[1]任春梅,程兆榜,杨柳,等.江苏省葫芦科作物6种病毒的多重RT-PCR方法及应用[J].江苏农业学报,2015,(04):756-763.[doi:10.3969/j.issn.1000-4440.2015.04.008]
　REN Chun-mei,CHENG Zhao-bang,YANG Liu,et al.Establishment and application of multiplex RT-PCR assays for simultaneous detection of six viruses on Cucurbit crops in Jiangsu province[J].,2015,(04):756-763.[doi:10.3969/j.issn.1000-4440.2015.04.008]
点击复制

江苏省葫芦科作物6种病毒的多重RT-PCR方法及应用()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年04期
页码:
756-763
栏目:
植物保护
出版日期:
2015-08-31

文章信息/Info

Title:
Establishment and application of multiplex RT-PCR assays for simultaneous detection of six viruses on Cucurbit crops in Jiangsu province
作者:
任春梅; 程兆榜; 杨柳; 缪倩; 周益军
（江苏省农业科学院植物保护研究所，江苏南京210014）
Author(s):
REN Chun-mei; CHENG Zhao-bang; YANG Liu; MIAO Qian; ZHOU Yi-jun
(Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
关键词:
葫芦科作物; 病毒; 多重RT-PCR
Keywords:
cucurbit crop; virus; multiplex RT-PCR
分类号:
S436.421.1
DOI:
10.3969/j.issn.1000-4440.2015.04.008
文献标志码:
A
摘要:
江苏省葫芦科作物上的病毒主要有小西葫芦黄花叶病毒（ZYMV）、西瓜花叶病毒（WMV）、黄瓜花叶病毒（CMV）、黄瓜绿斑驳花叶病毒（CGMMV）、烟草花叶病毒（TMV）和番木瓜环斑病毒（PRSV），通常发生复合侵染。基于GenBank中这6种病毒的核苷酸序列保守区设计特异性引物，在单重RT-PCR技术的基础上，通过对影响2种多重RT-PCR扩增的DNA聚合酶浓度、dNTPs及镁离子浓度、退火温度、延伸时间、循环次数等因素进行优化，对检测灵敏度进行测定，建立了2种PCR反应同时检测葫芦科作物上6种病毒的多重RT-PCR方法。结果显示：2种多重RT-PCR体系均能同时扩增出各自特异性片段，测序分析结果表明序列同源性在97%以上；优化的2种多重RT-PC体系检测灵敏度为总RNA稀释102～103倍，应用于江苏省210份样品的检测结果与单重RT-PCR一致，体现了检测的可靠性；检测结果表明近年来江苏省葫芦科作物以种子传播的ZYMV和CGMMV侵染为主，病毒的复合侵染率达75.24%。
Abstract:
Cucurbit crops are frequently co-infected by zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), cucumber mosaic virus (CMV), cucumber green mottle mosaic virus (CGMMV), tobacco mosaic virus (TMV) and papaya ringspot virus (PRSV) in Jiangsu province. Based on the RT-PCR for the detection of single virus, the two multiplex RT-PCR (mRT-PCR) systems were established for simultaneous detection of the six viruses by using six sets of specific primers designed on the basis of conserved sequences of the viruses in GenBank. The main factors suches concentrations of DNA polymerase, Mg2+ and dNTPs, PCR conditions of annealing temperature, extension time and amplification cycles, sensitivity of the detection were examined and optimized. The results showed that expected fragments of the six viruses were amplified in the two mRT-PCR systems and the sequences shared above 97 percent similarities with those of other relative viruses. The optimized mRT-PCR could detect diluted 100 to 1000 times. The results of the mRT-PCR were consistent with sRT-PCR on detecting 210 samples of cucurbit crops in Jiangsu province which reflects the reliability of the protocol. The results also revealed that in recent years the seed transmission viruses ZYMV and CGMMV were the dominant infectious viruses on cucurbit crops in Jiangsu province, with the co-infection rate of 75.24%. 

参考文献/References:


[1]PROVIIDENTI R. Resistence to viral disease of cucurbits. Resistence to viral disease of vegetables: genetics and breeding [M]. Oregon: Timer Press,1993:8-43.
[2]ABOU J Y, SOBH H E, ZAMMAR S, et al. Incidence and management of virus disease of cucurbits in Lebanon [J]. Crop Protection, 2000, 19(4):217-224.
[3]陈洁云,陈集双,柴立红,等. 两种葫芦科病毒的分子检测和致病性研究[J]. 植物病理学报, 2003,33(5): 449-455.
[4]古勤生. 葫芦科作物主要病毒病防治研究进展[J]. 中国西瓜甜瓜, 2002(4): 27-30.
[5]任春梅,程兆榜,缪倩,等. 江苏西瓜花叶病毒的分子鉴定和序列分析[J]. 南方农业学报, 2013, 44(9):1464-1470.
[6]LIN S S, HOU R F, YEH S D. Hetrodup lexmobility and sequence analyses for assessment of variability of zucchini yellow mosaic virus [J]. Phytopathology, 2000, 90 (3):228-235.
[7]LIN H X, RUBIO L, SMYTHE A, et al. Genetic diversity and biological variation among California isolates of cucumber mosaic virus [J]. Journal of General Virology, 2003, 84:249-258.
[8]任春梅,程兆榜,缪倩,等. 江苏黄瓜绿斑驳花叶病毒的鉴定[J]. 江苏农业学报, 2013, 29(1):65-70.
[9]黄金光,邓丛良,范在丰,等. 烟草花叶病毒丁香分离物的分离与鉴定[J]. 植物病理学报, 2004, 34(3):215-222.
[10]KO S J, LEE Y H, CHO M S, et al. Theincidence of virus diseases on melon in Jeonnam Province during 2000-2002 [J]. Plant Pathol J, 2007, 23:215-218.
[11]CHAMBERIAN J S, GIBBS R A, RANIER J E, et al. Detection screeninn of the duchenne muscular dystrophy locus via multiplex DNA amplifieation [J]. Nuel Acids Res, 1988, 16:1141-1156.
[12]DAI J, CHENG J L, HUANG T, et al. A multiple reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants [J]. Journal of Virological Methods, 2012, 183:57-62.
[13]BHAT A I, SIJU S. Development of a single-tube multiplex RT-PCR for the simultaneous detection of cucumber mosaic virus and piper yellow mottle virus associated with stunt disease of black pepper [J]. Current Science, 2007, 93:973-976.
[14]UGA H, TSUDA S. A one-step reverse transcription-polymerase chain reaction system for the simultaneous detection and identification of multiple topovirus infections [J]. Phytopathology, 2005, 95:166-171.
[15]SUGIYAMA S, MASUTA C， SEKIGUCHI H, et al. A simple, sensitive, specific detection of mixed infection of multiple plant viruses using macroarray and microtube hybridization [J]. Journal of Virological methods, 2008, 153:241-244.
[16]牛建新,马兵钢,何梅,等. 库尔勒香梨主要病毒多重RT-PCR检测技术研究[J]. 植物病理学报, 2006, 36(1): 12-21.
[17]YU C, WU J X, ZHOU X P. Detection and subgrouping of cucumber mosaic virus isolates by TAS-ELISA and immunocapture RT-PCR [J]. Journal of Virological Methods, 2005, 23:155-161.
[18]王威麟,张昊,于祥泉,等. 侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重 RT-PCR检测体系的建立与检测应用[J]. 植物病理学报, 2010, 40(1):27-32.
[19]赵丽,古勤生,陈红运,等. 葫芦科作物3种主要病毒的多重RT-PCR方法的建立[J]. 果树学报, 2008, 25(5):703-707.
[20]KWON J Y, HONG J S, KIM M J, et al. Simultaneous multiplex PCR detection of seven cucuibit-infecting viruses [J]. Journal of Virological Methods, 2014, 206:133-139.
[21]NIE X H, SINGH R P. Detection of multiple potato viruses using an oligo(dT) as a common cDNA primer in mutiple RT-PCR [J]. Journal of Virological Methods, 2000, 86:155-161.〖ZK）〗〖FL）〗

相似文献/References:

[1]杨柳,况佳颖,任春梅,等.江苏省主要葫芦科作物病毒种类及分布[J].江苏农业学报,2022,38(01):65.[doi:doi:10.3969/j.issn.1000-4440.2022.01.007]
　YANG Liu,KUANG Jia-ying,REN Chun-mei,et al.Variety and distribution of major viruses infecting Cucurbitaceae crops in Jiangsu province[J].,2022,38(04):65.[doi:doi:10.3969/j.issn.1000-4440.2022.01.007]

备注/Memo

备注/Memo:
收稿日期：2015-02-04 基金项目：国家公益性行业科研专项（201303028）；江苏省农业科技自主创新基金项目[CX(13)3023] 作者简介：任春梅（1981-），女，江苏大丰人，硕士，助理研究员，主要从事植物病毒研究。 通讯作者：程兆榜，（E-mail）onlyone8501@126.com
更新日期/Last Update: 2015-08-31