Reverse Transcription Digital PCR Detection Method for NAi-Based Transgenic Maize

doi:10.13560/j.cnki.biotech.bull.1985.2019-0897

Abstract

Abstract: Aiming at the transgenic maize based on RNAi technology,this paper studied and established a set of reverse transcription chip digital PCR（dPCR）method of quantitatively detecting dsRNA in this transgenic maize. The research contents mainly included exploration and optimization of RNA extraction methods,primer probe design,reverse transcription method,dPCR reaction conditions and compositions,etc. The limit of absolute quantification of this method is 2.5 copies/μL,and the RSD is 12.4%. The relative deviation is 2.7%,and the RSD is 14.6%. When detecting the actual sample of low concentration is 21.7 copies/μL,which meets the international requirement of transgenic quantitative result RSD≤25%. Applying the method in the quantitative detection of genetically modified crops based on RNAi technology will provide accurate and reliable technical means for the safety evaluation of related genetically modified crops in China.

Key words: RNAi, digital PCR, genetically modified maize

YANG Zhen-zhou, LIU Gang, XU Li. Reverse Transcription Digital PCR Detection Method for NAi-Based Transgenic Maize[J]. Biotechnology Bulletin, 2020, 36(5): 56-63.

References

[1] 国际农业生物技术应用服务组织. 2018 年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 2019, 39(8):1-6.
[2] 徐琳杰, 刘培磊, 熊鹂, 等. 国际上主要国家和地区农业转基因产品的标识制度[J]. 生物安全学报, 2014, 23(3):301-304.
[3] Andrew F.RNA-triggered gene silencing[J]. Trends in Genetics, 1999, 15(9):358-363.
[4] Schwind N, Zwiebel M, Itaya A, et al.RNAi-mediated resistance to potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct[J]. Molecular Plant Pathology, 2010, 10(4):459-469.
[5] Yang Y, Sherwood T, Patte C, et al.Use of tomato yellow leaf curl virus(TYLCV)rep gene sequences to engineer TYLCV resistance in tomata[J]. Phytopathology, 2004, 94(5):490-496.
[6] Tenlado F, Dlaz-Rufz JR.Double-stranded RNA-mediated interference with plant virus infection[J]. J Virol, 2001, 75(24):12288-12297.
[7] 李夏荧, 高鸿飞, 刘鹏程, 等. 转基因作物快速检测技术的研究进展[J]. 江苏农业科学, 2018, 46(3):5-9.
[8] 王荣谈, 刘冬儿, 厉建萌, 等. 实时荧光定量PCR技术及其在植物转基因产品检测中的应用[J]. 植物生理学通讯, 2009, 45(6):591-597.
[9] Quan PL, Sauzade M, Brouzes E. dPCR:A technology review[J]. Sensors(Basel), 2018, 18(4):1271.
[10] Tigst D, David D.Critical assessment of digital PCR for the detec-tion and quantification of genetically modified organisms[J]. Anal BioanalChem, 2018, 410(17):4039-4050.
[11] European Network of GMO Laboratories. Definition of minimum performance requirements for analytical methods of GMO testing[J]. ResearchGate, 2015, 5:1-24.
[12] 左洪亮, 李海怡, 高璐, 等. RNAi作用机制及其在害虫防治领域的应用前景[J]. 公共植保与绿色防护, 2010:379-384.
[13] The European Parliament and The Council of The European Union. Regulation(EC)NO 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed[J]. Official Journal of the European Communities, 2003, L268:1-23.

[1]	ZHOU Jia-yan, ZOU Jian, CHEN Wei-ying, WU Yi-chao, CHEN Xi-tong, WANG Qian, ZENG Wen-jing, HU Nan, YANG Jun. Construction of Multi-gene Interference System for Plant and Analysis of Its Application Efficiency [J]. Biotechnology Bulletin, 2023, 39(1): 115-126.
[2]	LI Hui-jie, DONG Lian-hua, CHEN Gui-fang, LIU Si-yuan, YANG Jia-yi, YANG Jing-ya. Establishment of Droplet Digital PCR Assay for Quantitative Detection of Pseudomonas cocovenenans in Foods [J]. Biotechnology Bulletin, 2023, 39(1): 127-136.
[3]	LUO Hao-tian, WANG Long, WANG Yu-qian, WANG Yue, LI Jia-zhen, YANG Meng-ke, ZHANG Jie, DENG Xin, WANG Hong-yan. Genome-wide Identification and Expression Analysis of the RNAi-related Gene Families in Setaria viridis [J]. Biotechnology Bulletin, 2023, 39(1): 175-186.
[4]	HU Xue-ying, ZHANG Yue, GUO Ya-jie, QIU Tian-lei, GAO Min, SUN Xing-bin, WANG Xu-ming. Comparison in Antibiotic Resistance Genes Carried by Bacteriophages and Bacteria in Farmland Soil Amended with Different Fertilizers [J]. Biotechnology Bulletin, 2022, 38(9): 116-126.
[5]	CHENG Shen-wei, ZHANG Ke-qiang, LIANG Jun-feng, LIU Fu-yuan, GAO Xing-liang, DU Lian-zhu. Establishment of a Triple Droplet Digital PCR Quantitative Detection Method for Typical Pathogenic Bacteria in Livestock and Poultry Manure [J]. Biotechnology Bulletin, 2022, 38(9): 271-280.
[6]	JIAO Yue, HAN Yu, YANG Qiao, HUANG Yao-hui, AN Ji-cui, YANG Ya-zhou, YE Ji-ming. Commercialization Development Trend of Genetically Modified Maize and the Enlightenment [J]. Biotechnology Bulletin, 2021, 37(4): 164-176.
[7]	WEN Hong-tao, LI Xia-ying, YANG Yang, CHEN Zi-yan, DING Yi-jia, ZHANG Xiu-jie, ZHANG Rui-ying. Strategy of Screening Genetically Modified Maize [J]. Biotechnology Bulletin, 2020, 36(5): 39-47.
[8]	WANG Hao-qian, XIAO Fang, YANG Lei, MIAO Qing-mei, ZHANG Xu-dong, ZHANG Xiu-jie. Analysis of Verification Results by PCR Methods for Genetically Modified Double-resistant 12-5 Event-specific Maize [J]. Biotechnology Bulletin, 2020, 36(5): 48-55.
[9]	JI Yi, XU Xiao-li, JIANG Yuan-yuan, WANG Xiao-fu, XU Jun-feng, LI Yue-ying, CHEN Xiao-yun. Copy Number Variations of Mitochondrial DNA and Genomic DNA from Different Tissues of Duck Based on Digital PCR [J]. Biotechnology Bulletin, 2020, 36(5): 86-91.
[10]	YANG Wen-wen, NI Jia-yao, HU Rui-jie, WANG Hua-zhong. A Sequencing Strategy for Inverted Repeats in RNAi Vectors [J]. Biotechnology Bulletin, 2020, 36(5): 205-210.
[11]	XU Xiang, DONG Wei-peng, ZHANG Shao-hua, FENG Chen-yi, LIU Tian-fu, YAN Jiong. Construction of Fsp27 Gene Silencing Vector and Its Effect on Cell Lipolysis [J]. Biotechnology Bulletin, 2020, 36(1): 88-94.
[12]	LIU Xiao, ZHU Peng-yu, WANG Yao, ZHU Shui-fang, FU Wei. Development Progress of Digital PCR in the Precise Detection of Functional Nucleic Acid [J]. Biotechnology Bulletin, 2018, 34(9): 149-162.
[13]	ZHANG Ting, YANG Xuan-xuan, LEI Qin, LIU Juan-juan, LI Ji-gang. Effects of Phoxim,Methomyl and Feeding dsRNA on the Expression of ace Genes in Helicoverpa armigera [J]. Biotechnology Bulletin, 2018, 34(7): 160-165.
[14]	WANG Yong, LAN Qing-kuo, ZHAO Xin, CHEN Rui, SHEN Xiao-ling, LI Wen, ZHANG Yao-zhong, LI Liang, WANG Qin-ying. Estimation of the Copy Number of Exogenous Genes in Genetically Modified Rice by Droplet Digital PCR [J]. Biotechnology Bulletin, 2018, 34(3): 53-58.
[15]	CHEN Jing, ZHANG Dao-wei, QIAN Zheng-min. Structure and Characteristics Analysis of Chitin Synthase 1 from Sogota furcifera [J]. Biotechnology Bulletin, 2018, 34(1): 195-201.