GUO Meng-ru,XIA Yi-miao,CHEN Fu-sheng.Application Progress of PCR Technology in Transgenic Detection of Soybean Products[J].Soybean Science,2022,41(04):490-497.[doi:10.11861/j.issn.1000-9841.2022.04.0490]
大豆制品转基因检测中PCR技术的应用进展及展望
- Title:
- Application Progress of PCR Technology in Transgenic Detection of Soybean Products
- 关键词:
- 聚合酶链式反应（PCR）; 转基因大豆; 大豆制品; 转基因检测; 标识
- Keywords:
- polymerase chain reaction(PCR); genetically modified soybean; soybean products; transgenic detection; label
- 文献标志码:
- A
- 摘要:
- 近年来，世界范围内转基因大豆的应用率已高达78%左右，转基因大豆主要用于大豆油生产，其制品已越来越广泛地参与到消费者的饮食生活中。我国自产大豆量不足进口大豆量的20%，进口大豆以转基因大豆为主。为规范转基因大豆的流通、加工和消费等环节，我国对转基因制品采用按目录强制性标识管理，这就对转基因大豆制品的检测技术提出了较高要求。目前广泛采用的检测方法是核酸扩增检测技术，其中聚合酶链式反应（Polymerase Chain Reaction，PCR）技术应用最普遍，主要包括普通定性PCR、巢式PCR、实时荧光定量PCR、多重PCR、数字PCR等。本文对不同PCR技术在大豆制品转基因检测中的应用进行综述，对其优缺点进行了对比，对不同加工程度大豆制品适用的PCR技术进行了探究，最后对大豆制品相关PCR技术未来潜在的研究方向进行了展望，以期提高PCR检测效率，助力我国转基因大豆制品生物安全管理工作的推进实施。
- Abstract:
- In recent years， the application rate of genetically modified soybeans in the world has reached about 78%. Genetically modified (GM) soybeans are mainly used for soybean oil production， and their products have been more and more widely involved in consumers′ dietary life. The quantity of Chinese self-produced soybeans is less than one-fifth of the quantity of imported soybeans， and the imported soybeans are mainly GM soybeans. In order to standardize the circulation， processing and consumption of GM soybeans， China adopts mandatory labeling management for GM soybean products， which puts forward higher requirements for the detection technology of GM products. At present， the most commonly used detection method is nucleic acid amplification detection technology， of which the polymerase chain reaction (PCR) technology is the most widely used， mainly including ordinary qualitative PCR， nested PCR， real-time fluorescent quantitative PCR， multiplex PCR， digital PCR， etc. This paper reviewed the application of different PCR technologies in the detection of GM soybean products， and compared their advantages and disadvantages. The applicability of PCR technologies to GM soybean products with different processing degrees was explored， and the future development direction of PCR technology for GM soybean products was prospected. This paper aims to provide a theoretical reference for the selection of the most suitable PCR technology in the detection of GM soybean products， and to help promote the implementation of the biosafety management of GM soybean products in China.
参考文献/References:
［1］陈元春. 中国大豆国际贸易及其影响因素研究[D]. 北京:中国社会科学院研究生院， 2020. (CHEN Y C. Research on China′s soybean international trade and its influencing factors[D]. Beijing: Graduate School of Chinese Academy of Social Sciences， 2020.)[2]Foreign Agricultural Service and United States Department of Agriculture. Oilseeds: World markets and trade[EB/OL]. (2022-03-9)[2022-03-22]. https: //www.fas.usda.gov/data/oilseeds-world-markets-and-trade.[3]农业部农业转基因生物安全管理办公室.农业转基因生物标识管理办法[EB/OL]. (2017-11-30)[2022-01-26]. http: //www.moa.gov.cn/ztzl/ zjyqwgz. (Agricultural GMO Safety Management Office of the Ministry of Agriculture. Measures for the administration of agricultural GMO labels[EB/OL]. (2017-11-30).[2022-01-26]. http: //www.moa.gov.cn/ztzl /zjyqwgz.)[4]LOU Y Y， CHEN C X， LONG X L， et al. Detection and quantification of chimeric antigen receptor transgene copy number by droplet digital PCR versus Real-Time PCR[J]. The Journal of Molecular Diagnostics， 2020， 22(5): 699-707.[5]孟静， 孙潇慧， 钟立霞， 等. 豆制品中转基因成分检测的研究进展[J]. 大豆科学， 2019， 38(1): 148-152. (MENG J， SUN X H， ZHONG L X， et al. Research progress on detection of genetically modified components in soy products[J]. Soybean Science， 2019， 38(1): 148-152.)[6]王柳. 用于食品的核酸扩增检测技术研究[D]. 浙江: 浙江大学， 2018. (WANG L. Research on nucleic acid amplification and detection technology for food[D]. Zhejiang: Zhejiang University， 2018.)[7]吴姗， 吴志毅， 张晓峰， 等. 普通PCR法、荧光定量PCR法及微流控芯片法检测大豆产品中外源基因灵敏度的比较[J]. 中国食品学报， 2009， 9(2): 176-186. (WU S， WU Z Y， ZHANG X F， et al. Comparison of the sensitivity of ordinary PCR， fluorescence quantitative PCR and microfluidic chip method for detecting foreign genes in soybean products[J]. Chinese Journal of Food Science， 2009， 9(2): 176-186.)[8]ANKLAM E， GADANI F， HEINZE P， et al. Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products[J]. European Food Research & Technology， 2002， 214(1): 3-26.[9]QIU Y W， ZHANG M H， YU Y B， et al. The construction of pMD18-HT-soybean as a calibrator plasmid and nested PCR assay for herbicide-tolerant soybeans[J]. European Food Research and Technology， 2014， 238(3): 375-386.[10]BROD F C A， FERRARI C D S， VALENTE L L， et al. Nested PCR detection of genetically modified soybean in soybean flour， infant formula and soymilk[J]. LWT-Food Science and Technology， 2005， 40(4): 748-751.[11]姚芹， 徐泽鹏， 曹灿灿， 等. 食用大豆油加工过程DNA降解及检测研究[J]. 食品科技， 2018， 43(4): 184-188. (YAO Q， XU Z P， CAO C C， et al. Study on DNA degradation and detection during edible soybean oil processing[J]. Food Science and Technology， 2018， 43(4): 184-188.)[12]PAULI U， LINIGER M， ZIMMERMANN A. Detection of DNA in soybean oil[J]. Zeitschrift Für Lebensmitteluntersuchung Und -Forschung A， 1998， 207(4): 264-267.[13]BUBNER〖KG(0.8mm〗 B， BALDWIN I T. Use of real-time PCR for determining copy number and zygosity in transgenic plants[J]. Plant Cell Reports， 2004， 23(5): 263-271.[14]王小花， 李建祥， 王国卿， 等. SYBR Green实时荧光定量PCR检测大豆转基因成分[J]. 食品科学， 2009， 30(8): 171-176. (WANG X H， LI J X， WANG G Q， et al. SYBR Green real-time fluorescent quantitative PCR for detection of genetically modified soybean components[J]. Food Science， 2009， 30(8): 171-176.)[15]MA H， LI H， LI J， et al. High-throughput， low-cost， and event-specific polymerase chain reaction detection of herbicide tolerance in genetically modified soybean A2704-12[J]. Genetics & Molecular Research， 2014， 13(1): 696-703.[16]ZHEN Z， LV W， TANG Z F， et al. SYBR Green qPCR screening methods for detection of anti-herbicide genes in genetically modified processed products[J]. Journal of Northeast Agricultural University(English Edition)， 2016， 23(1): 57-64.[17]XIA Y M， CHEN F S， JIANG L Z，et al. Development of an efficient method to extract DNA from refined soybean oil[J]. Food Analytical Methods， 2021， 14(1): 1-12.[18]SONG J， SONGQ C， WANG D， et al. Monitoring the prevalence of genetically modified soybeans in tofu in Chengdu， China using real-time and conventional PCR[J]. Journal of Food Composition and Analysis， 2018， 67: 172-177.[19]PARK S B， KIM J Y， LEE D G， et al. Development of a systematic qPCR array for screening GM soybeans[J]. Foods， 2021， 10(3): 610.[20]国家市场监督管理总局、国家标准化管理委员会. 转基因产品通用检测方法: GB/T 38505-2020[S]. 北京: 全国生化检测标准化技术委员会， 2020. (State Administration for Market Regulation， National Standardization Administration. General testing methods for genetically modified products: GB/T 38505-2020[S]. Beijing: National Biochemical Testing Standardization Technical Committee， 2020.)[21]国家市场监督管理总局，中国国家标准化管理委员会. 转基因产品检测 实时荧光定性聚合酶链式反应（PCR）检测方法: GB/T 19495.4-2018[S]. 北京: 全国植物检疫标准化技术委员会， 2018. (State Administration for Market Regulation， China National Standardization Administration. Detection of genetically modified products. Real-time fluorescent qualitative polymerase chain reaction (PCR) detection method: GB/T 19495.4-2018[S]. Beijing: National Phytosanitary Standardization Technical Committee， 2018.)[22]国家市场监督管理总局，中国国家标准化管理委员会. 转基因产品检测 实时荧光定量聚合酶链式反应（PCR）检测方法: GB/T 19495.5-2018[S]. 北京: 全国植物检疫标准化技术委员会， 2018. (State Administration for Market Regulation， China National Standardization Administration. Detection of genetically modified products-real-time fluorescence quantitative polymerase chain reaction (PCR) detection method: GB/T 19495.5-2018[S]. Beijing: National Phytosanitary Standardization Technical Committee， 2018.)[23]国家质量监督检验检疫总局. 植物及其加工产品中转基因成分实时荧光PCR定性检验方法: SN/T 1204-2016[S]. 北京: 国家认证认可监督管理委员会， 2016. (General Administration of Quality Supervision， Inspection and Quarantine. Real-time fluorescent PCR qualitative detection method for transgenic components in plants and their processed products: SN/T 1204-2016[S]. Beijing: National Certification and Accreditation Administration Commission， 2016.)[24]国家质量监督检验检疫总局. 饲料中转基因植物成分PCR 检测方法: SN/T 1201-2014[S]. 北京: 国家认证认可监督管理委员会， 2014. (General Administration of Quality Supervision， Inspection and Quarantine. PCR detection method of genetically modified plant components in feed: SN/T 1201-2014[S]. Beijing: 国家认证认可监督管理委员会， 2014.)[25]国家质量监督检验检疫总局. 调味品中转基因植物成分实时荧光PCR定性检测方法: SN/T 2705-2010[S]. 北京: 国家认证认可监督管理委员会， 2010. (General Administration of Quality Supervision， Inspection and Quarantine. Real-time fluorescent PCR qualitative detection method for transgenic plant components in condiments: SN/T 2705-2010[S]. Beijing: National Certification and Accreditation Administration Commission， 2010.)[26]国家质量监督检验检疫总局. 转基因植物品系特异性检测方法: SN/T 2668-2010[S]. 北京:国家认证认可监督管理委员会， 2010. (General Administration of Quality Supervision， Inspection and Quarantine. Strain-specific detection method of transgenic plants: SN/T 2668-2010[S]. Beijing: National Certification and Accreditation Administration Commission， 2010.)[27]天津市市场和质量监督管理委员会. 转基因耐除草剂大豆GTS40-3-2及其衍生品种定量检测实时荧光PCR方法: DB12/T 651-2016[S]. 天津: 天津市农业标准化技术委员会， 2016. (Tianjin Municipal Market and Quality Supervision and Administration Commission. Tianjin Municipal Bureau of Quality and Technical Supervision. Real-time PCR method for quantitative detection of transgenic herbicide-tolerant soybean GTS40-3-2 and its derivatives: DB12/T 651-2016[S]. Tianjin: Tianjin Agricultural Standardization Technical Committee， 2016.)[28]天津市市场和质量监督管理委员会. 转基因耐除草剂大豆DAS-68416-4及其衍生品 种定性检测实时荧光PCR法: DB12/T 652-2016[S]. 天津: 天津市农业标准化技术委员会， 2016. (Tianjin Municipal Market and Quality Supervision and Administration Commission. Real-time PCR method for qualitative detection of transgenic herbicide-tolerant soybean DAS-68416-4 and its derivatives: DB12/T 652-2016[S]. Tianjin: Tianjin Agricultural Standardization Technical Committee， 2016.)[29]农村农业部. 转基因植物及其产品成分检测 耐除草剂大豆GTS40-3-2及其衍生品种定量PCR方法: 农业农村部 公告第323号 10-2020[S]. (Ministry of Rural Agriculture. Detection of components of genetically modified plants and their products: Quantitative PCR method for herbicide-tolerant soybean GTS40-3-2 and its derivatives: Ministry of Agriculture and Rural Affairs Announcement No. 323 10-2020[S].)[30]PARK S B， KIM H Y， KIM J H. Multiplex PCR system to track authorized and unauthorized genetically modified soybean events in food and feed[J]. Food Control， 2015， 54: 47-52.[31]ZHOU Y， LI Y， PEI X. Determination of genetically modified soybean by multiplex PCR and CGE with LIF detection[J]. Chromatographia， 2007， 66(9-10): 691-696.[32]NIKOLI〖XCc2.TIF,JZ〗 Z， MILOEVI〖KG-*5〗〖XCc2.TIF,JZ〗 M， VUJAKOVI〖XCc2.TIF,JZ〗 M， et al. Qualitative triplex PCR for the detection of genetically modified soybean and maize[J]. Biotechnology & Biotechnological Equipment， 2008， 22(3): 801-803.[33]董立明， 李葱葱， 邢珍娟， 等. 利用多重PCR技术快速检测五个转基因大豆品系[J]. 大豆科学， 2016， 35(6): 1002-1007. (DONG L M， LI C C， XING Z J， et al. Rapid detection of five transgenic soybean lines using multiplex PCR[J]. Soybean Science， 2016， 35(6): 1002-1007.)[34]XIAO B， NIU C， SHANG Y， et al. A ′Turn-on′ ultra-sensitive multiplex real-time fluorescent quantitative biosensor mediated by a universal primer and probe for the detection of genetically modified organisms[J]. Food Chemistry， 2020， 330(5): 127247.[35]周方永， 李岩， 薄新文， 等. 单因子和正交设计法建立分枝杆菌多重PCR体系[J]. 石河子大学学报: 自然科学版， 2013， 31(4): 449-456. (ZHOU F Y， LI Y， BO X W， et al. Establishment of mycobacterium multiplex PCR system by single factor and orthogonal design[J]. Journal of Shihezi University: Natural Science Edition， 2013， 31(4): 449-456.)[36]LIU W W，WANG X N， TAO J， et al. A multiplex PCR assay mediated by universal primers for the detection of adulterated meat in mutton[J]. Journal of Food Protection， 2019， 82(2): 325-330.[37]DEMEKE T， DOBNIK D. Critical assessment of digital PCR for the detection and quantification of genetically modified organisms[J]. Analytical and Bioanalytical Chemistry， 2018， 410(17): 4039-4050.[38]MARGARET E H，ROBERT M D， JOHN S S B， et al. Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA[J]. Molecular Ecology Resources， 2017， 17(2): 221-229.[39]斯能武， 李俊， 武玉花， 等. 数字PCR在转基因定量检测中的研究进展[J]. 中国油料作物学报， 2021， 43(1): 40-50. (SI N W， LI J， WU Y H， et al. Research progress of digital PCR in quantitative detection of transgenes[J]. Chinese Journal of Oil Crops， 2021， 43(1): 40-50.)[40]DEMEKE T， GRFENHAN T， HOLIGROSKI M， et al. Assessment of droplet digital PCR for absolute quantification of genetically engineered OXY235 canola and DP305423 soybean samples[J]. Food Control， 2014， 46: 470-474.[41]KOSIR A B， DEMSAR T， STEBIH D， et al. Digital PCR as an effective tool for GMO quantification in complex matrices[J]. Food Chemistry， 2019， 294: 73-78.[42]魏霜， 周广彪， 刘津， 等. 多重串联式PCR基因碟片技术检测转基因大豆GTS 40-3-2[J]. 中国食品学报， 2018， 18(2): 244-249. (WEI S， ZHOU G B， LIU J， et al. Detection of genetically modified soybean GTS 40-3-2 using multiple tandem PCR gene disc technology[J]. Chinese Journal of Food Science， 2018， 18(2): 244-249.)[43]杨清华， 董德坤， 操晶， 等. 转GmWRKY70基因大豆的PCR检测及其T-DNA侧翼序列分析[J]. 农业生物技术学报， 2020， 28(7): 1203-1210. (YANG Q H， DONG D K， CAO J， et al. PCR detection and T-DNA flanking sequence analysis of GmWRKY70 transgenic soybean[J]. Journal of Agricultural Biotechnology， 2020， 28(7): 1203-1210.)
相似文献/References:
[1]林凡敏,柏锡,樊超,等.转GsGST14耐盐碱基因大豆的农艺性状调查[J].大豆科学,2013,32(01):56.[doi:10.3969/j.issn.1000-9841.2013.01.013]
LIN Fan-min,BAI Xi,FAN Chao,et al.Investigation and Analysis of the Main Agronomic Traits of Different Transgenic Soybean Lines with GsGST14 Gene[J].Soybean Science,2013,32(04):56.[doi:10.3969/j.issn.1000-9841.2013.01.013]
[2]芦春斌,周文,刘标.喂食转基因大豆对子代雄鼠生殖系统的影响[J].大豆科学,2013,32(01):119.[doi:10.3969/j.issn.1000-9841.2013.01.028]
LU Chun-bin,ZHOU Wen,LIU Biao.Effects of Transgenic Soybean on Reproductive System in Male Mice[J].Soybean Science,2013,32(04):119.[doi:10.3969/j.issn.1000-9841.2013.01.028]
[3]王 东,宋 君,叶先林,等.转基因大豆外源基因NOS终止子定量测定的不确定度分析[J].大豆科学,2013,32(05):601.[doi:10.11861/j.issn.1000-9841.2013.05.0601]
WANG Dong,SONG Jun,YE Xian-lin,et al.[J].Soybean Science,2013,32(04):601.[doi:10.11861/j.issn.1000-9841.2013.05.0601]
[4]程 遥.中国大豆种植业发展的思考[J].大豆科学,2013,32(05):711.[doi:10.11861/j.issn.1000-9841.2013.05.0711]
CHENG Yao.Consideration on the Development of China Soybean Industry[J].Soybean Science,2013,32(04):711.[doi:10.11861/j.issn.1000-9841.2013.05.0711]
[5]周 洁,于 崧,王珊珊,等.抗盐碱转基因大豆对根际土壤固氮细菌多样性的影响[J].大豆科学,2013,32(06):801.[doi:10.11861/j.issn.1000-9841.2013.06.0801]
ZHOU Jie,YU Song,WANG Shan-shan,et al.Effects of Salinization Resistance Transgenic Soybeans on Rhizosphere Soil Nitrogen-fixing Bacterial Diversity[J].Soybean Science,2013,32(04):801.[doi:10.11861/j.issn.1000-9841.2013.06.0801]
[6]厉 志,王曙明,刘 佳,等.广适性转bar基因大豆除草剂草丁膦筛选浓度的研究[J].大豆科学,2013,32(06):810.[doi:10.11861/j.issn.1000-9841.2013.06.0810]
LI zhi,WANG Shu-ming,LIU Jia,et al.Study on Screening Concentration of Wide Adaptability Herbicide Resistant? bar Transgenic Soybean[J].Soybean Science,2013,32(04):810.[doi:10.11861/j.issn.1000-9841.2013.06.0810]
[7]何龙凉,胡红东,李小琴,等.防城港口岸进境转基因大豆贸易概况及检验检疫分析[J].大豆科学,2013,32(04):539.[doi:10.11861/j.issn.1000-9841.2013.04.0539]
HE Long-liang,HU Hong-dong,LI Xiao-qin,et al.General Situation of Imported Genetically Modified Soybean in Fangchenggang Port and Its Inspection and Quarantine Analysis[J].Soybean Science,2013,32(04):539.[doi:10.11861/j.issn.1000-9841.2013.04.0539]
[8]周广彪,蔡 颖,陈文婉,等.QuickGene-810型自动核酸提取仪在转基因大豆检测中的应用研究[J].大豆科学,2014,33(03):434.[doi:10.11861/j.issn.1000-9841.2014.03.0434]
ZHOU Guang-biao,CAI Ying,CHEN Wen-wan,et al.Application of Quick Gene810 Automated Nucleic Acid Extraction Instrument on Detection of Genetically Modified Soybean[J].Soybean Science,2014,33(04):434.[doi:10.11861/j.issn.1000-9841.2014.03.0434]
[9]张彬彬,李永光,盖江南,等.转TaDREB3基因大豆基因漂流距离及频率的研究[J].大豆科学,2011,30(04):563.[doi:10.11861/j.issn.1000-9841.2011.04.0563]
ZHANG Bin-bin,LI Yong-guang,GAI Jiang-nan,et al.Distance and Frequency of Gene Flow in Transgenic Soybean Overexpressing TaDREB3[J].Soybean Science,2011,30(04):563.[doi:10.11861/j.issn.1000-9841.2011.04.0563]
[10]陈晟,郭丽琼,宋景深,等.T5代γ-亚麻酸转基因大豆的遗传稳定性分析[J].大豆科学,2012,31(01):24.[doi:10.3969/j.issn.1000-9841.2012.01.006]
CHEN Sheng,GUO Li-qiong,SONG Jing-shen,et al.Genetic Stability Analysis of the Fifth Generation of Transgenic Soybeans Expressing γ-linolenic Acid[J].Soybean Science,2012,31(04):24.[doi:10.3969/j.issn.1000-9841.2012.01.006]
备注/Memo
收稿日期：2022-01-29