西番莲TeMV和CMV双重RT-PCR检测体系的建立及应用
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摘要
【目的】建立西番莲夜来香花叶病毒(Telosma mosaic virus, TeMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)的双重RT-PCR检测体系。【方法】根据TeMV和CMV的外壳蛋白(coat protein, CP)基因序列,分别设计两对特异性引物,并对引物组合浓度、退火温度和循环次数进行优化。【结果】优化结果显示:TeMV和CMV最佳引物组合浓度分别为2.0 mmol·L~(-1)和8.0 mmol·L~(-1);最佳退火温度为57℃;最佳循环次数为35次。【结论】灵敏度分析结果显示,在样品c DNA原液稀释到10-5时,仍能检测到两种病毒扩增出的目的条带。同时,以健康组培苗作为对照组,应用建立的双重RT-PCR检测技术对采集于福建省不同产地的36份西番莲样品进行了病毒检测,发现有8份样品同时感染两种病毒,8份样品仅感染TeMV,10份样品仅感染CMV。本研究可为西番莲TeMV和CMV的检测提供便利。
【Objective】Passion fruit is rich in nutrition with unique flavor and more than 130 kinds of aromatic substances, covering most of the aroma in fruits. Passionfruit is an important raw material for juice making. At present, there are a large number of cultivated purple(P. edulis), yellow(P. edulis var.flavicarpa) and hybrid passionfruits in China. Passionfruit has been planted as a new fruit species in Fujian Province in recent years. However, severe passionfruit virus disease leads to tree decline as well as yield and quality decline, which seriously hinders the development of passionfruit industry. Cucumber mosaic virus, East Asian Passiflora virus, Passionfruit mottle virus, Papaya leaf curl Guangdong virus,Euphorbia leaf curl virus and Telosma mosaic virus have been found in China. Among them, Cucumber mosaic virus is the main infection virus. Therefore, it is of great significance to establish a rapid and efficient virus detection system for passionfruit.【Methods】From June to September 2018, 34 samples of passionfruit leaf were collected from different areas of Fujian Province. The samples were frozen in liquid nitrogen and stored in-80 ℃ refrigerator. According to the conserved region of TeMV, CMV coat protein gene in NCBI database, a pair of specific primers for the two viruses were designed using Primer5.0 software. Extraction of total RNA from passionfruit leaves was carried out using Trizol Kit(TRANS) according to the instruction manual. Synthesis of cDNA was done using reverse transcription Kit(TaKaRa). With the cDNA from the reverse transcription as a template, TeMV and CMV specific primers were used for RT-PCR amplification. The amplified product was recovered and ligated to the pMD18-T vector, then transformed into Escherichia coli DH5α. The positive clones were collected and sequenced after PCR detection. Based on single RT-PCR technology, the concentration of primer combination, annealing temperature and cycle times of Duplex RT-PCR system were optimized.【Results】The results of single RT-PCR amplification showed that when annealing temperature was 57 ℃, the band specificity was strongest, and the specific band was brighter and clearer than other bands. Therefore,57 ℃ was the best annealing temperature for TeMV and CMV. The nucleotide sequences of TeMV and CMV subjected to Blast alignment using NCBI database. The results showed that the amplified products were the partial sequences of TeMV and CMV of passionfruit. The optimization results of Duplex RT-PCR showed that the best primer combinations of TeMV and CMV were 2.0 mmol·L~(-1) and 8.0 mmol·L~(-1), respectively with the optimum annealing temperature of 57 ℃ and the optimum cycle number of 35. The sensitivity analysis results showed that when the total RNA solution was diluted to 10-5 the target bands amplified by the two viruses, 323 bp and 227 bp, could still be detected. Using healthy tissue culture seedlings as control group, 34 samples of passionfruit collected from different areas of Fujian Province were detected by using the established dual RT-PCR detection technique. Both TeMV and CMV could be detected at the same time.【Conclusion】In this study, a virus detection system for simultaneous detection of TeMV and CMV in passionfruit was established. When the optimized detection system is used to detect passionflower samples collected in the field, the detection effect is good. It provides technical support for rapid detection of passionflower virus diseases.
【Objective】Passion fruit is rich in nutrition with unique flavor and more than 130 kinds of aromatic substances, covering most of the aroma in fruits. Passionfruit is an important raw material for juice making. At present, there are a large number of cultivated purple(P. edulis), yellow(P. edulis var.flavicarpa) and hybrid passionfruits in China. Passionfruit has been planted as a new fruit species in Fujian Province in recent years. However, severe passionfruit virus disease leads to tree decline as well as yield and quality decline, which seriously hinders the development of passionfruit industry. Cucumber mosaic virus, East Asian Passiflora virus, Passionfruit mottle virus, Papaya leaf curl Guangdong virus,Euphorbia leaf curl virus and Telosma mosaic virus have been found in China. Among them, Cucumber mosaic virus is the main infection virus. Therefore, it is of great significance to establish a rapid and efficient virus detection system for passionfruit.【Methods】From June to September 2018, 34 samples of passionfruit leaf were collected from different areas of Fujian Province. The samples were frozen in liquid nitrogen and stored in-80 ℃ refrigerator. According to the conserved region of TeMV, CMV coat protein gene in NCBI database, a pair of specific primers for the two viruses were designed using Primer5.0 software. Extraction of total RNA from passionfruit leaves was carried out using Trizol Kit(TRANS) according to the instruction manual. Synthesis of cDNA was done using reverse transcription Kit(TaKaRa). With the cDNA from the reverse transcription as a template, TeMV and CMV specific primers were used for RT-PCR amplification. The amplified product was recovered and ligated to the pMD18-T vector, then transformed into Escherichia coli DH5α. The positive clones were collected and sequenced after PCR detection. Based on single RT-PCR technology, the concentration of primer combination, annealing temperature and cycle times of Duplex RT-PCR system were optimized.【Results】The results of single RT-PCR amplification showed that when annealing temperature was 57 ℃, the band specificity was strongest, and the specific band was brighter and clearer than other bands. Therefore,57 ℃ was the best annealing temperature for TeMV and CMV. The nucleotide sequences of TeMV and CMV subjected to Blast alignment using NCBI database. The results showed that the amplified products were the partial sequences of TeMV and CMV of passionfruit. The optimization results of Duplex RT-PCR showed that the best primer combinations of TeMV and CMV were 2.0 mmol·L~(-1) and 8.0 mmol·L~(-1), respectively with the optimum annealing temperature of 57 ℃ and the optimum cycle number of 35. The sensitivity analysis results showed that when the total RNA solution was diluted to 10-5 the target bands amplified by the two viruses, 323 bp and 227 bp, could still be detected. Using healthy tissue culture seedlings as control group, 34 samples of passionfruit collected from different areas of Fujian Province were detected by using the established dual RT-PCR detection technique. Both TeMV and CMV could be detected at the same time.【Conclusion】In this study, a virus detection system for simultaneous detection of TeMV and CMV in passionfruit was established. When the optimized detection system is used to detect passionflower samples collected in the field, the detection effect is good. It provides technical support for rapid detection of passionflower virus diseases.
引文
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[12]丁芳,曹庆,王国平,易干军,钟云.柑橘黄龙病、裂皮病和衰退病病原的多重RT-PCR检测[J].园艺学报,2006,33(5):947-952.DING Fang,CAO Qing,WANG Guoping,YI Ganjun,ZHONG Yun. Studies on the smiultaneous detection of citrus Huanglongbing Pathogen,Citrus exocortis viroid,Citrus tristeza virus by multiplex RT-PCR[J]. Acta Horticulturae Sinica,2006,33(5):947-952.
[13]郑轩,成巨龙,赵震,李正男,王威麟,张昊,吴云锋.五种烟草病毒TMV、CMV、TEV、PVY及TVBMV的多重RT-PCR同步检测[J].植物病理学报,2011,41(2):146-153.ZHENG Xuan,CHENG Julong,ZHAO Zhen,LI Zhengnan,WANG Weilin,ZHANG Hao,WU Yunfeng. Simultaneous detection of five viruses(TMV,CMV,TEV,PVY and TVBMV)infecting tobacco by multiplex RT-PCR[J]. Acta Phytopathologica Sinica,2011,41(2):146-153.
[14]沈建国,高芳銮,虞赟,蔡伟,李敏,于文涛,廖富荣.应用RTPCR检测水仙潜隐病毒[J].热带作物学报,2012,33(10):1808-1811.SHEN Jianguo,GAO Fangluan,YU Yun,CAI Wei,LI Min,YU Wentao,LIAO Furong. Detection of Narcissus latent virus by RT-PCR[J]. Chinese Journal of Tropical Crops,2012,33(10):1808-1811.
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[16]刘欢,刘斐,赵磊,郝兴安,吴云锋. 4种番茄病毒多重RT-PCR检测及应用[J].植物病理学报,2016,46(5):716-720.LIU Huan,LIU Fei,ZHAO Lei,HAO Xing’an,WU Yunfeng.Multiplex RT-PCR detection and application of four tomato viruses[J]. Acta Phytopathologica Sinica,2016,46(5):716-720.
[17]李唯,许蕊,栗孟飞,王雅琳.应用多重RT-PCR检测甘肃‘成县迟蒜’中的大蒜潜隐病毒和洋葱黄矮病毒[J].甘肃农业大学学报,2013,48(2):46-49.LI Wei,XU Rui,LI Mengfei,WANG Yalin. Detection of garlic latent virus and onion yellow dwarf virus in‘Chengxianchisuan’of Gansu by multiplex RT-PCR[J]. Journal of Gansu Agricultural University,2013,48(2):46-49.
[18]苏静,陈佰鸿,毛娟,左存武,胡紫璟,王凯.酿酒葡萄4种病毒多重RT-PCR检测体系的建立[J].果树学报,2017,34(5):632-638.SU Jing,CHEN Baihong,MAO Juan,ZUO Cunwu,HU Zijing,WANG Kai. Establishment of multiple RT-PCR detection system for four virus in the cultivars of wine grape[J]. Journal of Fruit Science,2017,34(5):632-638.
[19]董代幸,张祥林,罗明,韩剑,冯世强,唐德贤,岳仲海.马铃薯病毒一步法多重RT-PCR检测技术的构建[J].微生物学通报,2011,38(1):131-137.DONG Daixing,ZHANG Xianglin,LUO Ming,HAN Jian,FENG Shiqiang,TANG Dexian,YUE Zhonghai. Development of one-step multiplex RT-PCR for simultaneous detection of four potato viruses[J]. Microbiology,2011,38(1):131-137.
[20]蔡志翔,马瑞娟,俞明亮,沈志军,许建兰,张妤艳.利用多重RT-PCR检测桃潜隐性病毒[C]//中国园艺学会桃分会第二届学术年会论文集.中国园艺学会桃分会,中国成都,2009:290-296.CAI Zhixiang,MA Ruijuan,YU Mingliang,SHEN Zhijun,XU Jianlan,ZHANG Shuyan. Detection of latent virus in peach by multiplex RT-PCR[C]//Papers Collection of the Second Annual meeting of Peach Branch of Chinese Horticulture Society. Chinese Horticultural Society Peach Branch,China Chengdu,2009:290-296.
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[23]范旭东,董雅凤,张尊平,任芳,李亚惠.葡萄4种病毒多重RT-PCR检测体系的建立[J].园艺学报,2012,39(5):949-956.FAN Xudong,DONG Yafeng,ZHANG Zunping,REN Fang,LI Yahui. Multiplex RT-PCR for simultaneous detection of four grapevine viruses[J]. Acta Horticulturae Sinica,2012,39(5):949-956.
[2]韦晓霞,潘少霖,吴如健,王小安,周丹蓉,叶新福.‘黄金’黄果西番莲在福建引种初报及栽培要点[J].东南园艺,2016(6):6-8.WEI Xiaoxia,PAN Shaolin,WU Rujian,WANG Xiao’an,ZHOU Danrong,YE Xinfu. A preliminary report on introduction of passiflora edulis cv.'Gold'in Fujian Province and its key cultivation points[J]. Fujian Fruits,2016(6):6-8.
[3]严佳文,袁启凤,彭志军,王立娟,解璞,陈楠,马玉华.西番莲病毒病害研究进展[J].热带农业科学,2018,38(4):85-94.YAN Jiawen,YUAN Qifeng,PENG Zhijun,WANG Lijuan,XIE Pu,CHEN Nan,MA Yuhua. Research progress on viruses of passionfruit[J]. Chinese Journal of Tropical Agriculture,2018,38(4):85-94.
[4]徐平东,李梅,柯冲.福建西番莲病毒病的发生及其病原黄瓜花叶病毒亚组鉴定[J].植物保护学报,1999,26(1):50-54.XU Pingdong,LI Mei,KE Chong. Occurrence of passionflower virus disease and identification of its pathogenic subgroup of Cucumber Mosaic virus in Fujian Province[J]. Journal of Plant Protection,1999,26(1):50-54.
[5]谢丽雪,张小艳,郑姗,张立杰,李韬.侵染西番莲的夜来香花叶病毒的分子鉴定及特异性检测[J].中国农业科学,2017,50(24):4725-4734.XIE Lixue,ZHANG Xiaoyan,ZHENG Shan,ZHANG Lijie,LI Tao. Molecular identification and specific detection of Telosma mosaic virus infecting passionfruit[J]. Scientia Agricultura Sinica,2017,50(24):4725-4734.
[6] CHANG C A. Characterization and comparison of passionfruit mottle virus,a newly recognized potyvirus,with passionfruit woodiness virus[J]. Phytopathology,1992,82(11):1358-1363.
[7] CHENG Y H,DENG T C,CHEN C C,CHANG C H,CHANG C A. First report of Euphorbia leaf curl virus and Papaya leaf curl Guangdong virus on passion fruit in Taiwan[J]. Plant Disease,2014,98(12):1746.
[8] FISCHER I,REZENDE J. Diseases of Passion flower(Passiflora spp.)[J]. Pest Technology,2008,2(1):1-19.
[9] POLSTON J E,LONDONO M A,COHEN A L,PADILLA-RODRIGUEZ M,ROSARIO K,BREITBART M. Genome Sequence of Euphorbia mosaic virus from Passionfruit and Euphorbia heterophylla in Florida[J]. Genome Announc,2017,5(9):e01714-16.
[10] SPIEGEL S,ZEIDAN M,SOBOLEY I,BECKELMAN Y,HOLDENGREBER V,TAM Y. The complete nucleotide sequence of Passiflora latent virus and its phylogenetic relationship to other carlaviruses[J]. Archives of Virology,2007,152(1):181-189.
[11]邹迎春,杨朝柱,覃大吉,赵清华,向极钎,殷红清,杨永康,黄建民,皮磊.百合病毒的多重RT-PCR检测技术[J].湖北农业科学,2013,52(20):5051-5053.ZOU Yingchun,YANG Chaozhu,QIN Daji,ZHAO Qinghua,XIANG Jiqian,YIN Hongqing,YANG Yongkang,HUANG Jianmin,PI Lei. Studies on multiplex RT-PCR detection on viruses of Lily[J]. Hubei Agricultural Sciences,2013,52(20):5051-5053.
[12]丁芳,曹庆,王国平,易干军,钟云.柑橘黄龙病、裂皮病和衰退病病原的多重RT-PCR检测[J].园艺学报,2006,33(5):947-952.DING Fang,CAO Qing,WANG Guoping,YI Ganjun,ZHONG Yun. Studies on the smiultaneous detection of citrus Huanglongbing Pathogen,Citrus exocortis viroid,Citrus tristeza virus by multiplex RT-PCR[J]. Acta Horticulturae Sinica,2006,33(5):947-952.
[13]郑轩,成巨龙,赵震,李正男,王威麟,张昊,吴云锋.五种烟草病毒TMV、CMV、TEV、PVY及TVBMV的多重RT-PCR同步检测[J].植物病理学报,2011,41(2):146-153.ZHENG Xuan,CHENG Julong,ZHAO Zhen,LI Zhengnan,WANG Weilin,ZHANG Hao,WU Yunfeng. Simultaneous detection of five viruses(TMV,CMV,TEV,PVY and TVBMV)infecting tobacco by multiplex RT-PCR[J]. Acta Phytopathologica Sinica,2011,41(2):146-153.
[14]沈建国,高芳銮,虞赟,蔡伟,李敏,于文涛,廖富荣.应用RTPCR检测水仙潜隐病毒[J].热带作物学报,2012,33(10):1808-1811.SHEN Jianguo,GAO Fangluan,YU Yun,CAI Wei,LI Min,YU Wentao,LIAO Furong. Detection of Narcissus latent virus by RT-PCR[J]. Chinese Journal of Tropical Crops,2012,33(10):1808-1811.
[15]张业辉,张振臣蒋士君,秦艳红,张德胜,乔奇,王永江. 3种甘薯病毒多重RT-PCR检测方法的建立[J].植物病理学报,2010,40(1):95-98.ZHANG Yehui,ZHANG Zhenchen,JIANG Shijun,QIN Yanhong,ZHANG Desheng,QIAO Qi,WANG Yongjiang. Development of a multiplex RT-PCR detection method for three sweet potato potyviruses[J]. Acta Phytopathologica Sinica,2010,40(1):95-98.
[16]刘欢,刘斐,赵磊,郝兴安,吴云锋. 4种番茄病毒多重RT-PCR检测及应用[J].植物病理学报,2016,46(5):716-720.LIU Huan,LIU Fei,ZHAO Lei,HAO Xing’an,WU Yunfeng.Multiplex RT-PCR detection and application of four tomato viruses[J]. Acta Phytopathologica Sinica,2016,46(5):716-720.
[17]李唯,许蕊,栗孟飞,王雅琳.应用多重RT-PCR检测甘肃‘成县迟蒜’中的大蒜潜隐病毒和洋葱黄矮病毒[J].甘肃农业大学学报,2013,48(2):46-49.LI Wei,XU Rui,LI Mengfei,WANG Yalin. Detection of garlic latent virus and onion yellow dwarf virus in‘Chengxianchisuan’of Gansu by multiplex RT-PCR[J]. Journal of Gansu Agricultural University,2013,48(2):46-49.
[18]苏静,陈佰鸿,毛娟,左存武,胡紫璟,王凯.酿酒葡萄4种病毒多重RT-PCR检测体系的建立[J].果树学报,2017,34(5):632-638.SU Jing,CHEN Baihong,MAO Juan,ZUO Cunwu,HU Zijing,WANG Kai. Establishment of multiple RT-PCR detection system for four virus in the cultivars of wine grape[J]. Journal of Fruit Science,2017,34(5):632-638.
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