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(06):980-983,987.[doi:10.3969/j.issn.1000-9841.2012.06.026]
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
- Title:
- Detection of Tobacco Ringspot Virus in Soybean Seeds by the Reverse Transcription Loop-mediated Isothermal Amplification Method
- 文章编号:
- 1000-9841(2012)06-0980-04
- 关键词:
- 烟草环斑病毒; 逆转录环介导等温扩增; 检测
- Keywords:
- Tobacco ringspot virus(TRSV); Reverse transcription loop-mediated isothermal amplification(RT-LAMP); Detection
- 分类号:
- S412
- 文献标志码:
- A
- 摘要:
- 根据烟草环斑病毒(TRSV)外壳蛋白基因序列设计并合成了1组RT-LAMP引物,利用实时浊度仪监测反应过程,初步建立了烟草环斑病毒的RT-LAMP检测方法,并进行了特异性与灵敏度检测。结果显示,RT-LAMP灵敏度与普通RT-PCR法相当,但检测时间明显缩短,1 h即可完成反应。此外,体系中加入钙黄绿素,反应结束后,可通过裸眼观察荧光的有无来判断是否有扩增。对大豆种子中携带的烟草环斑病毒进行了RT-LAMP检测,裸眼判定与仪器监测结果一致。结果证明所建立的TRSV RT-LAMP方法具有快速、特异、灵敏,操作简单的特点,不需复杂仪器设备,适合于TRSV的现场快速检测。
- Abstract:
- A sets of primers were designed and synthesized based on the coat protein coding region of tobacco ringspot virus(TRSV)for reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay.The amplification was detected using the real time turbidimeter.An accelerated RT-LAMP procedure with one step was developed for the detection of TRSV.The specificity and sensitivity of the RT-LAMP assay were tested.RT-PCR compared to RT-LAMP by serial dilutions of total RNA extracts,they had the same sensitivities,but RT-LAMP was quicker,and the reaction could be finished within 1 hour.In addition,the presence or absence of the fluorescent display in daylight allowed naked easy detection of the amplification of TRSV genomic RNA using Calcein.The RT-LAMP assay was applied successfully to detect TRSV in soybean seeds,and the result by the addition of Calcein was consistent with the result detected by the real time turbidimeter.The method is rapid,specific,sensitive and suitable for rapid field detection of TRSV.
参考文献/References:
[1]Fauquet C M,Mayo M A,Maniloff J,et al.Virus taxonomy[R].San Diego:Academic Press,2004:813-818. [2]陈青,林石明,杨翠云,等.GB/T 28081-2011 烟草环斑病毒检疫鉴定方法[S].中华人民共和国国家标准.(Chen Q,Lin S M,Yang C Y,et al.GB/T 28081-2011 Detection and identification of tobacco ringspot virus[S].National standards of the People’s Republic of China.) [3]陈枝楠,邓琼,郑耘,等.SN/T 1146-2010烟草环斑病毒检疫鉴定方法[S].中华人民共和国出入境检验检疫行业标准.(Chen Z N,Deng Q,Zheng Y,et al.SN/T 1146-2010 Detection and identification of tobacco ringspot virus[S].Entry-Exit Inspection and Quarantine of the People’s Republic of China industry standards.) [4]杨翠云,于翠,魏梅生,等.SN/T 1146.2-2009烟草环斑病毒分子生物学检测方法[S].中华人民共和国出入境检验检疫行业标准.(Yang C Y,Yu C,Wei M S,et al.SN/T 1146.2-2009 Molecular detection of tobacco ringspot virus[S].Entry-Exit Inspection and Quarantine of the People’s Republic of China industry standards) [5]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):e63. [6]Lee M S,Yang M J,Hseu Y C,et al.One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of Cymbidium mosaic virus[J].Journal of Virological Methods,2011,173:43-48. [7]Boubourakas I N,Fukuta S,Kyriakopoulou P E.Sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification[J].Journal of Virological Methods,2009,160:63-68. [8]Qiao Y M,Guo Y C,Zhang X E,et al.Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis?spores[J].Biotechnology Letters,2007,29(12):1939-1946. [9]张裕君,刘跃庭,廖芳,等.基于LAM P方法的苜蓿疫霉根腐病菌分子检测研究[J].中国植保导刊,2011,31(10):7-9.(Zhang Y J,Liu Y T,Liao F,et al.Research on molecular detection of?Phytophthora medicaginis?based on LAMP[J].China Plant Protection,2011,31(10):7-9.) [10]张晓雷,檀根甲,魏梅生,等.大豆种传南方菜豆花叶病毒和烟草环斑病毒的GICA-RT-PCR检测[J].安徽农业大学学报,2009,36(2):319-322.(Zhang X L,Tan G j,Wei M S,et al.Detection of southern bean mosaic virus and tobacco ringspot virus in soybean seed by GICA-RT-PCR[J].Journal of Anhui Agricultural University,2009,36(2):319-322.) [11]Nagamine K,Hase T,Notomi T.Accelerated reaction by loop-mediated isothermal amplification using loop primers[J].Molecular and Cellular Probes,2002,16:223-229.
相似文献/References:
[1]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(06):748.[doi:10.3969/j.issn.1000-9841.2007.05.023]
[2]魏霜,袁俊杰,杨卓瑜,等.双重DPO-RT-PCR检测南方菜豆花叶病毒及烟草环斑病毒[J].大豆科学,2021,40(02):265.[doi:10.11861/j.issn.1000-9841.2021.02.0265]
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(06):265.[doi:10.11861/j.issn.1000-9841.2021.02.0265]
备注/Memo
基金项目:宁波检验检疫局科研项目(甬K07-2011);国家质检总局课题(2011IK169);质检公益性行业科研专项项目(201010256);浙江省重点科技创新团队项目(2010R50028)。