Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

doi:10.1016/S2095-3119(14)60892-3

Journal of Integrative Agriculture ›› 2015, Vol. 14 ›› Issue (5): 900-908.DOI: 10.1016/S2095-3119(14)60892-3

Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou

1、Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Provincial Engineering Research Center
for Potatoes, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, P.R.China
2、Potato Research Centre, Agriculture and Agri-Food Canada, Fredericton, E3B 4Z7, New Brunswick, Canada

收稿日期:2014-05-19 出版日期:2015-05-01 发布日期:2015-05-13
通讯作者: XIONG Xing-yao, Tel/Fax: +86-731-84635295,E-mail: xiongxy@hunau.net; NIE Xian-zhou, Tel: +1-506-4604514,Fax: +1-506-4604377, E-mail: xianzhou.nie@agr.gc.ca
作者简介:HU Xin-xi, Tel: +86-731-84618171, E-mail: huxinxi163@163.com; LEI Yan, Tel: +86-731-84618171, E-mail: leiyan5181@sina.com;
基金资助:
This research was supported by the Non-Profit Industry Financial Program of Ministry of Agriculture of China (20130328) to Prof. Liu Yong and Prof. Dai Liangying, and the Natural Science Foundation of Hunan Province, China to Prof. Hu Xinxi (11JJ2018).

Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou

1、Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Provincial Engineering Research Center
for Potatoes, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, P.R.China
2、Potato Research Centre, Agriculture and Agri-Food Canada, Fredericton, E3B 4Z7, New Brunswick, Canada

Received:2014-05-19 Online:2015-05-01 Published:2015-05-13
Contact: XIONG Xing-yao, Tel/Fax: +86-731-84635295,E-mail: xiongxy@hunau.net; NIE Xian-zhou, Tel: +1-506-4604514,Fax: +1-506-4604377, E-mail: xianzhou.nie@agr.gc.ca
About author:HU Xin-xi, Tel: +86-731-84618171, E-mail: huxinxi163@163.com; LEI Yan, Tel: +86-731-84618171, E-mail: leiyan5181@sina.com;
Supported by:
This research was supported by the Non-Profit Industry Financial Program of Ministry of Agriculture of China (20130328) to Prof. Liu Yong and Prof. Dai Liangying, and the Natural Science Foundation of Hunan Province, China to Prof. Hu Xinxi (11JJ2018).

摘要/Abstract

摘要： A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription- polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.

关键词: garlic , garlic viruses , RT-PCR , detection , multiplex PCR

Abstract: A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription- polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.

Key words: garlic , garlic viruses , RT-PCR , detection , multiplex PCR

HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou. Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses[J]. Journal of Integrative Agriculture, 2015, 14(5): 900-908.

参考文献

Arya M, Baranwal V K, Ahlawat Y S, Singh L. 2006. RT-PCRdetection and molecular characterization of Onion yellowdwarf virus associated with garlic and onion. CurrentScience, 91, 1230-1234

Canning E S, Penrose M J, Barker I, Coates D 1996. Improveddetection of Barley yellow dwarf virus in single aphids usingRT-PCR. Journal of Virological Methods, 56, 191-197

Chen J, Chen J. 2001. Sequencing and phylogenetic analysisof Garlic virus E-A new member of allexivirus. ChineseScience Bulletin, 46, 1463-1468 (in Chinese)

Chen J, Chen J, Adams M J. 2001. Molecular characterization ofa complex mixture of viruses in garlic with mosaic symptomsin China. Archives of Virology, 146, 1841-1853

Cohen J, Zeidan M, Rosner A, Gera A. 2000. Biological andmolecular characterization of a new carlavirus isolated froman Aconitum sp. Phytopathology, 90, 340-344

Conci V C, Canavelli A, Lunello P. 2003. Yield losses associatedwith virus-infected garlic plants during five successive years.Plant Disease, 87, 1411-1415

Dai J, Cheng J, Huang T, Zheng X, Wu Y. 2012. A multiplexreverse transcription PCR assay for simultaneous detectionof five tobacco viruses in tobacco plants. Journal ofVirological Methods, 183, 57-62

van Dijk P. 1993. Carlavirus isolates from cultivatedAllium species represent three viruses. NetherlandsJournal of Plant Pathology, 99, 233-257

van Dijk P. 1994. Virus disease of Allium species and productsfor their control. Acta Horticulturae, 358, 299-305

Dovas C I, Hatzibukas E, Salomon R, Barg E, Shiboleth Y M,Katis N I. 2001. Comparison of methods for virus detectionin Allium species. Journal of Phytopathology, 149, 731-737

Dovas C I, Katis N I. 2003. A spot nested RT-PCR methodfor the simultaneous detection of members of the Vitivirusand Foveavirus genera in grapevine. Journal of VirologicalMethods, 107, 99-106

Figueira A R, Domier L L, D’Arcy C J. 1997. Comparison oftechniques for detection of Barley yellow dwarf virus-PAVIL.Plant Disease, 81, 1236-1240

Hadidi A, Montasser M S, Levy L, Goth R W, Converse R H,Madkour M A, Ckrzeckowski L J. 1993. Detection of potatoleafroll and strawberry mild yellow-edge luteovirusesby reverse transcription-polymerase chain reactionamplification. Plant Disease, 77, 595-601

Ito T, Ieki H, Ozaki K. 2002. Simultaneous detection of six citrusviroids and Apple stem grooving virus from citrus plants bymultiplex reverse transcription polymerase chain reaction.Journal of Virological Methods, 106, 235-239

Kanyuka K V, Vishnichenko V K, Levay K E. 1992.Nucleotide sequence of Shallot virus X RNA reveals a5´-proximal cistron closely related to those of potexvirusesand a unique arrangement of the 3´-proximal cistrons.Journal of General Virology, 73, 2553-2560

Kobayashi K, Rabinowicz P, Bravo-Almonacid F, HelgueraM, Conci V, Lot H, Mentaberry A. 1996. Coat proteingene sequences of garlic and onion isolates of the Onionyellow dwarf potyvirus (OYDV). Archives of Virology, 141,2277-2287

Liu X, Cheng Z. 2013. Research progress on cryopreservationof garlic germplasm and its virus eradication technique.Chinese Vegetables, 2, 12-19 (in Chinese)

Majumder S, Baranwal V K, Joshi S. 2008. Simultaneousdetection of Onion yellow dwarf virus and Shallot latent virusin infected leaves and cloves of garlic bu duplex RT-PCR.Journal of Plant Pathology, 90, 371-374

Marys E, Carballo O, Izaguirre-Mayoral M L. 1994. Isolation andcharacterization of viruses present in four clones of garlic(Allium sativum) in Venezuela. Journal of Phytopathology,142, 227-234

Nagakubo T, Kubo M, Oeda K.1994. Nucleotide sequences ofthe 3´ regions of two major viruses from mosaic-diseasedgarlic: Molecular evidence of mixed infection by a potyvirusand a carlavirus. Phytopathology, 84, 640-645Nie X, Singh R P

2001. A novel usage of random primers formultiplex RT-PCR detection of virus and viroid in aphids,leaves, and tubers. Journal of Virological Methods, 91,37-49

Pan Y. 2012. Analysis and prospects of Chinese garlic industrydevelopment. Food and Nutrition in China, 18, 22-26 (inChinese)

Park K S, Bae Y J, Jung E J, Kang S J. 2005. RT-PCR-baseddetection of six garlic viruses and their phylogeneticrelationships. Journal of Microbiology and Biotechnology,15, 1110-1114

Pramesh D, Baranwal V K. 2013. Molecular characterization ofcoat protein gene of Garlic common latent virus isolates fromIndia: an evidence for distinct phylogeny and recombination.Virus Genes, 47, 189-193

Shiboleth Y M, Gal-On A, Koch M, Rabinowitch H D, SalomonR. 2001. Molecular characterisation of Onion yellow dwarfvirus (OYDV) infecting garlic (Allium sativum L.) in Israel:Thermotherapy inhibits virus elimination by meristem tipculture. Annals of Applied Biology, 138, 187-195

Singh R P, Nie X, Singh M. 2000. Duplex RT-PCR: Reagentconcentrations at reverse transcription stage affect the PCRperformance. Journal of Virological Methods, 86, 121-129

Song S I, Song J T, Kim C H, Lee J S, Choi Y D. 1998. Molecularcharacterization of the garlic virus X genome. Journal ofGeneral Virology, 79, 155-159

Sumi S I, Tsuneyoshi T, Furutani H. 1993. Novel rod-shapedviruses isolated from garlic, Allium sativum, possessing aunique genome organization. Journal of General Virology,74, 1879-1885

Sward R J, Brennan A P. 1994. Diagnosis and control Alliumvirus disease in Victoria, Australia. Acta Horticulturae, 358,295-298

Takaichi M, Yamamoto M, Nagakubo T, Oeda K. 1998. Fourgarlic viruses identified by reverse transcription-polymerasechain reaction and their regional distribution in NorthernJapan. Plant Disease, 82, 694-698

Takaichi M, Yamamoto M, Nagakubo T, Oeda K. 2001. Mixedvirus infections of garlic determined by a multivalentpolyclonal antiserum and virus effects on diseasesymptoms. Plant Disease, 85, 71-75

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.2011. MEGA5: Molecular evolutionary genetics analysis usingmaximum likelihood, evolutionary distance, and maximumparsimony methods. Molecular Biology and Evolution, 28,2731-2739

Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, HigginsD G. 1997. The CLUSTAL_X windows interface: Flexiblestrategies for multiple sequence alignment aided by qualityanalysis tools. Nucleic Acids Research, 25, 4876-4882

Tsuneyoshi T, Sumi S. 1996. Differentiation among garlicviruses in mixed infections based on RT-PCR proceduresand direct tissue blotting immunoassays. Phytopathology, 86, 253-259

Tsuneyoshi T, Matsumi T, Deng T C, Sako I, Sumi S. 1998a.Differentiation of Allium carlaviruses isolated from differentparts of the world based on the viral coat protein sequence.Archives of Virology, 143, 1093-1107

Tsuneyoshi T, Matsumi T, Natsuaki K T, Sumi S. 1998b.Nucleotide sequence analysis of virus isolates indicatesthe presence of three potyvirus species in Allium plants.Archives of Virology, 143, 97-113

Uga H, Tsuda S. 2005. A one-step reverse transcriptionpolymerasechain reaction system for the simultaneousdetection and identification of multiple topovirus infections.Phytopathology, 95, 166-171

Van der Vlugt R A A, Steffens P, Cuperus C, Barg E, LesemannD E, Bos L, Vetten H J. 1999. Further evidence thatShallot yellow stripe virus (SYSV) is a distinct potyvirusand reidentification of Welsh Onion yellow stripe virus as aSYSV strain. Phytopathology, 89, 148-155

Vunsh R, Rosner A E, Stein A. 1990. The use of the polymerasechain reaction (PCR) for the detection of Bean yellowmosaic virus in gladiolus. Annals of Applied Biology, 117,561-569

Walkey D G A, Antil D N. 1989. Agronomic evaluation of virusfreeand virus infected garlic (Allium sativum L.). Journal ofHorticultural Science, 64, 53-60

Wei Y, Wei C, Yang Y, Hua J. 2012. Cloning and phylogeneticanalysis of six allexivirus CP genes from China isolates.Chinese Journal of Bioinformatics, 10, 79-82 (in Chinese)

Yamashita K, Sakai J, Hanada K. 1996. Characterization of anew virus from garlic (Allium sativum L.), Garlic mite-bornemosaic virus. Annals of the Phytopathologicial Society ofJapan, 62, 483-489

Zhang W, Bai Y, Shen Y, Gao Y, Fan G, Geng H, Meng X.2010. Study on pathogen identification and virus diseaseinvestigation of garlic virus in Heilongjiang Province. Journalof Northeast Agricultural University, 41, 21-26 (in Chinese)

[1]	LING Jian, ZHANG Ji-xiang, ZENG Feng, CAO Yue-xia, XIE Bing-yan, YANG Yu-hong. Comparative genomics provide a rapid detection of Fusarium oxysporum f. sp. conglutinans[J]. Journal of Integrative Agriculture, 2016, 15(4): 822-831.
[2]	CHENG Bo, LIAN Hai-feng, LIU Ying-ying, YU Xin-hui, SUN Ya-li, SUN Xiu-dong, SHI Qing-hua, LIU Shi-qi. Effects of selenium and sulfur on antioxidants and physiological parameters of garlic plants during senescence[J]. Journal of Integrative Agriculture, 2016, 15(3): 566-572.
[3]	ZHAO Fu-ping, WEI Cai-hong, ZHANG Li, LIU Jia-sen, WANG Guang-kai, ZENG Tao, DU Li-xin. A genome scan of recent positive selection signatures in three sheep populations[J]. Journal of Integrative Agriculture, 2016, 15(1): 162-174.
[4]	ZHAO Yue, MAO Jing-jing, SHE Rui-ping, HU Feng-jiao, Majid H Soomro, LIANG Rui-ping, YANG Yi-fei, DU Fang, WANG Tong-tong, GUO Zhao-jie, CHENG Min-heng. Hepatitis associated with hepatitis B virus in broilers[J]. Journal of Integrative Agriculture, 2016, 15(1): 191-199.
[5]	CHANG Wei-hua, ZHANG Yong, CHENG Zhang-rui, ZHAO Xing-xu, WANG Juan-hong, MA You-ji, HU Jun-jie, ZHANG Quan-wei. Identification of novel and differentially expressed microRNAs in ovine ovary and testis tissues using solexa sequencing and bioinformatics[J]. Journal of Integrative Agriculture, 2015, 14(8): 1604-1616.
[6]	ZHANG Yong-qiang, LIU Hai-sheng, WU Xiao-dong, WANG Xiao-zhen, LI Jin-ming, ZHAO Yonggang, Lü Yan, REN Wei-jie, GE Sheng-qiang, WANG Zhi-liang. A novel real-time RT-PCR with TaqMan-MGB probes and its application in detecting BVDV infections in dairy farms[J]. Journal of Integrative Agriculture, 2015, 14(8): 1637-1643.
[7]	Jonathan Nimal Selvaraj, ZHOU Lu, WANG Yan, ZHAO Yue-ju, XING Fu-guo, DAI Xiao-feng, LIU Yang. Mycotoxin detection- Recent trends at global level[J]. Journal of Integrative Agriculture, 2015, 14(11): 2265-2281.
[8]	SHI Ping, Shu Geng, LI Ting-ting, LI Yu-shui, FENG Ting, WU Hua-nan. Methods to detect avian influenza virus for food safety surveillance[J]. Journal of Integrative Agriculture, 2015, 14(11): 2296-2308.
[9]	LIU Huan, SONG Xi-jiao, NI Yue-qun, LU Li-na, ZHOU Xue-ping , WU Jian-xiang. Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors[J]. Journal of Integrative Agriculture, 2014, 13(9): 1943-1951.
[10]	WU Shi-wen, WANG Hong-wei, YANG Zai-dong , KONG Ling-rang. Expression Comparisons of Pathogenesis-Related (PR) Genes in Wheat in Response to Infection/Infestation by Fusarium, Yellow dwarf virus (YDV) Aphid-Transmitted and Hessian Fly[J]. Journal of Integrative Agriculture, 2014, 13(5): 926-936.
[11]	LIU Guo-qing, LIAN Ying-qi, GAO Chao, YU Xiao-feng, ZHU Ming, ZONG Kai, CHEN Xuejiao. In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes[J]. Journal of Integrative Agriculture, 2014, 13(5): 1121-1129.
[12]	Zheng gui-jie, Yang Yong-qing, Ma Ying, Yang Xiao-feng, Chen Shan-yu, Ren Rui, Wang Da-gang, Yang Zhong-lu , ZhI hai-jian. Fine Mapping and Candidate Gene Analysis of Resistance Gene RSC3Q to Soybean mosaic virus in Qihuang 1[J]. Journal of Integrative Agriculture, 2014, 13(12): 2608-2615.
[13]	WENG Chang-mei, LU Jun-xing, WAN Hua-fang, WANG Shu-wen, WANG Zhen, LU Kun, LIANG Ying. Over-Expression of BnMAPK1 in Brassica napus Enhances Tolerance to Drought Stress[J]. Journal of Integrative Agriculture, 2014, 13(11): 2407-2415.
[14]	LIU Tai-guo, WANG Xi, GAO Li, LIU Bo, CHEN Wan-quan , XIANG Wen-sheng. A FIASCO-Based Approach for Detection and Diagnosis of Puccinia graminis f. sp. tritici in China[J]. Journal of Integrative Agriculture, 2014, 13(11): 2438-2444.
[15]	ZHOU Hai-bo, CHEN Ju-lian, LIU Yong, Frédéric Francis, Eric Haubruge, Claude Bragard, SUN Jingrui, CHENG Deng-fa. Influence of Garlic Intercropping or Active Emitted Volatiles in Releasers on Aphid and Related Beneficial in Wheat Fields in China[J]. Journal of Integrative Agriculture, 2013, 12(3): 467-473.

Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics