基于数字PCR的转基因水稻LL62品系精准定量检测方法建立

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.011

摘要/Abstract

摘要： 为了促进转基因产品标识制度的顺利实施,对未获我国农业部批准的转基因水稻LL62品系进行精准定量检测方法研究,以保障我国进出口转基因农产品的检测监管。基于转基因水稻LL62品系外源插入片段和水稻基因组DNA的3'端旁侧序列设计数字PCR检测体系,建立了精准定量检测方法。结果显示,设计的引物探针反应效率高,基于此建立的数字PCR方法高度特异于转基因水稻LL62品系检测;方法可重复性好,生成微滴数相对标准偏差（RSD）值介于0.60%-11.11%;准确度高,3个混合样品（10%、1%和0.1%）测定值与真实值之间的偏差（bias）分别为0.12、0.09和0.10,相对标准偏差（RSD）介于0.09%-10.31%。建立的转基因水稻LL62品系微滴数字PCR定量检测方法简便快捷、特异性强、重复性好、准确度高,可准确有效地对农产品和食品中转基因水稻LL62成分进行精准定量分析。

关键词: 转基因水稻LL62品系, 微滴数字PCR, 品系特异性, 定量

Abstract: In order to promote the smooth implementation of labeling policy for genetically modified（GM）products,we study an precise quantitative detection method for the GM rice LL62 that has not been approved by the Ministry of Agriculture in China,aiming at guarantee the detection and supervision of GM agricultural products in export and import. A digital PCR（dPCR）detection system were designed based on the inserted exogenous fragment of LL62 and 3’ flanking sequence of DNA of rice genome,and a precisely quantitative method was established. The results showed that the designed probes presented high efficiency,the developed dPCR method was highly specific for GM rice LL62 detection. The method was highly repeated,and the relative standard deviation（RSD）values of droplets’ number ranged from 0.60%-11.11%. Results of the quantification of three blind samples showed that the bias between the true value and the measured value was 0.12,0.09 and 0.10,the RSD was 0.09%-10.31%,indicating that the accuracy was high. In conclusion,the established droplet dPCR method for GM rice LL62 detection is simple and convenient with high specificity,solid repeatability and high accuracy,and is suitable for precisely and effectively quantitative analysis of ingredients of GM rice or peeled rice LL62 in agricultural products and foods.

Key words: genetically modified rice LL62, droplet digital PCR, event-specific, quantification

任怡菲, 高琴, 邓婷婷, 李想, 黄文胜, 陈舜胜, 陈颖. 基于数字PCR的转基因水稻LL62品系精准定量检测方法建立[J]. 生物技术通报, 2016, 32(8): 69-76.

REN Yi-fei, GAO Qin, DENG Ting-ting, LI Xiang, HUANG Wen-sheng, CHEN Shun-sheng, CHEN Ying. Establishment of Precisely Quantitative Method of Genetically Modified Rice LL62 Based on Digital PCR[J]. Biotechnology Bulletin, 2016, 32(8): 69-76.

参考文献

[1] Clive J. 2014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, 2015, 35（1）：1-14.
[2] European Commission. 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 Union, 2003, L268：1-23.
[3] Russian Federation. SanPiN 2. 3. 2. 2227- 07“Additions and Changes #5 to the Sanitary- Epidemiological Rules SanPiN 2. 3. 2. 1078- 01 “Hygiene Requirements to Safety and Nutrition Value of Food Products”. 2007.
[4] Ministry of Agriculture, Forestry and Fisheries of Japan. Notification 1775. Food and Marketing Nureau[S]. Tokyo, Japan：2000.
[5] Chiueh LC, Chen YL, Shih DY. Study on the detection method of six varieties of genetically modified maize and processed foods[J]. Journal of Food and Drug Analysis, 2002, 10（1）：25-33.
[6] Ministry of Agriculture and Forestry of Korea（MAF）. Notification No. 2000- 31, Guidelines for Labeling of Genetically Modified Agricultural Products[S]. Seoul, Korea：2000.
[7] 中华人民共和国农业部. 农业部第10号令《农业转基因生物标识管理办法》[S]. 2002.
[8] 宋君, 雷绍荣, 刘勇, 等. 转基因玉米NK603品系特异定量PCR检测方法的建立[J]. 生物技术通讯, 2012, 23（2）：238-241.
[9] 王渭霞, 赖凤香, 洪利英, 等. 实时定量PCR检测转基因水稻科丰6号插入拷贝数和转基因含量[J]. 农业生物技术学报, 2012, 20（1）：9-15.
[10] 敖金霞, 高学军, 仇有文, 等. 实时荧光定量PCR技术在转基因检测中的应用[J]. 东北农业大学学报, 2009, 40（6）：141-144.
[11] Li X, Pan LW, Li JY, et al. Establishment and application of event- specific polymerase chain reaction methods for two genetically modified soybean events, A2704-12 and A5547-127[J]. Journal of Agricultural and Food Chemistry, 2011, 59（24）：13188-13194.
[12] 潘良文, 田风华, 张舒亚. 转基因抗草丁膦油菜籽中Barnase基因的实时荧光定量PCR检测[J]. 中国油料作物学报, 2006, 28（2）：194-198.
[13] Burns M, Burrell A, Foy C. The applicability of digital PCR for the assessment of detection limits in GMO analysis[J]. European Food Research and Technology, 2010, 231（3）：353-362.
[14] Heyries KA, Carolina T, Michael C, et al. Megapixel digital PCR[J]. Nat Methods, 2011, 8（8）：649-651.
[15] Corbisier P, Somanath B, Lina P, et al. Absolute quantification of genetically modified MON810 maize（Zea mays L. ）by digital polymerase chain reaction[J]. Anal Bioanal Chem, 2010, 396（6）：2143-2150.
[16] Terry CF, Shanahan DJ, Ballam LD, et al. Real-time detection of genetically modified soya using Lightcycler and ABI 7700 platforms with TaqMan, Scorpion, and SYBR GreenⅠchemistries[J]. J AOAC International, 2002, 85（4）：938-944.
[17] Bhat S, Herrmann J, Armishaw P, et al. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number[J]. Anal Bioanal Chem, 2009, 394（2）：457-467.
[18] 李春勇. 数字PCR技术原理及应用[J]. 生物技术与世界, 2014：9-13.
[19] Sanders R, Huggett JF, Bushell CA, et al. Evaluation of digital PCR fo rabsolute DNA quantification[J]. Anal Chem, 2011, 83（17）：6474-6484.
[20] 李亮, 隋志伟, 王晶, 等. 基于数字PCR的单分子DNA定量技术研究进展[J]. 生物化学与生物物理进展, 2012, 39（10）：1017-1023.
[21] 赵卫东, 郑文杰, 贺艳. 荧光 PCR 方法定性和定量检测BT63 转基因大米[J]. 食品研究与开发, 2009, 30（3）：133-135.
[22] 汪秀秀, 杨捷琳, 宋青, 等. 转基因棉花GHB119品系特异性定量PCR检测方法的建立[J]. 农业生物技术学报, 2014, 22（3）：380-388.
[23] Joint Research Centre Institute for Health and Consumer Protection Biotechnology & GMOs Unit. Event-specific method for the quantification of rice line LLRICE62 using Real-time PCR（CRLVL05/04VP）. 2006.
[24] National Center for Biotechnology Information. Oryza sativa（rice）. [OL]http://www. ncbi. nlm. nih. gov/genome?term=txid4530%5Borgn%5D
[25] Liu J, Guo JC, Zhang HB, et al. Development and in-house validation of the event-specific polymerase chain reaction detection methods for genetically modified soybean MON89788 based on the cloned integration flanking sequence[J]. Journal of Agricultural and Food Chemistry, 2009, 57（22）：10524-10530.
[26] European Network of GMO Laboratories（ENGL）. Definition of minimum performance requirements for analytical methods of GMO testing. 2008.
[27] 苏长青, 谢家建, 王奕海, 等. 转基因水稻Bt汕优63的整合结构和品系特异性定量PCR方法[J]. 农业生物技术学报, 2011, 19（3）：434-441.
[28] 吴永彬, 肖维威, 张宝, 等. 转基因大豆、玉米、油菜和水稻品系特异性检测质粒标准分子的快速构建及应用[J]. 中国生物化学与分子生物学报, 2011, 27（8）：775-782.
[29] Burns M, Burrell A, Foy C. The applicability of digital PCR for the assessment of detection limits in GMO analysis[J]. European Food Research and Technology, 2010, 231（3）：353-362.
[30] White AK, Michael V, Oleh IP, et al. High-throughput micro fluidic single-cell RT-qPCR[J]. Proc Natl Acad Sci USA, 2011, 108 （34）：13999-14004.
[31] Jones MA, Bhide S, Chin E, et al. Targeted polymerase chain reaction-based enrichment and next generation sequencing for diagnostic testing of congenital disorders of glycosylation[J]. Genet Med, 2011, 13（11）：921-932.
[32] 胡伟, 陈荣华, 张晨, 等. 微滴式数字PCR技术用于生物样品种属鉴定和绝对定量[J]. 法医学杂志, 2014, 30（5）：342-345.
[33] 姜羽, 胡佳莹, 杨立桃, 等. 利用微滴数字PCR分析转基因生物外源基因拷贝数[J]. 农业生物技术学报, 2014, 22（10）：1298-1305.