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

doi:10.3969/j.issn.1674-7968.2020.12.016

Abstract
Figure/Table
References
Related Citation (1)

Download: PDF (3033 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract False-negative result is a major factor affecting the results of nucleic acid testing. The internal amplification control (IAC) plays an important role in monitoring false negative results during PCR. In order to solve the false-negative result, the application of the IAC in reverse transcription- recombinase polymerase amplification (RT-RPA) was established. The IAC was constructed by the composite primer method and applied to the detection of three soybean viruses, Soybean mosaic virus (SMV), Bean pod mottle virus (BPMV) and Southern bean mosaic virus (SBMV), during RT-RPA. The results showed that the methods were able to achieve specific amplification of the target gene while indicating false negative results. Sensitivity experiments showed that the detection limit of this assay for SMV and BPMV was 0.05 ng, when the content of the IAC was 1.83×10⁴ copies in 50 μL reaction system. And when the content of the IAC was 1.83×10³ copies in 50 μL reaction system, the detection limit of this assay for SBMV was 0.05 ng. The methods were used to test 10 soybean (Glycine max) samples and the results were consistent with those of the corresponding reverse transcription-PCR (RT-PCR), indicating its good applicability. In conclusion, the IAC-RT-PRA methods for SMV, BPMV and SBMV were established in this study could indicate false negatives and improve credibility of test results.

Key words： Soybean mosaic virus (SMV) Bean pod mottle virus (BPMV) Southern bean mosaic virus (SBMV) Recombinase polymerase amplification (RT-RPA) Internal amplification control (IAC)

Received: 02 April 2020

ZTFLH:

S41-30

Corresponding Authors: *278063657@qq.com

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	YUAN Jun-Jie
	LONG Yang
	WEI Ting-Yu
	LIU Er-Long
	FENG Li-Xia
	LIU Si-Si
	CHEN Wen
	WEI Shuang

Cite this article:

YUAN Jun-Jie,LONG Yang,WEI Ting-Yu, et al. RT-RPA Technology Included with IAC for Detection of Three Soybean (Glycine max) Viruses[J]. 农业生物技术学报, 2020, 28(12): 2261-2269.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2020.12.016 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2020/V28/I12/2261

[1] 陈艳玲. 2019. 环介导等温扩增技术在快速病原菌检测中的价值[J]. 实用检验医师杂志, 11(03): 131-133.
(Chen Y L.2019. The value of loop mediated isothermal amplification technology in rapid pathogen detection[J]. Chinese Journal of Clinical Pathologist, 11(03): 131-133.)
[2] 丁钿, 魏霜, 李新浩, 等. 2019.马铃薯斑纹片病菌IAC-PCR检测方法[J]. 植物检疫, 33(05): 22-27.
(Ding T, Wei S, Li X H, et al.2019. Detection of Candidatus Liberibacter solanacearum by PCR method with internal amplification control[J]. Plant Quarantine, 33(05): 22-27.)
[3] 贺卫华, 杨伦, 姜敏, 等. 2016. 含扩增内标的犬巴贝斯虫PCR检测方法的建立[J]. 中国兽医科学, 46(11): 1376-1382.
(He W H, Yang L, Jiang M, et al.2016. Detection of canine babesiosis by PCR assay with an internal amplification control[J]. Chinese Veterinary Science, 46(11): 1376-1382.)
[4] 胡文多, 宋志刚, 赵曙国. 2004. 进口大豆中杂草种子含量检测及分析[J]. 植物检疫, 18(03): 173-174.
(Hu W D, Song Z G, Zhao S G.2004. Detection and analysis the weed seeds content in imported soybean[J]. Plant Quarantine, 18(03): 173-174.)
[5] 李林杰, 王凯, 高尧华, 等. 2018. 2017年山东口岸进口大豆中有害生物截获分析[J]. 大豆科学, 37(03): 429-437.
(Li L J, Wang K, Gao Y H, et al.2018. Analysis and suggestion on imported soybean pest intercepted in shandong port in 2017[J]. Soybean Science, 37(03): 429-437.)
[6] 李伟,庄濠宇,温尔英,等. 2019. 添加扩增内标的重组酶聚合酶扩增技术(RPA-IAC)检测创伤弧菌[J].检验检疫学刊, 29(03): 5-8.
(Li W, Zhuang H Y, Wen E Y, et al.2019. Recombinant enzyme polymerase amplification (RPA-IAC) with amplified internal standard for detection of Vibrio Vulnificus[J]. Journal of Inspection and Quarantine, 29(03): 5-8.)
[7] 李雪玲, 徐慧敏. 2013. 扩增内标在食源性致病菌PCR检测中应用的研究进展[J]. 中国卫生检验杂志, 23(02): 523-524.
(Li X L, Xu H M.2013. Progress on development of internal amplification control in PCR detection of foodborne pathogens[J]. Chinese Journal of Health Laboratory Technology, 23(02): 523-524.)
[8] 梁海燕, 刘文鑫, 杨志刚. 2017. 等温核酸扩增技术进展[J]. 中国医学创新, 14(16): 145-148.
(Liang H Y, Liu W X, Yang Z G.2017. Progress of isothermal nucleic acid amplification techniques[J]. Medical Innovation of China, 14(16): 145-148.)
[9] 彭丹丹, 张源明, 舒灿伟, 等. 2017. 基于环介导等温扩增技术的植物病原物检测技术的应用研究[C]//, 彭友良, 李向东(编著), 中国植物病理学会2017年学术年会论文集. 中国农业科学技术出版社, 北京, pp. 513.
(Peng D D, Zhang Y M, Shu C W, et al.2017. Research and application of Loop-mediated isothermal amplification in plant pathogen detection technology[C]//, Peng Y L, Li X D(eds.). Proceedings of the annual meeting of Chinese society for plant pathology [2017]. China Agricultural Science and Technology Press, Beijing, pp. 513.)
[10] 申进玲,赵丽娜,蒋原,等. 2019. 含内标的多重实时PCR法同时检测空肠弯曲菌和结肠弯曲菌[J].食品安全质量检测学报, 10(07): 1844-1852.
(Shen J L, Zhao L N, Jiang Y, et al.2019. Simultaneous detection of Campylobacter jejuni and Campylobacter coli using multiplex real-time PCR containing internal amplification control[J]. Journal of Food Safety and Quality, 10(07): 1844-1852.)
[11] 王杰,王晓鸣,黄丽丽. 2005. 大豆干种子中大豆花叶病毒的RT-PCR检测[J].植物病理学报, (03): 214-220.
(Wang J, Wang X M, Huang L L. 2005. Detection of Soybean mosaic virus in soybean dry seed using RT-PCR[J]. Acta Phytopathologica Sinica, (03):214-220.)
[12] 魏霜, 袁俊杰, 李桂芬, 等. 2018. 南方菜豆花叶病毒RT-RPA检测方法的建立[J]. 植物检疫, 32(01): 50-53.
(Wei S, Yuan J J, Li G F, et al.2018. One-step reverse transcription recombinase polymerase amplification (RT-RPA) for the detection of Southern bean mosaic virus[J]. Plant Quarantine, 32(01): 50-53.)
[13] 肖霞. 2017. 含扩增内标的PCR技术在犬、猫5种病毒检测中的应用研究[D]. 硕士学位论文, 南京农业大学, 导师:姚大伟, pp.15-17.
(Xiao X.2017. Application of PCR technology included with IAC in the detection of dogs and cats five viruses[D]. Thesis for M.S, Nanjing Agriculture University, Supervisor: Yao D W, pp. 15-17.)
[14] 袁俊杰, 魏霜, 龙阳, 等. 2018. 一步法逆转录重组酶聚合酶常温扩增技术(RT-RPA)检测大豆花叶病毒[J]. 检验检疫学刊, 28(02): 1-4.
(Yuan J J, Wei S, Long Y, et al.2018. One-step reverse transcription recombinase polymerase amplification (RT-RPA) for the detection of SMV[J]. Journal of Inspection and Quarantine, 28(02): 1-4.)
[15] 张永江, 魏霜, 袁俊杰, 等. 2018. 一步法逆转录重组酶聚合酶常温扩增(RT-RPA)技术检测菜豆荚斑驳病毒[J]. 江苏农业科学, 46(21): 96-98.
(Zhang Y J, Wei S, Yuan J J, et al.2018. Detection of bean pod mottle virus (BPMV)by RT-RPA method with internal amplification control[J]. Jiangsu Agricultural Sciences, 46(21): 96-98.)
[16] 周莹, 杨丽梅, 刘梅, 等. 2019. 重组酶聚合酶扩增技术在番茄黄化曲叶病毒检测中的应用[J]. 中国蔬菜, (01): 36-40.
(Zhou Y, Yang L M, Liu M, et al. 2019. Application of recombinase polymerase amplification technology in detecting TYLCV[J]. China Vegetables, (01): 36-40.)
[17] Basto A P, Portugal R S, Nix R J, et al.2006. Development of a nested PCR and its internal control for the detection of African swine fever virus (ASFV) in Ornithodoros erraticus[J]. Archives of virology, 151(4): 819-826.
[18] Herwegh S, Carnoy C, Wallet F, et al.2005. Development and use of an internal positive control for detection of Bordetella pertussis by PCR[J]. Journal of Clinical Microbiology, 43(5): 2462-2464.
[19] Li J, Macdonald J, Von SF.2018. Review: A comprehensive summary of a decade development of the recombinase polymerase amplification[J]. The Analyst, 144(1): 31-67.
[20] Malorny B, Hoorfar J, Bunge C, et al.2003. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard[J]. Applied and Environmental Microbiology, 69(1): 290-296.
[21] Piepenburg O, Williams C, Stemple D N.2006. DNA detection using recombination proteins[J]. PLOS Biology, 4(7): 1115-1121.