CN106995841B - A kind of multiplex PCR kit for detection of transgenic soybean and detection method - Google Patents

Abstract

Translated fromChinese

本发明公开了一种转基因大豆检测用多重PCR试剂盒及检测方法，可快速检测我国农业部批准进口的12种转基因大豆，包括MON87701、GTS40‑3‑2、MON89788、CV127、A2704、A5547、DP356043、DP305423、MON87769、MON87708、305423×40‑3‑2、MON87701×MON89788；本发明公开的多重PCR试剂盒基于“公共引物”介导的多重PCR方法，包括公共引物、多重引物、多重PCR体系检测；两步退火温度法提高多重PCR体系的特异性、敏感性，该多重PCR方法具有包容性，可继续增加检测靶标，有利于提高多重PCR的检测通量。本发明具有灵敏度高，经济、快速、操作简单等优点，可应用于食品检测。

The invention discloses a multiplex PCR kit and detection method for detecting transgenic soybeans, which can rapidly detect 12 kinds of transgenic soybeans approved for import by the Ministry of Agriculture of my country, including MON87701, GTS40-3-2, MON89788, CV127, A2704, A5547, DP356043 , DP305423, MON87769, MON87708, 305423×40-3-2, MON87701×MON89788; the multiplex PCR kit disclosed in the present invention is based on the multiplex PCR method mediated by "common primers", including common primers, multiplex primers, multiplex PCR system detection The two-step annealing temperature method improves the specificity and sensitivity of the multiplex PCR system. The multiplex PCR method is inclusive and can continue to increase the detection target, which is beneficial to improve the detection throughput of the multiplex PCR. The invention has the advantages of high sensitivity, economy, rapidity, simple operation and the like, and can be applied to food detection.

Description

Translated fromChinese

一种转基因大豆检测用多重PCR试剂盒及检测方法A kind of multiplex PCR kit for detection of transgenic soybean and detection method

技术领域technical field

本发明属于食品/基因检测领域，具体涉及转基因大豆检测用多重PCR试剂盒及检测方法。The invention belongs to the field of food/gene detection, and particularly relates to a multiplex PCR kit and a detection method for detection of transgenic soybeans.

背景技术Background technique

目前，国内外报道的转基因作物检测技术主要分为三类，一类是基于核酸的检测技术；二是基于蛋白质的酶学和免疫学检测技术；第三类是转基因检测新技术，包括向自动化技术发展的生物传感器与生物芯片、基于现代分析仪器的近红外光谱和质谱分析技术等。第二类基于蛋白质的检测技术是对转基因植物中外源基因表达的蛋白进行检测，这些蛋白在加工过程中易受到破坏而失活、分解或消失，检查结果易出现假阴性且方法的重复性差。近些年发展的转基因检测新技术如生物传感器、色谱法和近红外光谱法等，此类技术目前仍处于研究阶段，存在一些暂时无法有效解决或客观存在的缺点，如色谱法只适用于转基因植物及其产品的成分发生重大变化时的定性检测分析，该类检测技术短期内仍无法有效推广应用。因此，我国农业部发布的转基因成分检测方法标准以核酸检测技术为主，该类检测技术包括PCR技术、恒温扩增技术、southern杂交、基因芯片技术、荧光定量PCR等。At present, the detection technologies of genetically modified crops reported at home and abroad are mainly divided into three categories, one is nucleic acid-based detection technology; the second is protein-based enzymatic and immunological detection technology; the third is new technology of genetically modified detection, including automatic Technology development of biosensors and biochips, near-infrared spectroscopy and mass spectrometry based on modern analytical instruments, etc. The second type of protein-based detection technology is the detection of proteins expressed by foreign genes in transgenic plants. These proteins are easily damaged during processing and are inactivated, decomposed or disappeared. The test results are prone to false negatives and the method has poor reproducibility. New technologies for GMO detection developed in recent years, such as biosensors, chromatography, and near-infrared spectroscopy, are still in the research stage, and there are some shortcomings that cannot be effectively resolved or exist objectively. For example, chromatography is only suitable for GMOs. Qualitative detection and analysis when the composition of plants and their products undergoes major changes, this type of detection technology cannot be effectively promoted and applied in the short term. Therefore, the detection method standards for genetically modified components issued by the Ministry of Agriculture of my country are mainly based on nucleic acid detection technology, which includes PCR technology, constant temperature amplification technology, southern hybridization, gene chip technology, fluorescence quantitative PCR, etc.

多重PCR是在普通PCR的基础上，于同一个反应体系中利用多对引物同时扩增多段靶序列的PCR技术。自多重PCR报道以来，由于其高效性、灵敏性和经济简便性，已广泛应用于核酸诊断的多个领域，如遗传疾病诊断、突变和多态性分析、病原体检测、转基因鉴定等；多重PCR体系，一般包括以下试剂：PCR缓冲液、Taq酶、dNTP、MgCl₂、多重引物、模板和去离子水。Multiplex PCR is a PCR technology that uses multiple pairs of primers to simultaneously amplify multiple target sequences in the same reaction system on the basis of ordinary PCR. Since the report of multiplex PCR, due to its high efficiency, sensitivity and economic simplicity, it has been widely used in many fields of nucleic acid diagnosis, such as genetic disease diagnosis, mutation and polymorphism analysis, pathogen detection, transgene identification, etc.; multiplex PCR The system generally includes the following reagents: PCR buffer, Taq enzyme, dNTP, MgCl₂ , multiplex primers, template and deionized water.

我国目前允许进口的转基因大豆高达12种（具体包括GTS-40-3-2、MON87701、MON89788、MON87701×MON89788、CV127-9、A2704-12、DP356043、DP305423、A5547、305423×40-3-2、MON87769、MON87708，其中MON87701×MON89788和305423×40-3-2为复合性状转基因大豆）。my country currently allows import of up to 12 kinds of genetically modified soybeans (including GTS-40-3-2, MON87701, MON89788, MON87701×MON89788, CV127-9, A2704-12, DP356043, DP305423, A5547, 305423×40-3-2 , MON87769, MON87708, of which MON87701×MON89788 and 305423×40-3-2 are compound trait transgenic soybeans).

转基因作物是利用基因工程技术将外源目的基因转入植物中培育而出的，它们具有抗虫、抗除草剂、抗逆和营养改良等优良特性。全球转基因作物的种植面积自1996年的0.17亿公顷增加到2015年的1.797亿公顷。随着转基因技术不断发展，转基因作物产业化与商业化的程度不断加深，我国陆续针对转基因作物制定了相关法律法规，然而，公众对转基因食品安全的担忧及对所食用食品是否含有转基因成分的知情权仍与日俱增，因此，快速、准确、敏感的转基因食品检测技术是所有转基因安全性评价等法律法规真正落到实处的基础和关键之一。Transgenic crops are cultivated by using genetic engineering technology to transfer exogenous target genes into plants. They have excellent characteristics such as insect resistance, herbicide resistance, stress resistance and nutritional improvement. The global area of biotech crops has increased from 17 million hectares in 1996 to 179.7 million hectares in 2015. With the continuous development of genetically modified technology, the degree of industrialization and commercialization of genetically modified crops continues to deepen, and my country has successively formulated relevant laws and regulations on genetically modified crops. The rights are still increasing day by day. Therefore, fast, accurate and sensitive genetically modified food testing technology is one of the foundations and keys for the implementation of all laws and regulations such as safety evaluation of genetically modified foods.

发明内容SUMMARY OF THE INVENTION

本发明的目的是提供一种检测转基因大豆的多重PCR试剂盒，用于我国目前批准进口（即已颁发安全证书）的12种转基因大豆的单一或者同时多重PCR检测，通过设计公共引物及转基因大豆特异性引物，限定多重PCR检测体系和条件，可检测食品（比如大豆、大豆制品、含有大豆成份的食物等）中是否含有转基因大豆成分。The purpose of the present invention is to provide a multiplex PCR kit for detecting transgenic soybeans, which can be used for single or simultaneous multiplex PCR detection of 12 kinds of transgenic soybeans currently approved for import (that is, safety certificates have been issued) in my country. By designing public primers and transgenic soybeans Specific primers, which define the multiplex PCR detection system and conditions, can detect whether foods (such as soybeans, soybean products, foods containing soybean ingredients, etc.) contain genetically modified soybean ingredients.

为达到上述发明目的，本发明采用的技术方案是：In order to achieve the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is:

一种转基因大豆检测用多重PCR试剂盒，包括公共引物、针对转基因大豆的嵌合引物；所述公共引物的序列为SEQ ID NO.1；所述针对转基因大豆的嵌合引物为SEQ ID NO.2至SEQ ID NO.18所示的核苷酸序列中的一种或几种。A multiplex PCR kit for detection of transgenic soybeans, comprising public primers and chimeric primers for transgenic soybeans; the sequence of the public primers is SEQ ID NO.1; the chimeric primers for transgenic soybeans are SEQ ID NO. 2 to one or more of the nucleotide sequences shown in SEQ ID NO.18.

上述技术方案中，所述转基因大豆检测用多重PCR试剂盒还包括Taq 聚合酶、dNTP、MgCl₂、PCR缓冲液、去离子水、阳性对照引物对；其中所述阳性对照引物对的序列优选为SEQ ID NO.19、SEQ ID NO.20；可用来检测DNA模板质量和反应体系扩增效率；In the above technical scheme, the multiplex PCR kit for detection of transgenic soybean further comprises Taq polymerase, dNTP, MgCl₂ , PCR buffer, deionized water, and a positive control primer pair; wherein the sequence of the positive control primer pair is preferably SEQ ID NO.19, SEQ ID NO.20; can be used to detect DNA template quality and reaction system amplification efficiency;

SEQ ID NO.19：GCCCTCTACTCCACCCCCATCCSEQ ID NO. 19: GCCCTCTACTCCACCCCCATCC

SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG 。SEQ ID NO. 20: GCCCATCTGCAAGCCTTTTTGTG.

上述技术方案中，进行PCR反应时，采用两步退火温度法，前10个循环退火温度为61℃；后30个循环退火温度为54℃。第一步退火温度高于第二步的退火温度5～10℃；比如前10个循环的退火温度高于后30个循环退火温度7℃，可有效提高检测的特异性；进行多重PCR反应时，针对转基因大豆的每条嵌合引物的终浓度在8～80nM之间，公共引物的终浓度为1600nM。In the above technical solution, when performing PCR reaction, a two-step annealing temperature method is adopted, and the annealing temperature for the first 10 cycles is 61°C; the annealing temperature for the last 30 cycles is 54°C. The annealing temperature of the first step is 5-10°C higher than the annealing temperature of the second step; for example, the annealing temperature of the first 10 cycles is higher than the annealing temperature of the last 30 cycles by 7°C, which can effectively improve the specificity of detection; when performing multiple PCR reactions , the final concentration of each chimeric primer for transgenic soybean is between 8 and 80 nM, and the final concentration of the common primer is 1600 nM.

本发明还公开了一种检测产品中转基因大豆成分的方法，提取待检测产品基因组DNA作为模板（比如转基因大豆的插入基因与大豆基因组的连接区为靶标序列），在嵌合引物与公共引物存在下采用两步退火温度法进行PCR反应，对PCR产物进行电泳分析即完成产品中转基因大豆成分的检测；最后对PCR产物扩增进行电泳分析，即完成产品中转基因大豆成分的检测；所述公共引物的序列为SEQ ID NO.1；所述嵌合引物为SEQ ID NO.2至SEQ IDNO.18所示的核苷酸序列中的一种或几种。The invention also discloses a method for detecting the components of transgenic soybean in a product. The genomic DNA of the product to be detected is extracted as a template (for example, the junction region between the inserted gene of the transgenic soybean and the soybean genome is the target sequence), and the chimeric primer and the public primer exist in the presence of the chimeric primer and the public primer. The PCR reaction is carried out by a two-step annealing temperature method, and the detection of the transgenic soybean components in the product is completed by electrophoresis analysis of the PCR product; finally, the detection of the transgenic soybean component in the product is completed by performing electrophoresis analysis on the amplification of the PCR product; the public The sequence of the primer is SEQ ID NO.1; the chimeric primer is one or more of the nucleotide sequences shown in SEQ ID NO.2 to SEQ ID NO.18.

上述技术方案中，第一步（即前10个循环）多重PCR扩增的退火温度高于第二步（即后30个循环）多重PCR扩增的退火温度5～10℃；每条嵌合引物的终浓度在8～80nM之间，公共引物的终浓度为1600nM；所述产品为包含大豆成分的食品。具体可包括以下步骤：95℃预变性 5min；95℃ 变性30s，61℃退火 30s，72℃ 延伸30s，共10个循环；72℃ 延伸2min；95℃ 变性30s，54℃退火 30s，72℃ 延伸30s，共30个循环；最后72℃延伸7min。前10个循环，在高退火温度下，低浓度的嵌合引物中特异引物扩增靶标序列，产生带有公共引物末端的PCR产物；后30个循环，在低退火温度下，高浓度的公共引物以早期产生的带有公共引物末端的PCR产物为模板进行大量扩增。根据本发明的方法可以有效、简便的检测产品中，尤其是食品中是否含有转基因大豆成分，为国民知情权以及食品安全做出巨大贡献。In the above technical scheme, the annealing temperature of the first step (ie, the first 10 cycles) multiple PCR amplification is higher than the annealing temperature of the second step (ie, the last 30 cycles) multiple PCR amplification by 5-10 °C; The final concentration of the primers is between 8-80 nM, and the final concentration of the common primer is 1600 nM; the product is a food containing soybean ingredients. Specifically, it may include the following steps: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30s, annealing at 61°C for 30s, extension at 72°C for 30s, a total of 10 cycles; extension at 72°C for 2min; denaturation at 95°C for 30s, annealing at 54°C for 30s, extension at 72°C 30 s, a total of 30 cycles; the final extension at 72 °C for 7 min. For the first 10 cycles, at high annealing temperatures, specific primers in low concentrations of chimeric primers amplify the target sequence, resulting in PCR products with common primer ends; for the next 30 cycles, at low annealing temperatures, high concentrations of common primers The primers use the PCR products generated earlier with the common primer ends as templates for bulk amplification. The method according to the present invention can effectively and simply detect whether products, especially foods, contain genetically modified soybean ingredients, which makes great contributions to the people's right to know and food safety.

本发明公开的转基因大豆成分核酸检测方法准确，可同时检测12中目标基因，而且检测结果准确，可解决公众对转基因食品安全的担忧及对所食用食品是否含有转基因成分的知情权。The nucleic acid detection method for genetically modified soybean components disclosed by the invention is accurate, can detect 12 target genes at the same time, and has accurate detection results, which can solve the public's concerns about the safety of genetically modified foods and the right to know whether the food they eat contains genetically modified ingredients.

本发明进一步公开了一种转基因大豆检测用引物，包括公共引物、针对转基因大豆的嵌合引物；所述公共引物的序列为SEQ ID NO.1；所述针对转基因大豆的嵌合引物为SEQ ID NO.2至SEQ ID NO.18所示的核苷酸序列中的一种或几种。The invention further discloses a primer for detection of transgenic soybean, including a public primer and a chimeric primer for transgenic soybean; the sequence of the public primer is SEQ ID NO. 1; the chimeric primer for transgenic soybean is SEQ ID One or more of the nucleotide sequences shown in NO.2 to SEQ ID NO.18.

本发明还公开了上述转基因大豆检测用引物在制备检测转基因大豆的多重PCR试剂盒中的应用或者权利要求8所述转基因大豆检测用引物在检测转基因大豆中的应用。The invention also discloses the application of the above-mentioned primers for detection of transgenic soybeans in preparing a multiplex PCR kit for detecting transgenic soybeans or the application of the primers for detection of transgenic soybeans in the detection of transgenic soybeans.

本发明中，所述公共引物的序列SEQ ID NO.1及 所述嵌合引物的核苷酸序列如下所示：In the present invention, the sequence SEQ ID NO.1 of the public primer and the nucleotide sequence of the chimeric primer are as follows:

所述嵌合引物为针对12种转基因大豆的嵌合引物；A2704与 A5547共用上游引物SEQ ID NO.11，MON89788、MON87769和MON87708共用下游引物SEQ ID NO.18。305423×40-3-2、MON87701×MON89788是两种基因堆叠的转基因大豆，若样品中同时检测出转基因大豆DP305423和GTS40-3-2，则样品中有可能含有转基因大豆305423×40-3-2；若样品中同时检测出转基因大豆MON87701和MON89788，则样品中有可能含有转基因大豆MON87701×MON89788。The chimeric primers are chimeric primers for 12 kinds of transgenic soybean; A2704 and A5547 share the upstream primer SEQ ID NO.11, MON89788, MON87769 and MON87708 share the downstream primer SEQ ID NO.18. 305423×40-3-2, MON87701×MON89788 are two genetically stacked transgenic soybeans. If transgenic soybeans DP305423 and GTS40-3-2 are detected in the sample at the same time, the sample may contain transgenic soybean 305423×40-3-2; Transgenic soybeans MON87701 and MON89788, the samples may contain transgenic soybeans MON87701×MON89788.

上述技术方案中，公共引物与转基因大豆相关基因组无同源性，当以转基因大豆基因组为模板进行扩增时，不论条件如何，均应没有特异性产物；当以嵌合引物的扩增产物为模板时，公共引物可以扩增出特异性产物；并且公共引物的Tm比转基因大豆靶特异性引物的Tm值低5～10 ℃。In the above technical scheme, the public primers have no homology with the relevant genome of the transgenic soybean, and when the transgenic soybean genome is used as a template for amplification, no matter what the conditions are, there should be no specific product; when the amplification product of the chimeric primer is When used as a template, the common primer can amplify a specific product; and the Tm of the common primer is 5-10°C lower than the Tm value of the transgenic soybean target-specific primer.

上述技术方案中，进行PCR反应时，公共引物的终浓度为1600nM，针对转基因大豆的嵌合引物的终浓度为8～80nM，用于扩增12种转基因大豆MON87701、GTS40-3-2、MON89788、CV127、A2704、A5547、DP356043、DP305423、MON87769、MON87708、305423×40-3-2、MON87701×MON89788 ，比如SEQ ID NO.2/3:80nM，SEQ ID NO.4/5: 60nM，SEQ ID NO.6/7: 64nM，SEQ ID NO.8/9: 8nM，SEQ ID NO.10: 32nM，SEQ ID NO.11: 48nM，SEQ IDNO.12: 16nM，SEQ ID NO.13/14: 24nM，SEQ ID NO.15/16: 8nM，SEQ ID NO.17: 16nM,SEQ ID NO.18: 32nM；进行PCR反应时，采用两步退火温度法，第一步退火温度比第二步退火温度高5～10 ℃。In the above technical scheme, when performing PCR reaction, the final concentration of the common primer is 1600nM, and the final concentration of the chimeric primer for transgenic soybean is 8-80nM, which is used to amplify 12 kinds of transgenic soybean MON87701, GTS40-3-2, MON89788 , CV127, A2704, A5547, DP356043, DP305423, MON87769, MON87708, 305423×40-3-2, MON87701×MON89788, such as SEQ ID NO.2/3: 80nM, SEQ ID NO.4/5: 60nM, SEQ ID NO. NO.6/7: 64nM, SEQ ID NO.8/9: 8nM, SEQ ID NO.10: 32nM, SEQ ID NO.11: 48nM, SEQ ID NO.12: 16nM, SEQ ID NO.13/14: 24nM , SEQ ID NO.15/16: 8nM, SEQ ID NO.17: 16nM, SEQ ID NO.18: 32nM; During PCR reaction, a two-step annealing temperature method was adopted, and the annealing temperature of the first step was higher than the annealing temperature of the second step. High 5 ~ 10 ℃.

本发明公开的试剂盒可用于产品尤其是食品中转基因大豆成分的检测，本发明公开的多重PCR是公共引物介导的多重PCR结合两步退火温度法。具体比如采用两步退火温度法，以转基因大豆插入基因序列与大豆基因连接区序列为靶标序列，用嵌合引物先扩增10个循环，退火温度为61℃，进行第一步多重PCR扩增，可产生公共引物末端的PCR产物；公共引物以第一步扩增的带有公共引物末端的PCR产物为模板，进行后30个循环的第二步多重PCR扩增，退火温度为54℃；即完成转基因大豆的多重PCR扩增；前10个循环的多重PCR扩增退火温度比后面30个循环的多重PCR扩增退火温度高5～10 ℃。循环早期，在高退火温度下，低浓度的嵌合引物扩增靶标序列，产生带有公共引物末端的PCR产物；循环后期，在低退火温度下，高浓度的公共引物以早期产生的PCR产物为模板进行大量扩增。多重PCR采用的25μL反应体系包括：10μM的公共引物4μL，不同浓度的嵌合引物混合物2μL，2×Dream TaqGreen Mix 12.5μL，DNA模板 1μL，灭菌ddH₂O 4.5μL；PCR反应条件为：95℃预变性 5min；95℃ 变性30s，61℃退火 30s，72℃ 延伸30s，共10个循环；72℃ 延伸2min；95℃ 变性30s，54℃退火 30s，72℃ 延伸30s，共30个循环；最后72℃延伸7min。最后进行琼脂糖凝胶电泳分析，结果表明本发明的检测转基因大豆的多重PCR试剂盒可同时或者独立特异性检测10种转基因大豆的基因组；利用构建的质粒模板，本发明的检测转基因大豆的多重PCR试剂盒的敏感性最高可达0.1%（w/w）转基因大豆成分含量，即0.1ng/100ngDNA样品。The kit disclosed in the invention can be used for the detection of transgenic soybean components in products, especially food, and the multiplex PCR disclosed in the invention is a public primer-mediated multiplex PCR combined with a two-step annealing temperature method. Specifically, for example, a two-step annealing temperature method is used. The transgenic soybean insertion gene sequence and the soybean gene junction region sequence are used as the target sequence, and chimeric primers are used to amplify for 10 cycles. The annealing temperature is 61 °C, and the first step of multiple PCR amplification is performed. , the PCR product at the end of the common primer can be generated; the common primer uses the PCR product with the end of the common primer amplified in the first step as a template, and carries out the second-step multiple PCR amplification of the last 30 cycles, and the annealing temperature is 54 ℃; That is, the multiplex PCR amplification of the transgenic soybean is completed; the annealing temperature of the multiplex PCR amplification of the first 10 cycles is 5-10°C higher than the annealing temperature of the multiplex PCR amplification of the next 30 cycles. Early in the cycle, at high annealing temperatures, low concentrations of chimeric primers amplify the target sequence, resulting in PCR products with the ends of the common primers; later in the cycle, at low annealing temperatures, high concentrations of common primers produce PCR products at early stages Perform bulk amplification for the template. The 25 μL reaction system used in multiplex PCR included: 4 μL of 10 μM public primers, 2 μL of chimeric primer mixtures with different concentrations, 12.5 μL of 2×Dream TaqGreen Mix, 1 μL of DNA template, and 4.5 μL of sterilized ddH₂ O; PCR reaction conditions were: 95 10 cycles of denaturation at 95°C for 30s, annealing at 61°C for 30s, and extension at 72°C for 30s; extension at 72°C for 2min; denaturation at 95°C for 30s, annealing at 54°C for 30s, and extension at 72°C for 30s, a total of 30 cycles; A final extension at 72°C for 7 min. Finally, agarose gel electrophoresis analysis is performed, and the results show that the multiplex PCR kit for detecting transgenic soybeans of the present invention can simultaneously or independently and specifically detect the genomes of 10 kinds of transgenic soybeans; using the constructed plasmid template, the multiplex PCR kit for detecting transgenic soybeans of the present invention The sensitivity of the PCR kit can reach up to 0.1% (w/w) GM soybean component content, that is, 0.1ng/100ng DNA sample.

由于上述技术方案运用，本发明与现有技术相比具有以下优点：Due to the application of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

（1）本发明公开的检测转基因大豆的多重PCR试剂盒，可同时也可单独检测我国批准进口的的12种转基因大豆，即MON87701、GTS40-3-2、MON89788、CV127、A2704、A5547、DP356043、DP305423、MON87769、MON87708、305423×40-3-2、MON87701×MON89788，不仅可以节约试剂使用量和降低检测成本，同时提高了检测效率，具有灵敏度高，经济、快速、操作简单等优点；(1) The multiplex PCR kit for detecting transgenic soybeans disclosed in the present invention can simultaneously and independently detect 12 kinds of transgenic soybeans approved for import in my country, namely MON87701, GTS40-3-2, MON89788, CV127, A2704, A5547, DP356043 , DP305423, MON87769, MON87708, 305423×40-3-2, MON87701×MON89788, not only can save the amount of reagents used and reduce the detection cost, but also improve the detection efficiency, with high sensitivity, economy, fast, simple operation and other advantages;

（2）本发明公开的试剂盒进行检测时，降低嵌合引物浓度，最低至8nM/每条，公共引物浓度为1600nM；降低嵌合引物的浓度有利于加入更多对嵌合引物，提高多重PCR检测通量；采用单条公共引物，有利于减少二聚体的形成，高浓度有利于扩增特异性产物的量，进而提高检测敏感性；(2) When the kit disclosed in the present invention is used for detection, the concentration of chimeric primers is reduced to a minimum of 8nM/each, and the concentration of common primers is 1600nM; reducing the concentration of chimeric primers is conducive to adding more pairs of chimeric primers and increasing multiplexing. PCR detection throughput; using a single public primer is beneficial to reduce the formation of dimers, and high concentrations are beneficial to amplify the amount of specific products, thereby improving detection sensitivity;

（3）本发明公开的试剂盒采用一个PCR反应中的两步退火温度法，前10个循环扩增采用高退火温度（61℃）有利于嵌合引物的特异性结合，减少非特异性产物；后30个循环扩增低退火温度（54℃）使公共引物发挥放大作用；中间额外增加72℃延伸2 min，有利于为公共引物获得足够的模板；(3) The kit disclosed in the present invention adopts a two-step annealing temperature method in a PCR reaction, and the first 10 cycles of amplification adopt a high annealing temperature (61°C), which is conducive to the specific binding of chimeric primers and reduces non-specific products; The low annealing temperature (54°C) for the last 30 cycles of amplification enables the common primer to play a role in amplification; an additional extension of 72°C for 2 min in the middle is beneficial to obtain enough template for the common primer;

（4）本发明公开的试剂盒中，公共引物与每条多重PCR特异引物的5' 末端连接，一起构成了嵌合引物；公共引物介导的多重PCR可以降低引物对的浓度，减少二聚体的形成，从而提高多重PCR的检测通量；通过创造性的设计克服了现有多重PCR体系存在的非特异性扩增、易存在二聚体的缺陷；(4) In the kit disclosed in the present invention, the common primer is connected to the 5' end of each multiplex PCR-specific primer to form a chimeric primer; the multiplex PCR mediated by the common primer can reduce the concentration of primer pairs and reduce dimerization It can improve the detection throughput of multiplex PCR; it overcomes the defects of non-specific amplification and easy existence of dimers in the existing multiplex PCR system through creative design;

（5）本发明的多重PCR检测体系特异性高，敏感性高达0.1%（w/w）(转基因大豆成分含量)，即0.1ng/100ngDNA样品；单管进行，操作简单，且具有灵敏度高、经济、快速等优点，具有实际应用价值；(5) The multiplex PCR detection system of the present invention has high specificity, and the sensitivity is as high as 0.1% (w/w) (transgenic soybean component content), that is, 0.1ng/100ng DNA sample; it is performed in a single tube, the operation is simple, and it has high sensitivity, It has the advantages of economy and speed, and has practical application value;

（6）本发明公开的多重PCR(mutiplex PCR)在一个PCR管中同时检测多个靶标分子，比southern杂交、基因芯片方法更加简便快捷，比荧光定量PCR更经济，与恒温扩增技术（包括环介导等温扩增法）相比不易污染及高通量。由于该技术所具有的高效性、敏感性和经济简便性等优点，可以在核酸检测技术中受到广泛关注。(6) The multiplex PCR (mutiplex PCR) disclosed in the present invention simultaneously detects multiple target molecules in one PCR tube, which is simpler and faster than southern hybridization and gene chip methods, and more economical than fluorescence quantitative PCR. Loop-mediated isothermal amplification method) is less prone to contamination and high-throughput. Due to the advantages of high efficiency, sensitivity and economic simplicity, this technology can be widely concerned in nucleic acid detection technology.

附图说明Description of drawings

图1是本发明公共引物的特异性检测电泳图；Fig. 1 is the specificity detection electrophoresis figure of the public primer of the present invention;

图2是本发明公共引物敏感性结果图；Fig. 2 is the public primer sensitivity result graph of the present invention;

图3是本发明多重体系的特异性检测的电泳结果图；Fig. 3 is the electrophoresis result figure of the specificity detection of multiple system of the present invention;

图4是本发明检测转基因大豆测序结果图；4 is a diagram showing the sequencing results of the detection of transgenic soybeans according to the present invention;

图5是本发明多重检测体系的敏感性结果图。Figure 5 is a graph of the sensitivity results of the multiplex detection system of the present invention.

具体实施方式Detailed ways

下面结合实施例与附图对本发明作进一步的描述The present invention will be further described below in conjunction with the embodiments and the accompanying drawings

实施例Example

1. 公共引物的筛选及其检测的敏感性1. Screening of public primers and the sensitivity of their detection

公共引物作为整个技术体系中的核心技术，其质量好坏直接影响着技术的成败。本发明所采用的公共引物SEQ ID NO.1与转基因大豆和非转基因大豆基因组DNA进行PCR扩增，在退火温度梯度45℃--65℃范围内均不能扩增特异产物，如图1，a-k为公共引物与转基因大豆和非转基因大豆基因组DNA扩增结果，结果显示，公共引物均不能扩增特异产物，其中，泳道0为空白对照，泳道M为100bp DNA Marker，泳道1-8为退火温度梯度45℃--65℃；a模板为非转基因大豆基因组DNA，b模板为GTS 40-3-2基因组DNA，c模板为A2704-12基因组DNA，d模板为MON89788基因组DNA，e模板为DP356043基因组DNA，f模板为DP305423基因组DNA，g模板为CV127-9基因组DNA，h模板为MON87701基因组DNA，i模板为A5547-127基因组DNA，j模板为MON87708基因组DNA，k模板为MON87769基因组DNA。As the core technology of the whole technical system, the quality of public primers directly affects the success or failure of the technology. The public primer SEQ ID NO.1 used in the present invention performs PCR amplification with the genomic DNA of transgenic soybean and non-transgenic soybean, and can not amplify specific products in the range of annealing temperature gradient of 45°C--65°C, as shown in Figure 1, a-k are the amplification results of the public primers and the genomic DNA of transgenic soybean and non-transgenic soybean. The results show that the public primers cannot amplify the specific products.Lane 0 is the blank control, lane M is the 100bp DNA Marker, and lanes 1-8 are the annealing temperatures Gradient 45℃--65℃; a template is non-transgenic soybean genomic DNA, b template is GTS 40-3-2 genomic DNA, c template is A2704-12 genomic DNA, d template is MON89788 genomic DNA, e template is DP356043 genomic DNA DNA, f template is DP305423 genomic DNA, g template is CV127-9 genomic DNA, h template is MON87701 genomic DNA, i template is A5547-127 genomic DNA, j template is MON87708 genomic DNA, and k template is MON87769 genomic DNA.

通过构建的重组质粒模板，进一步检测公共引物SEQ ID NO.1检测的敏感性，其检测敏感性达到100个拷贝，如图2，泳道 M 为100bp DNA marker，泳道0为空白对照，泳道1-8模板对应的质粒拷贝数依次为10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸。Through the constructed recombinant plasmid template, the sensitivity of the public primer SEQ ID NO.1 was further tested, and its detection sensitivity reached 100 copies, as shown in Figure 2, lane M is a 100bp DNA marker,lane 0 is a blank control, lane 1- The plasmid copy numbers corresponding to the 8 templates were 10¹ , 10² , 10³ , 10⁴ , 10⁵ , 10⁶ , 10⁷ , and 10⁸ in sequence.

本发明中，当以转基因大豆和非转基因大豆基因组DNA为模板进行扩增时，不论扩增条件如何优化，均应没有扩增产物；当带有公共引物的多重嵌合引物有扩增产物时，通过公共引物一定可以扩增出特异性产物。In the present invention, when transgenic soybean and non-transgenic soybean genomic DNA are used as templates for amplification, no matter how the amplification conditions are optimized, there should be no amplification products; when the multiple chimeric primers with common primers have amplification products , specific products can be amplified by common primers.

2. 检测转基因大豆的多重PCR试剂盒，包括常规多重PCR组件，还包括针对这10种转基因大豆的嵌合引物和公共引物；嵌合引物和公共引物序列见表1，常规多重PCR组件包括Taq 聚合酶、dNTP、MgCl₂、PCR缓冲液、去离子水、阳性对照引物对；阳性对照引物对可用来检测DNA模板质量和反应体系扩增效率，具体序列为：SEQ ID NO.19：GCCCTCTACTCCACCCCCATCC、2. The multiplex PCR kit for detecting transgenic soybeans includes conventional multiplex PCR components, as well as chimeric primers and public primers for these 10 types of transgenic soybeans; the sequences of chimeric primers and public primers are shown in Table 1, and the conventional multiplex PCR components include Taq Polymerase, dNTP, MgCl₂ , PCR buffer, deionized water, positive control primer pair; the positive control primer pair can be used to detect the quality of the DNA template and the amplification efficiency of the reaction system, the specific sequence is: SEQ ID NO. 19: GCCCTCTACTCCACCCCCATCC,

SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG。SEQ ID NO. 20: GCCCATCTGCAAGCCTTTTTGTG.

表1 针对我国进口的12种转基因大豆嵌合引物和公共引物Table 1 Chimeric primers and public primers for 12 transgenic soybeans imported from my country

3. 多重体系的特异性检测 3. Specific detection of multiplex systems

采用多引物+公共引物+单模板的PCR方法，扩增各种检测转基因大豆的基因组或靶标基因，DNA模板均采用提取的相对应的10种转基因大豆基因组DNA，浓度约为25ng/μL。多重PCR采用的25μL反应体系包括：10μM的公共引物4μL，不同终浓度（SEQ ID NO.2/3:80nM，SEQ ID NO.4/5: 60nM，SEQ ID NO.6/7: 64nM，SEQ ID NO.8/9: 8nM，SEQ ID NO.10:32nM，SEQ ID NO.11: 48nM，SEQ ID NO.12: 16nM，SEQ ID NO.13/14: 24nM，SEQ IDNO.15/16: 8nM，SEQ ID NO.17: 16nM, SEQ ID NO.18: 32nM）的嵌合引物混合物2μL，2×Dream Taq Green Mix 12.5μL，DNA模板 1μL，灭菌ddH₂O 5.5μL；PCR反应条件为：95℃预变性 5min；95℃ 变性30s，61℃退火 30s，72℃ 延伸30s，共10个循环；72℃ 延伸2min；95℃变性30s，54℃退火 30s，72℃ 延伸30s，共30个循环；最后72℃延伸7min。图3为多重体系的特异性检测的电泳结果图，其中0为空白对照，N为阴性对照，1-10泳道是PCR产物的琼脂糖凝胶电泳结果，依次为MON87701、DP356043、DP305423、CV127、MON87769、A2704、GTS40-3-2、MON87708、MON89788、A5547，N为阴性对照，M为DNA标准分子量（从下到上目的带大小依次为100、200、300、400、500、600、700、800、900和1000bp），PM为产物marker，条带大小见图3上标示；结果显示，运用本发明的多重体系检测靶标DNA，可以从相应模板中扩增出预期大小的特异性PCR产物，且条带清晰。扩增产物经进一步测序，证明为相对应的转基因大豆检测序列（图4）。本部分的实验结果表明，本发明的多重体系具有高特异性。The multi-primer + public primer + single-template PCR method was used to amplify the genomes or target genes of various detection transgenic soybeans. The DNA templates were all extracted from the corresponding 10 kinds of transgenic soybean genomic DNA, and the concentration was about 25ng/μL. The 25 μL reaction system used in multiplex PCR includes: 4 μL of 10 μM public primers, different final concentrations (SEQ ID NO.2/3: 80nM, SEQ ID NO.4/5: 60nM, SEQ ID NO.6/7: 64nM, SEQ ID NO. ID NO.8/9: 8nM, SEQ ID NO.10:32nM, SEQ ID NO.11: 48nM, SEQ ID NO.12: 16nM, SEQ ID NO.13/14: 24nM, SEQ ID NO.15/16: 8nM, SEQ ID NO.17: 16nM, SEQ ID NO.18: 32nM) chimeric primer mix 2μL, 2×Dream Taq Green Mix 12.5μL, DNA template 1μL, sterilized ddH₂ O 5.5μL; PCR reaction conditions are: : Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30s, annealing at 61°C for 30s, extension at 72°C for 30s, a total of 10 cycles; extension at 72°C for 2min; denaturation at 95°C for 30s, annealing at 54°C for 30s, extension at 72°C for 30s, a total of 30 cycles Cycle; final extension at 72°C for 7 min. Figure 3 shows the electrophoresis results of the specificity detection of the multiplex system, in which 0 is the blank control, N is the negative control, and lanes 1-10 are the agarose gel electrophoresis results of PCR products, followed by MON87701, DP356043, DP305423, CV127, MON87769, A2704, GTS40-3-2, MON87708, MON89788, A5547, N is the negative control, M is the DNA standard molecular weight (the size of the target band from bottom to top is 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 bp), PM is the product marker, and the band size is indicated in Figure 3; the results show that using the multiplex system of the present invention to detect the target DNA, the specific PCR product of the expected size can be amplified from the corresponding template, And the strips are clear. The amplified product was further sequenced and proved to be the corresponding transgenic soybean detection sequence (Figure 4). The experimental results in this section show that the multiplex system of the present invention has high specificity.

4. 多重检测体系的敏感性检测4. Sensitivity testing of multiple testing systems

体系敏感性检测采用10种转基因大豆A2704-12、MON89788、CV127、MON87701、A5547-127、MON87708、MON87769、DP356043、DP305423、GTS 40-3-2基因组DNA溶液等体积混合（其中 DP356043、DP305423和GTS40-3-2初始浓度为2.5ng/μL，其余7种初始浓度为25ng/μL），然后将此混合基因组DNA溶液采用TE缓冲液进行10倍梯度稀释，依次获得10、100、1000及10000倍基因组DNA稀释混合液，每种稀释液取4μL作为模板（每种转基因大豆基因组DNA模板浓度分别为10ng、1ng、0.1ng、0.01ng，而DP356043、DP305423、 GTS40-3-2这3种相对应浓度分别为1ng、0.1ng、0.01ng、0.001ng），分别对10个靶标基因进行模板梯度PCR反应，ddH₂O为模板作为阴性对照。多重PCR采用25μL反应体系包括：10μM的公共引物4μL，1μM针对相应品种转基因大豆的嵌合引物对混合物2μL，2×Dream Taq Green Mix 12.5μL，DNA模板4μL，灭菌ddH₂O 2.5μL；PCR反应条件为：95℃预变性 5min；95℃ 变性30s，61℃退火 30s，72℃ 延伸30s，共10个循环；72℃ 延伸2min；95℃ 变性30s，54℃退火 30s，72℃ 延伸30s，共30个循环；最后72℃延伸7min。PCR反应产物通过2%琼脂糖凝胶进行电泳，凝胶成像系统分析结果如图5，泳道M为100bpDNA Ladder，泳道0为去离子水为模板的空白阴性对照，1～4分别为基因组DNA模板分别为稀释10000、1000、100及10倍10种转基因大豆基因组DNA稀释混合液。结果显示，本发明建立的公共引物介导的多重PCR的敏感性可检测稀释1000倍10种转基因大豆基因组DNA稀释混合液，体系敏感性超过0.1%，优于国家对于转基因食品检测标准要求。The sensitivity of the system was tested using 10 kinds of transgenic soybean A2704-12, MON89788, CV127, MON87701, A5547-127, MON87708, MON87769, DP356043, DP305423, GTS 40-3-2 genomic DNA solutions mixed in equal volume (including DP356043, DP305423 and GTS40 -3-2 The initial concentration is 2.5ng/μL, and the initial concentration of the remaining 7 species is 25ng/μL), and then this mixed genomic DNA solution is diluted 10 times with TE buffer to obtain 10, 100, 1000 and 10000 times successively. Genomic DNA dilution mixture, take 4 μL of each dilution as template (concentrations of genomic DNA template of each transgenic soybean are 10ng, 1ng, 0.1ng, 0.01ng, respectively, while DP356043, DP305423, GTS40-3-2 are corresponding The concentrations were 1ng, 0.1ng, 0.01ng, and 0.001ng respectively), and template gradient PCR reactions were performed on 10 target genes respectively, and ddH₂ O was used as the template as a negative control. The multiplex PCR used a 25 μL reaction system including: 4 μL of 10 μM public primer, 2 μL of 1 μM chimeric primer pair mixture for the corresponding variety of transgenic soybean, 12.5 μL of 2×Dream Taq Green Mix, 4 μL of DNA template, sterilized ddH₂ O 2.5 μL; PCR The reaction conditions were: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 61 °C for 30 s, extension at 72 °C for 30 s, a total of 10 cycles; extension at 72 °C for 2 min; denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, A total of 30 cycles; a final extension at 72°C for 7 min. The PCR reaction products were electrophoresed on a 2% agarose gel. The analysis results of the gel imaging system are shown in Figure 5. Lane M is a 100bp DNA Ladder,lane 0 is a blank negative control with deionized water as a template, and 1 to 4 are genomic DNA templates, respectively. Dilution mixture of 10 kinds of transgenic soybean genomic DNA by 10000, 1000, 100 and 10 times respectively. The results show that the sensitivity of the public primer-mediated multiplex PCR established in the present invention can detect the dilution mixture of 10 kinds of genetically modified soybean genomic DNA diluted 1000 times, and the sensitivity of the system exceeds 0.1%, which is better than the national requirements for the detection of genetically modified food.

SEQUENCE LISTINGSEQUENCE LISTING

<110> 苏州大学<110> Soochow University

<120>一种转基因大豆检测用多重PCR试剂盒及检测方法<120> A multiplex PCR kit for detection of genetically modified soybean and detection method

<160> 20<160> 20

<210> 1<210> 1

<211> 17<211> 17

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 1<400> 1

CTCGTCAACTCCGCAAG 17CTCGTCAACTCCGCAAG 17

<210> 2<210> 2

<211> 42<211> 42

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 2<400> 2

CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG 42CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG 42

<210> 3<210> 3

<211> 40<211> 40

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 3<400> 3

CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT 40CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT 40

<210> 4<210> 4

<211> 37<211> 37

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 4<400> 4

CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG 37CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG 37

<210> 5<210> 5

<211> 39<211> 39

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 5<400> 5

CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG 39CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG 39

<210> 6<210> 6

<211>42<211>42

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 6<400> 6

CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA 42CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA 42

<210> 7<210> 7

<211> 42<211> 42

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 7<400> 7

CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA 42CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA 42

<210> 8<210> 8

<211> 41<211> 41

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 8<400> 8

CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG 41CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG 41

<210> 9<210> 9

<211> 39<211> 39

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 9<400> 9

CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT 39CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT 39

<210> 10<210> 10

<211> 43<211> 43

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 10<400> 10

CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG 43CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG 43

<210> 11<210> 11

<211> 40<211> 40

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 11<400> 11

CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC 40CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC 40

<210> 12<210> 12

<211> 42<211> 42

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 12<400> 12

CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG 42CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG 42

<210> 13<210> 13

<211> 39<211> 39

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 13<400> 13

CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC 39CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC 39

<210> 14<210> 14

<211> 39<211> 39

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 14<400> 14

CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT 39CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT 39

<210> 15<210> 15

<211> 42<211> 42

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 15<400> 15

CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC 42CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC 42

<210> 16<210> 16

<211>42<211>42

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 16<400> 16

CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT 42CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT 42

<210> 17<210> 17

<211> 43<211> 43

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 17<400> 17

CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG 43CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG 43

<210> 18<210> 18

<211> 36<211> 36

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 18<400> 18

CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG 36CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG 36

<210> 19<210> 19

<211> 22<211> 22

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 19<400> 19

GCCCTCTACTCCACCCCCATCC 22GCCCTCTACTCCACCCCCATCC 22

<210> 20<210> 20

<211> 23<211> 23

<212> DNA<212> DNA

<213> 人工合成<213> Synthetic

<400> 20<400> 20

GCCCATCTGCAAGCCTTTTTGTG 23GCCCATCTGCAAGCCTTTTTGTG 23

Claims (5)

Translated fromChinese

1.一种转基因大豆检测用多重PCR试剂盒，其特征在于：所述转基因大豆检测用多重PCR试剂盒包括公共引物、针对转基因大豆的嵌合引物；所述公共引物的序列为SEQ IDNO.1；所述针对转基因大豆的嵌合引物为SEQ ID NO.2至SEQ ID NO.18所示的核苷酸序列：1. A multiplex PCR kit for the detection of transgenic soybean, characterized in that: the multiplex PCR kit for the detection of the transgenic soybean comprises a public primer, a chimeric primer for the transgenic soybean; the sequence of the public primer is SEQ ID NO.1 ; The chimeric primer for transgenic soybean is the nucleotide sequence shown in SEQ ID NO.2 to SEQ ID NO.18:SEQ ID NO.1：CTCGTCAACTCCGCAAGSEQ ID NO. 1: CTCGTCAACTCCGCAAGSEQ ID NO.2：CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTGSEQ ID NO. 2: CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTGSEQ ID NO.3：CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCTSEQ ID NO. 3: CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCTSEQ ID NO.4：CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAGSEQ ID NO. 4: CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAGSEQ ID NO.5：CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTGSEQ ID NO. 5: CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTGSEQ ID NO.6：CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTASEQ ID NO. 6: CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTASEQ ID NO.7：CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTASEQ ID NO. 7: CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTASEQ ID NO.8：CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATGSEQ ID NO. 8: CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATGSEQ ID NO.9：CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGTSEQ ID NO. 9: CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGTSEQ ID NO.10：CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGGSEQ ID NO. 10: CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGGSEQ ID NO.11：CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGCSEQ ID NO. 11: CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGCSEQ ID NO.12：CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTGSEQ ID NO. 12: CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTGSEQ ID NO.13：CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGCSEQ ID NO. 13: CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGCSEQ ID NO.14：CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGTSEQ ID NO. 14: CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGTSEQ ID NO.15：CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAACSEQ ID NO. 15: CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAACSEQ ID NO.16：CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGTSEQ ID NO. 16: CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGTSEQ ID NO.17：CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATGSEQ ID NO. 17: CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATGSEQ ID NO.18：CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG。SEQ ID NO. 18: CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG.2.根据权利要求1所述转基因大豆检测用多重PCR试剂盒，其特征在于：所述转基因大豆检测用多重PCR试剂盒还包括Taq 聚合酶、dNTP、MgCl₂、PCR缓冲液、去离子水、阳性对照引物对。2. The multiplex PCR kit for detection of transgenic soybean according to claim 1, is characterized in that: the multiplex PCR kit for detection of said transgenic soybean also comprises Taq polymerase, dNTP, MgCl₂ , PCR buffer, deionized water, Positive control primer pair.3.根据权利要求2所述转基因大豆检测用多重PCR试剂盒，其特征在于：所述阳性对照引物对的序列为SEQ ID NO.19、SEQ ID NO.20；3. The multiplex PCR kit for detection of transgenic soybean according to claim 2, wherein the sequence of the positive control primer pair is SEQ ID NO.19 and SEQ ID NO.20;SEQ ID NO.19：GCCCTCTACTCCACCCCCATCCSEQ ID NO. 19: GCCCTCTACTCCACCCCCATCCSEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG。SEQ ID NO. 20: GCCCATCTGCAAGCCTTTTTGTG.4.转基因大豆检测用引物，其特征在于：所述转基因大豆检测用引物包括公共引物、针对转基因大豆的嵌合引物；所述公共引物的序列为SEQ ID NO.1；所述针对转基因大豆的嵌合引物为SEQ ID NO.2至SEQ ID NO.18所示的核苷酸序列：4. A primer for detection of transgenic soybean, characterized in that: said primer for detection of transgenic soybean comprises a public primer and a chimeric primer for transgenic soybean; the sequence of said public primer is SEQ ID NO. Chimeric primers are the nucleotide sequences shown in SEQ ID NO.2 to SEQ ID NO.18:SEQ ID NO.1：CTCGTCAACTCCGCAAGSEQ ID NO. 1: CTCGTCAACTCCGCAAGSEQ ID NO.2：CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTGSEQ ID NO. 2: CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTGSEQ ID NO.3：CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCTSEQ ID NO. 3: CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCTSEQ ID NO.4：CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAGSEQ ID NO. 4: CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAGSEQ ID NO.5：CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTGSEQ ID NO. 5: CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTGSEQ ID NO.6：CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTASEQ ID NO. 6: CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTASEQ ID NO.7：CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTASEQ ID NO. 7: CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTASEQ ID NO.8：CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATGSEQ ID NO. 8: CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATGSEQ ID NO.9：CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGTSEQ ID NO. 9: CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGTSEQ ID NO.10：CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGGSEQ ID NO. 10: CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGGSEQ ID NO.11：CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGCSEQ ID NO. 11: CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGCSEQ ID NO.12：CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTGSEQ ID NO. 12: CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTGSEQ ID NO.13：CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGCSEQ ID NO. 13: CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGCSEQ ID NO.14：CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGTSEQ ID NO. 14: CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGTSEQ ID NO.15：CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAACSEQ ID NO. 15: CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAACSEQ ID NO.16：CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGTSEQ ID NO. 16: CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGTSEQ ID NO.17：CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATGSEQ ID NO. 17: CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATGSEQ ID NO.18：CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG。SEQ ID NO. 18: CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG.5.权利要求4所述转基因大豆检测用引物在制备检测转基因大豆的多重PCR试剂盒中的应用；所述转基因大豆为GTS-40-3-2、MON87701、MON89788、MON87701×MON89788、CV127-9、A2704-12、DP356043、DP305423、A5547、305423×40-3-2、MON87769、MON87708。5. The application of the primers for detecting transgenic soybeans according to claim 4 in the preparation of a multiplex PCR kit for detecting transgenic soybeans; the transgenic soybeans are GTS-40-3-2, MON87701, MON89788, MON87701×MON89788, CV127-9 , A2704-12, DP356043, DP305423, A5547, 305423×40-3-2, MON87769, MON87708.

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