection of Southern Bean Mosaic Virus and Tobacco Ring Spot Virus[J].Soybean Science,2021,40(02):265-269.[doi:10.11861/j.issn.1000-9841.2021.02.0265]
双重DPO-RT-PCR检测南方菜豆花叶病毒及烟草环斑病毒
- Title:
- Dual Priming Oligonucleotide System for Duplex Detection of Southern Bean Mosaic Virus and Tobacco Ring Spot Virus
- Keywords:
- Southern bean mosaic virus; Tobacco ring spot virus; Imported soybean; Seed-borne virus; Detection; DPO-RT-PCR
- 文献标志码:
- A
- 摘要:
- 南方菜豆花叶病毒及烟草环斑病毒是中国进境植物检疫性有害生物，属于大豆种传病害，为提高在进口大豆中两种病毒的检出率，本研究针对SBMV及TRSV基因组中的保守序列，分别设计特异性DPO引物，建立了一种快速检测这两种植物病毒的双重DPO-RT-PCR方法，并对该方法的特异性、灵敏度及退火温度敏感性等性能进行评价。结果表明：该方法能准确地从10种植物病毒中鉴定出SBMV及TRSV，表现出良好的特异性，且检测灵敏度可达0.02 ng?μL-1，退火温度为在45~65 ℃时该方法检测结果无明显差异，表现出对退火温度不敏感的特性。研究结果为进出境植物病毒的高通量检测提供了新的思路。
- Abstract:
- Abstract: Southern bean mosaic virus (SBMV) and tobacco ring spot virus (TRSV) are quarantine pests in China, belonging to soybean seed-borne diseases. In order to improve the detection rate of these two viruses in imported soybeans, we designed two pairs of DPO (dual priming oligonucleotide) primers based on specific sequence of SBMV and TRSV in this research, and developed a duplex DPO-RT-PCR method for the detection of SBMV and TRSV. The method has been evaluated for its specificity, sensitivity and annealing temperature sensitivity. The result showed that this method could accurately identify SBMV and TRSV from 10 kinds of plant viruses, showing good specificity and detection sensitivity up to 0.02 ng?L-1. The detection results of this method had no significant difference at the annealing temperature of 45-65 ℃, showing the insensitivity characteristics to annealing temperature. The study provides a new method for high-throughput detection of plant pathogens.
参考文献/References:
［1］陈萍, 李盼畔, 刘明航, 等. 广州南沙口岸进口大豆截获有害生物疫情分析［J］.植物检疫, 2020, 34(1): 67-70. (Chen P, Li P P, Liu M H, et al. Analysis of pests intercepted from imported soybean in Guangzhou Nansha Port［J］. Plant Quarantine, 2020, 34(1): 67-70.)［2］魏尊苗, 艾嘉亮, 尤波, 等. 黑龙江口岸进口俄罗斯大豆疫情分析与建议［J］. 植物检疫, 2018, 32(4): 78-81. (Wei Z M, Ai J L, You B, et al. Analysis of pest interception from Russia soybean in Heilongjiang ports and managment suggestion［J］. Plant Quarantine, 2018, 32(4): 78-81.)［3］杨卓瑜, 卢乃会, 袁俊杰, 等. 2014—2018年湛江口岸进境大豆疫情分析与建议［J］. 植物检疫, 2020, 34(3): 80-84. (Yang Z Y, Lu N H, Yuan J J, et al. Analysis on epidemic situation of imported soybean at Zhanjiang Port and quarantine suggestions［J］. Plant Quarantine, 2020, 34(3): 80-84.)［4］朱煜建, 周伟光.关注进口大豆携带杂草传带植物病害的潜在可能性［J］.农业与技术, 2020, 40(5): 38-40. (Zhu Y J, Zhou W G. Concern about the potential of imported soybeans to carry weed-borne plant diseases［J］. Agriculture and Technology, 2020, 40(5): 38-40.)［5］马淑梅,丁俊杰,顾鑫,等.黑龙江省大豆主要病害发生危害调査［J］. 黑龙江农业科学, 2005(6): 48-52. (Ma S M, Ding J J, Gu X, et al. Survey on the occurrence and damage in main disease of soybean in Heilongjiang Province［J］. Heilongjiang Agrcultrual Sciences, 2005(6): 48-52.)［6］谷建夫, 范晓红, 陈建宏, 等.南方菜豆花叶病毒在大豆种子中的分布特点与检疫的关系［J］.天津农林科技, 1998(1): 3-5. (Gu J F, Fan X H, Chen J H, et al. The relationship between quarantine and distribution characteristics of mosaic virus in soybean seeds［J］. Science and Technology of Tianjin Agriculture and Forestry, 1998(1): 3-5.)［7］黄江华, 陈秀菊, 彭仁, 等. 烟草环斑病毒研究进展［J］. 现代农业科学, 2008(1): 24-27. (Huang J H, Chen X J, Peng R, et al. Research advance in tobacco ringspot virus［J］. Modern Agricultural Sciences, 2008(1): 24-27.)［8］国家质量监督检验检疫总局. SN/T 3438-2012, 南方菜豆花叶病毒检疫鉴定方法［S］. 北京:中国标准出版社, 2012. (General Administration of Quality Supervision, Inspection and Quarantine. SN/T 3438-2012, Detection and identification of southern bean mosaic virus［S］. Beijing: Standards Press of China, 2012.)［9］国家质量监督检验检疫总局. GB/T 28081-2011, 烟草环斑病毒检疫鉴定方法［S］. 北京:中国标准出版社, 2012. (General Administration of Quality Supervision, Inspection and Quarantine. GB/T 28081-2011, Detection and identification of tobacco ringspot virus［S］. Beijing: Standards Press of China, 2012.)［10］王亚琴, 田沂民, 于翠, 等. 基于单克隆抗体的南方菜豆花叶病毒血清学检测技术［J］. 植物保护学报, 2020, 47(2): 347-354. (Wang Y Q, Tian Y M, Yu C, et al. Monoclonal antibody-based serological technology for southern bean mosaic virus detection［J］. Journal of Plant Protection, 2020, 47(2): 347-354.)［11］Chun J Y, Kim K J, Hwang I T, et al. Dual priming oligonucleotide system for the duplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene［J］.Nucleic Acids Research, 2007, 35(6): e40．［12］苏琰, 李融, 黎华. 基于双启动寡聚核苷酸引物的PCR技术及其研究应用［J］.基因组学与应用生物学, 2018, 37(7): 2906- 2912. (Su Y, Li R, Li H. Research and application of PCR technique based on dual priming oligonucleotide primers［J］. Genomics and Applied Biology, 2018, 37(7): 2906-2912.)［13］魏霜, 马新华, 汪天杰, 等. 双重DPO-PCR检测副溶血弧菌和霍乱弧菌［J］. 食品工业科技, 2016, 37(22): 80- 83. (Wei S, Ma X H, Wang T J, et al. Multiplex DPO-PCR for the detection of Vibrio parahaemolyticus and Vibrio cholerae［J］. Science and Technology of Food Industry, 2016, 37(22): 80-83.)［14］张娜, 乾义柯, 魏霜, 等. 两种向日葵检疫性真菌病害的双重DPO-PCR检测方法［J］.植物检疫, 2015, 29(6): 35-38. (Zhang N, Qian Y K, Wei S, et al. Multiplex DPO-PCR detection of two quarantine fungal diseases of sunflower［J］. Plant Quarantine, 2015, 29(6): 35-38.)［15］乾义柯,张娜,魏霜,等.向日葵黄萎病病原菌轮枝菌的双重DPO-PCR检测方法［J］.植物保护学报,2017,44(1):115-120.(Qian Y K, Zhang N, Wei S, et al. Multiplex DPO-PCR detection of Verticillium dahliae and V. albo-atrum from sunflower Verticillium wilt［J］. Journal of Plant Protection, 2017, 44(1): 115-120.)［16］刘忠梅,罗佳,潘仲乐,等.应用DPO技术检测番茄斑萎病毒方法的建立［J］.河南农业科学,2017, 46(11): 93-97. (Liu Z M, Luo J, Pan Z L, et al. Development of DPO-PCR detection method for tomato spotted wilt virus［J］. Journal of Henan Agricultural Sciences, 2017, 46(11): 93-97.)［17］龙阳,袁俊杰,候翠丽,等.油菜茎基溃疡病菌DPO-PCR检测方法的建立［J］.植物检疫,2017,31(4):42-45.(Long Y, Yuan J J, Hou C L, et al. Development of DPO-PCR method for the detection of Leptosphaeria maculans［J］. Plant Quarantine, 2017, 31(4): 42-45.)［18］魏霜,袁俊杰,李桂芬,等.南方菜豆花叶病毒RT-RPA检测方法的建立［J］.植物检疫,2018,32(1):50-53.(Wei S, Yuan J J, Li G F, et al. One-step Reverse Transcription Recombinase Polymerase Amplification （RT-RPA）for the detection of southern bean mosaic virus［J］. Plant Quarantine, 2018, 32(1): 50-53.)［19］袁俊杰,龙阳,渭婷玉,等.添加扩增内标的逆转录重组酶聚合酶扩增技术检测三种大豆病毒［J］.农业生物技术学报, 2020, 28(12):2261-2269.（Yuan J J, Long Y, Wei T Y, et al. Application of RT-RPA technology included with IAC in the detection of soybean three viruses［J］. Journal of Agricultural Biotechnology, 2020, 28(12):2261-2269.）［20］郭立新,段维军,徐亚飞,等.逆转录环介导等温扩增技术检测南方菜豆花叶病毒［J］.植物病理学报,2014,44(4):349-356.(Guo L X, Duan W J, Xu Y F, et al. Detection of southern bean mosaic virus by the reverse transcription loop-mediated isothermal amplification method［J］. Acta Phytopathologica Sinica, 2014, 44(4): 349-356.)［21］张吉红,陈先锋,孙辉.逆转录环介导等温扩增技术检测烟草环斑病毒的研究［J］.植物病理学报,2014,44(6):629-633. (Zhang J H, Chen X F, Sun H. Research on detection of tobacco ringspot virus by RT-LAMP［J］. Acta Phytopathologica Sinica, 2014,44(6):629-633.)
相似文献/References:
[1]郭立新,陈先锋,徐亚飞,等.应用逆转录环介导等温扩增技术检测大豆种子中烟草环斑病毒[J].大豆科学,2012,31(06):980.[doi:10.3969/j.issn.1000-9841.2012.06.026]
GUO Li-xin,CHEN Xian-feng,XU Ya-fei,et al.Detection of Tobacco Ringspot Virus in Soybean Seeds by the Reverse Transcription Loop-mediated Isothermal Amplification Method[J].Soybean Science,2012,31(02):980.[doi:10.3969/j.issn.1000-9841.2012.06.026]
[2]闻伟刚,崔俊霞,盛蕾.进境大豆种子中烟草环斑病毒的快速检测[J].大豆科学,2007,26(05):748.[doi:10.3969/j.issn.1000-9841.2007.05.023]
WEN Wei-gang,CUI Jun-xia,SHENG Lei.RAPID DETECTION OF TOBACCO RINGSPOT VIRUS IN IMPORTED SOYBEAN SEEDS[J].Soybean Science,2007,26(02):748.[doi:10.3969/j.issn.1000-9841.2007.05.023]
备注/Memo
收稿日期：2020-12-03