WO2015067025A1 - Dna chip suitable for high-throughput detection of transgenic product - Google Patents

Dna chip suitable for high-throughput detection of transgenic product Download PDF

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WO2015067025A1
WO2015067025A1 PCT/CN2014/077945 CN2014077945W WO2015067025A1 WO 2015067025 A1 WO2015067025 A1 WO 2015067025A1 CN 2014077945 W CN2014077945 W CN 2014077945W WO 2015067025 A1 WO2015067025 A1 WO 2015067025A1
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dna
genetically modified
detection
probe
transgenic
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PCT/CN2014/077945
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Chinese (zh)
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杨立桃
陶生策
张大兵
邵宁
蒋诗萌
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上海交通大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a detection method in the field of bioengineering technology, in particular to a DNA chip suitable for high-throughput detection of transgenic products.
  • GMOs Genetically ly Modified Organisms
  • the genetically modified organism itself must be a biological individual that can propagate or transmit genetic material.
  • GM0 includes all life forms of GMOs. In the narrow sense, it refers to transgenic plants and transgenic animals. Because transgenic animals are not commercialized except for ornamental fish and pets, they are strictly referred to as transgenic plants (Li Quanfen et al., 2011, Chinese Animal Husbandry and Veterinary Medicine, 38, 152).
  • China's GM crop planting area ranks sixth in the world.
  • China's current commercial GM crops are only Bt cotton for textile materials, with an area of 4 million hectares, with an adoption rate of 80%.
  • Commercial varieties of flowers have a major impact on the Chinese market. These two varieties received a biosafety certificate issued by the Ministry of Agriculture in 2009 and are conducting field trials. Planting Bt rice will increase production by 8%, which can increase domestic food and bring about nearly 4 billion US dollars in economic income.
  • about 75% of rice is affected by pests, while genetically modified varieties can avoid pests.
  • transgenic phytase corn hopes to commercialize transgenic phytase corn, followed by glyphosate-resistant corn and Bt corn.
  • the field trial of transgenic phytase corn has entered the third phase.
  • transgenic wheat resistant to yellow mosaic virus species is in the field trial stage, 7 years from the last unapproved GM wheat, and other varieties are also in the development stage.
  • the detection methods of genetically modified products mainly include protein detection methods (mainly immunoassay) and collision detection methods (mainly PCR techniques).
  • Protein detection methods Genetically modified products can be detected by immunological and physicochemical techniques.
  • the enzyme-linked immunosorbent assay is the most widely used method. It relies on a specific antibody-binding chromogenic system to detect the target protein (antigen).
  • the biggest limitation of this method is that it can only be tested on primary processed or unprocessed, single-protein transgenic products (van Dui jn G; et al, 2002, AOAC Int, 85, 787-791), and is compatible with DNA detection methods.
  • the development is slower than two reasons: 1 The cost of producing specific antibodies is high; 2 The preparation of specific antibodies is more complicated than oligonucleotides.
  • DNA detection method mainly using PCR technology for detection.
  • PCR plays a leading role in the detection of genetically modified products and is widely used by global traders, food processing companies and law enforcement agencies.
  • the genetic modification of GM0 is mainly DNA modification
  • DNA detection is the highest level of detection of GM0 in terms of detection relative to transcriptional level (RNA), translational level (protein), and phenotypic level (biological identification).
  • the main processes of PCR detection include: 1 detection of DNA extraction in the sample; 2 PCR amplification; 3 amplification product detection.
  • traditional single-plex PCR has not been able to meet the ever-increasing detection requirements of the number and complexity of GM0 worldwide, and has also promoted the birth of new methods.
  • Real-time fluorescent PCR multi-target analysis method is to add a fluorescent substance based on qualitative PCR. Once the template is amplified, the fluorescent signal is collected once, and the fluorescence signal increases proportionally with the number of amplifications. The whole fluorescence is monitored in real time through accumulated fluorescence. The PCR process, finally quantitative analysis of the template by standard curve, without the need for agarose gel electrophoresis identification.
  • Real-time fluorescent quantitative PCR includes the SYBR Green fluorescent dye method and the TaqMan probe method, among which the TaqMan probe method is more commonly used (Querci M, et al, 2009, Food Anal. Methods, 2, 238-335). The disadvantage of this type of test is that it requires a matching instrument and is not used on a large scale.
  • Multiplex PCR-capillary electrophoresis is a PCR reaction in which two or more primers and two or more DNA templates are simultaneously amplified in a PCR reaction tube, and multiple target genes can be simultaneously detected.
  • Capillary electrophoresis is a type of liquid in which a gel or a buffer is filled in a capillary, a high-voltage electric field is used as a driving force, and a capillary is used as a separation channel to separate the enthalpy and distribution behavior between components in the sample. Phase separation technology.
  • the liquid phase chip technology combines flow cytometry and enzyme label detection technology to obtain more than 100 kinds of fluorescent combined spheres by blending the ratio of the two fluorescent dyes.
  • Each fluorescent combination sphere is labeled with a target DNA sequence.
  • the bound probe (Fantozzi A, et al, 2008, Food Anal. Methods, 1, 10-17), the detection result can be obtained by detecting the fluorescence signal and the sample signal of the microsphere after the probe is combined with the sample.
  • microspheres can be combined with any primer/probe combination to better accommodate high-throughput detection, but the shortcomings of liquid-phase chips are still in further research and the need to purchase expensive liquid-phase chip instruments.
  • Solid phase chip technology is a highly integrated DNA detection technology.
  • the known DNA oligonucleotide probe is arranged on a slide or a silicon wafer by using a special automatic device, and then the DNA to be tested is hybridized with the chip probe, and the corresponding hybridization signal is generated by the DNA corresponding to the target.
  • This allows the purpose of detecting multiple target DNAs in one experiment, with high throughput, high sensitivity, and high degree of automation.
  • the invention is a solid phase DNA chip technology, and has the unique advantages such as the experimental principle, the simple operation, the short time required, the low cost, and the low dependence on expensive equipment; compared with the above several methods; The increase can effectively solve the problem of simultaneous detection of a large number of transgenic targets.
  • An object of the present invention is to provide a DNA chip suitable for high-throughput detection of transgenic products, which improves detection efficiency and detection accuracy, reduces detection cost, and shortens detection time.
  • the DNA chip is suitable for high-throughput detection of global transgenic products and can detect about 97% of transgenic target sequences currently covered.
  • the invention relates to a DNA chip suitable for high-throughput detection of a transgenic product, wherein the DNA chip has a specific touch probe capable of detecting 97 transgenic targets, and the DNA probe is SEQ ID NO. ⁇ The nucleic acid sequence shown in SEQ ID NO.
  • an amino linker of 10 to 30 T bases is attached to the 5' end of the DNA probe core base.
  • the purpose of such a connection is to effectively connect and fix the probe to the carrier.
  • 15 T base amino link arms are attached to the 5' end of the DNA probe core base.
  • the DNA probe is specifically complementary to a PCR amplification product containing a transgenic component.
  • the carrier is a slide, a silicon wafer, a nitrocellulose membrane, a nylon membrane or polystyrene.
  • the invention also relates to a kit for high throughput detection of transgenic agricultural products, the kit comprising the DNA core described above.
  • the invention has the beneficial effects of: improving detection efficiency and detection accuracy, reducing detection cost, shortening detection time, and specifically hybridizing with amplification products of various transgenic target sequences, and the invention can simultaneously detect 97 transgenic targets
  • the sequence which covers approximately 97% of the target sequences of transgenic products, can be applied to the detection and labeling of various transgenic products, and as an important technical support for the quantitative experiments of genetically modified components and the safety management of genetically modified products.
  • Figure 1 is a schematic diagram of probe distribution patterns of 97 transgenic targets for detecting a touch chip
  • Figure 2 is a schematic diagram showing the results of hybridization of four kinds of transgenic soybean mixed samples in the laboratory
  • Figure 3 is a schematic diagram showing the results of hybridization of 8 kinds of transgenic rice mixed samples in the laboratory. detailed description
  • the invention utilizes biochip technology to fix a specific DNA probe capable of detecting 97 transgenic targets on a carrier such as a slide, a silicon wafer, a membrane or a polymer material to form a larger density (200/microscope slide). DNA probe array.
  • a specific probe hybridization method on the surface of the vector is used to detect the vast majority of exogenous inserts that may be present in the transgenic product. Specific steps are as follows:
  • GMDD GMO detection database, GMDD
  • GM Crop Database GM Crop Database
  • patent databases at home and abroad, a total of 97 target sequences of transgenic events and their commonly used transgenic components, which are widely distributed in the world, are analyzed and analyzed, among which 55 species are involved. 44 kinds of components, 8 endogenous genes, and compiled detailed information of the above 44 kinds of elements contained in 55 event target sequences, for analysis and verification of subsequent probe hybridization results.
  • UV-targeted probe After synthesis, it is purified by reverse column, and UV-targeted probe: a routine PCR is performed using the genomic template of the target sequence, and the product is diluted 1000 times after amplification, and then the universal primer is used for two-round PCR, and the amplified product and the designed product are designed. Oligonucleotide probes are hybridized to screen for probes with strong signal and specificity.
  • Probe preparation The oligonucleotide probe was dissolved in lOumol/L with 50% DMS0 spotting solution, and the oligonucleotide probe was spotted onto the aldehyde-treated glass slide using a gene chip spotter. The probes were repeatedly sampled 4 times to verify the repeatability of the test results. See Figure 1 for the spotting method. In the figure, there are 97 X 2 X 2 on the slide (4 times for each point, 2 for each row) The points are the same probe) the target sequence probe, the upper part, from left to right, in order; the lower part, for Right to left in order. The white point is the probe, the gray dot is blank, and the outer gray dot is landmark.
  • the prepared probe chip was fixed in a wet box at 37 ° C overnight, fixed after washing with 0.2% SDS for 3 min, washed with deionized water and then blocked solution (new configuration of 0.3% NaBH 4 solution + 1 X PBS) +25% absolute ethanol) was blocked for 5 min to remove free aldehyde groups, rinsed thoroughly with water, and finally centrifuged or blown dry with nitrogen.
  • the prepared DNA chip is stored at a normal temperature and in a dry place.
  • 3 uL of the fluorescently labeled PCR product was added to the 30 uL hybridization reaction system.
  • the components of the hybridization buffer include: 20 x SSC, 50 X Denhardt's reagent (production, Shanghai) and 0.2% SDS (w/v).
  • the hybridization solution was denatured at 95 °C for 5 min, then immediately placed on ice and quenched, ice bath 3 mir! .
  • a crystal core multi-sample chip cover slip (Beijing Boao Biotechnology Co., Ltd.) was used, and 30 uL of the treated sample was added to the DNA microarray through the sample well, and then placed in a wet box and reacted at 55 ° C for 3 h.
  • the chips were washed with the cleaning solution I and the cleaning solution I I, and slowly shaken for 5 minutes each time.
  • the cleaning solution I was 20 X SSC, 2% SDS, and preheated at 42 °C.
  • the cleaning solution II is 0.2% SDS. It is then rinsed with deionized water, dried by centrifugation or blown dry with nitrogen.
  • Chip scanner using GenePix TM 4200A Scanner (Molecular Devices, USA) and obtain image data, and scans channels using the 635 nm 532 nm, scanning resolution 5um, PMT, respectively 350 and 300, power is 100%, the scanning 1- 2 times.
  • the chip scan result is compared with the previously collated event and the component contains detailed information to judge the chip detection.
  • Designing the appropriate primers and probes is the key to accurate GM0 detection. Designed specific primers and probes for the 97 target sequences (41 commonly used inserts, 48 common events and 8 endogenous genes) contained in the current transgenic products. The designed primers were specifically verified by conventional PCR. Multiple probes were designed to be screened using genomic templates to determine the most accurate primers and probes.
  • the seed powders of the four transgenic soybean events (A2704-12, A5547-127, GTS40-3-2 and M0N89788) in the laboratory were extracted and purified using a commercial DNA extraction kit, and then NanoDrop was used. 1000 The measured concentration of the collision was uniformly diluted to 10 ng/ul, and each of the four templates was thoroughly mixed as a sample to be tested.
  • the chip PCR reaction was carried out using a hydrophobic microporous chip technique. That is, the gene chip spotter will be designed in advance.
  • the primers were inserted into the micropores of the chip, and the PCR reaction system was introduced by the drainage method, thereby performing the first round of chip PCR reaction.
  • the amplification products in the micropores are collected by centrifugation (50 ul of deionized water is added before centrifugation to facilitate the removal of part of the PCR product), and 2 ul is taken to the second round of the common PCR system, and the amplified primers are universal primers.
  • the purpose is to re-amplify each target segment in parallel for detection and analysis.
  • Probe chip detection hybridization detection using an on-chip oligonucleotide probe and an amplification product.
  • the hybridization system is configured, including hybridization buffer, amplification product, etc., and the hybridization solution is denatured at 95 °C for 5 min, then immediately placed on ice and quenched, ice bath 3 mir! .
  • a core multi-sample chip cover slip (Beijing Boao Biotechnology Co., Ltd.) was used, and 20 uL of the treated hybrid sample was added to the DNA microarray through the sample well, and then placed in a wet box and reacted at 55 ° C for 3 h.
  • the chips are washed with the cleaning solution I and the cleaning solution II, respectively, and shaken slowly for 5 minutes each time.
  • the cleaning solution I was 20 X SSC, 2% SDS, and preheated at 42 °C.
  • the cleaning solution II is 0.2% SDS. It is then rinsed with deionized water, dried by centrifugation or blown dry with nitrogen.
  • the probe signals in the hybridization diagram are respectively number: 1, 3, 6, 8, 11, 12, 29, 30, 34, 74, 75, 76, 77, 78 And 92, the corresponding target names are: P-CaMV35s, bla, T-nos, Pat, CP4 epsps, CTP2, CMoVb, E9, P-FMV/TSFU A2704-12, A5547-127, GTS40-3-2
  • the theoretical four events and the soybean endogenous signal were completely detected, and the contained component signals were also completely consistent, except for the hybridization of signals 3, 29 and 30.
  • the signal is weak, and the probe map is adjusted to be grayscale and the display is not obvious.
  • the hybridization results are highly consistent, and compared with the theoretical values, the contained events and endogenous information are 100% accurate, and the contained component information is also highly specific and accurate.

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Abstract

The present invention provides a DNA chip suitable for high-throughput detection of a transgenic product. A specific DNA probe capable of detecting 97 transgenic targets is fixed on a carrier of the DNA chip, and the nucleic acid sequences of the DNA probe are shown by SEQ ID NO. 1 to SEQ ID NO. 97.

Description

适用于转基因产品高通量检测的 DNA芯片 技术领域  DNA chip for high-throughput detection of genetically modified products
本发明涉及生物工程技术领域的检测方法， 具体涉及一种适用于转基因产品高通量检 测的 DNA芯片。 转基因生物 （Genetical ly Modified Organisms, GMOs) 是指一类生物有机体， 其遗 传物质的改变不是通过自然条件下的相互杂交或自然整合方式实现的。 转基因生物本身必 须是一个可进行繁殖或传递遗传物质的生物学个体。 广义上 GM0包括所有生命形式在内的 转基因生物， 狭义上指转基因植物和转基因动物， 又因为转基因动物除了观赏鱼和宠物外 还没有商品化， 所以严格意义上是指转基因植物 （李全芬等， 2011， 中国畜牧兽医， 38， 152 )。  The invention relates to a detection method in the field of bioengineering technology, in particular to a DNA chip suitable for high-throughput detection of transgenic products. Genetically ly Modified Organisms (GMOs) refer to a class of biological organisms whose genetic alterations are not achieved by mutual hybridization or natural integration under natural conditions. The genetically modified organism itself must be a biological individual that can propagate or transmit genetic material. In a broad sense, GM0 includes all life forms of GMOs. In the narrow sense, it refers to transgenic plants and transgenic animals. Because transgenic animals are not commercialized except for ornamental fish and pets, they are strictly referred to as transgenic plants (Li Quanfen et al., 2011, Chinese Animal Husbandry and Veterinary Medicine, 38, 152).
自从 1994年， 第一例转基因事件一转基因番茄 FLAVR SAVR的出现， 到 1996年孟山都 的转基因大豆 Roundup Ready被商业化以来， 全球转基因植物面积每年以两位数的百分比 迅速增加。据 ISAAA数据统计， 截止 2012年底， 全球转基因作物种植面积达到 1. 703亿公 顷， 比 2011年的 1. 6亿公顷增长了 6%， 即 1030万公顷。 从 1996年至 2011年， 转基因作 物通过以下方式为粮食安全、 可持续性以及气候变化做出贡献： 增加价值 982亿美元的农 作物产量； 节省 4. 73亿公斤的杀虫剂， 更好地维护了环境； 通过节省 1. 087亿公顷土地， 保护了生物多样性； 改善了超过 1500万小型农户即超过 5000万人口的生计。 美国仍是转 基因作物种植国中的领先者， 而加拿大的转基因油菜种植面积最大， 近几年巴西的转基因 种植发展也十分迅速， 种植 3660万公顷， 同比增长 21%。 2012年 28个种植转基因作物的 国家中， 20个发展中国家转基因作物的种植面积 （占全球的 52% ) 超过了 8个发达国家的 转基因作物种植面积 （占全球的 48%)  Since 1994, the first genetically modified event, the emergence of the transgenic tomato FLAVR SAVR, has been rapidly increasing in double-digit percentages per year since the commercialization of Monsanto's genetically modified soybean Roundup Ready in 1996. According to ISAAA statistics, by the end of 2012, the global GM crop planting area reached 170.3 billion hectares, an increase of 6% from the 160 million hectares in 2011, or 10.3 million hectares. From 1996 to 2011, GM crops contributed to food security, sustainability and climate change by: increasing crop yields by $98.2 billion; saving 473 million kg of pesticides for better maintenance The environment; protected biodiversity by saving 108.87 million hectares of land; improved livelihoods for more than 15 million small farmers, or more than 50 million people. The United States is still the leader in GM crop-growing countries, while Canada's GM canola is the largest. In recent years, Brazil's GM planting has also grown rapidly, planting 36.6 million hectares, up 21% year-on-year. Of the 28 countries that have planted GM crops in 2012, the area of GM crops in 20 developing countries (52% of the world) exceeds the area of GM crops in 8 developed countries (48% of the world).
截止 2012年， 中国的转基因作物种植面积位居全球第六， 我国目前商业化种植的转基 因作物仅有用于纺织材料的 Bt棉花， 种植面积达 400万公顷， 采用率为 80%， 平均每个农 民种植 0. 5公顷的 Bt棉花。 有关专家预测， Bt水稻和转基因植酸酶玉米可能成为继 Bt棉 花的商业化品种， 并对中国市场产生重大影响。这两个品种在 2009年就获得了农业部颁发 的生物安全证书， 并正在开展田间试验。 种植 Bt水稻将增加 8%的产量， 既可以增补国内 粮食也可以带来近 40亿美元的经济收入目前约 75%的水稻受到虫害影响， 而转基因品种能 避免虫害。 总部位于北京的 Origin Agritech公司希望将转基因植酸酶玉米商业化，继而是 抗草甘膦玉米和 Bt玉米， 目前转基因植酸酶玉米环境田间测试已经进入第三阶段。 目前， 抗黄色花叶病毒品种的转基因小麦正处在田间试验阶段， 距上一个未被批准的转基因小麦 已有 7年， 其他特性的品种也处于发展阶段。 As of 2012, China's GM crop planting area ranks sixth in the world. China's current commercial GM crops are only Bt cotton for textile materials, with an area of 4 million hectares, with an adoption rate of 80%. Planting 0.5 hectares of Bt cotton. Experts predict that Bt rice and transgenic phytase corn may become Bt cotton Commercial varieties of flowers have a major impact on the Chinese market. These two varieties received a biosafety certificate issued by the Ministry of Agriculture in 2009 and are conducting field trials. Planting Bt rice will increase production by 8%, which can increase domestic food and bring about nearly 4 billion US dollars in economic income. Currently, about 75% of rice is affected by pests, while genetically modified varieties can avoid pests. Beijing-based Origin Agritech hopes to commercialize transgenic phytase corn, followed by glyphosate-resistant corn and Bt corn. The field trial of transgenic phytase corn has entered the third phase. At present, transgenic wheat resistant to yellow mosaic virus species is in the field trial stage, 7 years from the last unapproved GM wheat, and other varieties are also in the development stage.
随着转基因作物的进一步推广和普及， 其生物安全问题已成为全球关注的热点。 有关 安全性的问题主要分为生态环境安全和食品安全性两个方面， 其中转基因食品的安全性至 今也没有定论。 公众的焦点忧虑有： 表型性状的改变； 营养成分的变化及抗营养因子的出 现（如蛋白酶抑制剂、 脂肪氧化酶的产生或含量的变化）； 出现新的过敏原； 天然有毒物的 产生（如茄碱、 葫芦素、 a-番茄素等）； 致病性， 即转基因植物产品食用后是否会治病； 标 记基因和报告基因的安全性等 （聂呈荣等， 2003， 生态学杂志， 22， 43-44)。 我国目前虽 然尚未发生严重的转基因食品安全性事件， 但在转基因安全性， 以及生物安全性问题上确 实存在着诸多不稳定因素亟待解决。。而对众多转基因事件和元件的快速， 高效检测则是转 基因作物安全监控的关键一步。 目前， 转基因产品的检测方法主要有蛋白质检测方法 (主要 是免疫分析法)和碰检测方法（主要是 PCR技术）。  With the further promotion and popularization of GM crops, its biosafety issue has become a hot spot of global concern. The issues related to safety are mainly divided into two aspects: ecological environment safety and food safety. The safety of genetically modified foods is still inconclusive. Public concerns include: changes in phenotypic traits; changes in nutrients and the emergence of anti-nutritional factors (such as protease inhibitors, production of lipoxygenase or changes in content); emergence of new allergens; production of natural toxicants (such as solanine, cucurbitacin, a-tomatonin, etc.); pathogenicity, that is, whether transgenic plant products will cure after consumption; safety of marker genes and reporter genes, etc. (Nie Chengrong et al., 2003, Journal of Ecology, 22 , 43-44). Although serious GM food safety incidents have not yet occurred in China, there are indeed many unstable factors in the safety of genetically modified organisms and biosafety issues that need to be resolved. . The rapid and efficient detection of many transgenic events and components is a key step in the safety monitoring of GM crops. At present, the detection methods of genetically modified products mainly include protein detection methods (mainly immunoassay) and collision detection methods (mainly PCR techniques).
蛋白质检测方法： 转基因产品可应用免疫和物理化学技术进行检测， 其中酶联免疫吸 附法应用最广泛， 它是依赖特异性抗体结合显色***对目的蛋白（抗原)进行检测。 该方法 最大的局限就是只能针对初步加工或未经加工、成分单一蛋白质转基因产品进行检测（van Dui jn G; et al ， 2002, AOAC Int, 85， 787-791 )， 且与 DNA检测方法相比发展较为缓慢， 原因有 2个方面： ①制作特异性抗体的成本高； ②特异性抗体的制备比寡核苷酸要复杂。 近年来， 蛋白检测方法逐步发展了一些其他方法， 如还有免疫磁性电化学传感器测定法 (Volpe G, et al , 2006, Anal Lett, 39， 1599-1601)、 二维电泳测定法（Kim V H， et al , 2006, Microbio Biotechnol , 16， 26-30)、 质谱测定法（Ocana M F， et al , 2007, Rapid Commun Mass Spectrom, 21 , 321-326)等， 但这些方法设备昂贵， 检测成本较高。  Protein detection methods: Genetically modified products can be detected by immunological and physicochemical techniques. The enzyme-linked immunosorbent assay is the most widely used method. It relies on a specific antibody-binding chromogenic system to detect the target protein (antigen). The biggest limitation of this method is that it can only be tested on primary processed or unprocessed, single-protein transgenic products (van Dui jn G; et al, 2002, AOAC Int, 85, 787-791), and is compatible with DNA detection methods. The development is slower than two reasons: 1 The cost of producing specific antibodies is high; 2 The preparation of specific antibodies is more complicated than oligonucleotides. In recent years, protein detection methods have gradually developed other methods, such as immunomagnetic electrochemical sensor assays (Volpe G, et al, 2006, Anal Lett, 39, 1599-1601), two-dimensional electrophoresis assays (Kim VH). , et al, 2006, Microbio Biotechnol, 16, 26-30), mass spectrometry (Ocana MF, et al, 2007, Rapid Commun Mass Spectrom, 21, 321-326), etc., but these methods are expensive and the cost of detection is higher. high.
DNA检测方法：主要是应用 PCR技术进行检测。 PCR在转基因产品检测中占据主导地位， 被全球贸易商、 食品加工企业和执法机关广泛采用。 GM0的遗传修饰主要是 DNA修饰， 所 以相对于转录水平 (RNA)、翻译水平（蛋白）和表型水平（生物学鉴定法）的检测方法来说， DNA 检测是检测 GM0的最高水平。 PCR检测的主要过程包括： ①检测样品中 DNA的提取； ② PCR 扩增； ③扩增产物检测。 但是随着商业化转基因作物的多样性不断增加， 传统的单重 PCR 已不能满足全球范围内 GM0数量和复杂度不断增加的检测要求， 同时也促进了新方法的诞 生。 如实时荧光 PCR多靶分析方法、 多重 PCR—毛细管电泳法、液相、 固相芯片技术等（李 全芬等， 2011， 中国畜牧兽医， 38， 154-155 )。 DNA detection method: mainly using PCR technology for detection. PCR plays a leading role in the detection of genetically modified products and is widely used by global traders, food processing companies and law enforcement agencies. The genetic modification of GM0 is mainly DNA modification, DNA detection is the highest level of detection of GM0 in terms of detection relative to transcriptional level (RNA), translational level (protein), and phenotypic level (biological identification). The main processes of PCR detection include: 1 detection of DNA extraction in the sample; 2 PCR amplification; 3 amplification product detection. However, as the diversity of commercial GM crops continues to increase, traditional single-plex PCR has not been able to meet the ever-increasing detection requirements of the number and complexity of GM0 worldwide, and has also promoted the birth of new methods. Such as real-time fluorescent PCR multi-target analysis methods, multiplex PCR-capillary electrophoresis, liquid phase, solid phase chip technology, etc. (Li Quanfen et al., 2011, Chinese Animal Husbandry and Veterinary Medicine, 38, 154-155).
实时荧光 PCR多靶分析方法， 是在定性 PCR基础上加入一种荧光物质， 模板每扩增一 次， 收集一次荧光信号， 荧光信号随着扩增次数成比例地增加， 通过累积的荧光实时监测 整个 PCR过程， 最后通过标准曲线对模板进行定量分析， 无需进行琼脂糖凝胶电泳鉴定。 实时荧光定量 PCR包括 SYBR Green荧光染料法和 TaqMan探针法等， 其中 TaqMan探针法更 为常用 ( Querci M， et al， 2009, Food Anal. Methods , 2, 238-335 )。 此种检测方法的 缺点是需要配套仪器且未大规模应用。  Real-time fluorescent PCR multi-target analysis method is to add a fluorescent substance based on qualitative PCR. Once the template is amplified, the fluorescent signal is collected once, and the fluorescence signal increases proportionally with the number of amplifications. The whole fluorescence is monitored in real time through accumulated fluorescence. The PCR process, finally quantitative analysis of the template by standard curve, without the need for agarose gel electrophoresis identification. Real-time fluorescent quantitative PCR includes the SYBR Green fluorescent dye method and the TaqMan probe method, among which the TaqMan probe method is more commonly used (Querci M, et al, 2009, Food Anal. Methods, 2, 238-335). The disadvantage of this type of test is that it requires a matching instrument and is not used on a large scale.
多重 PCR—毛细管电泳法， 多重 PCR是在一个 PCR反应管中同时加 2对以上引物和 2 种以上 DNA模板进行扩增的 PCR反应， 能同时检测多种靶基因。 毛细管电泳是在毛细管中 填入凝胶或缓冲液， 以高压电场为驱动力， 以毛细管为分离通道， 依据样品中各组分之间 淌度和分配行为上的差异而实现分离的一类液相分离技术。二者并用是 GM0检测的新趋势， 但多次实验发现， 待检样品超过 10种时易出现假阳性结果 ( Chaouachi M， et al , 2008, Agric. Food Chem. , 56， 11596-11606 )， 方法有待进一步完善。  Multiplex PCR-capillary electrophoresis. Multiplex PCR is a PCR reaction in which two or more primers and two or more DNA templates are simultaneously amplified in a PCR reaction tube, and multiple target genes can be simultaneously detected. Capillary electrophoresis is a type of liquid in which a gel or a buffer is filled in a capillary, a high-voltage electric field is used as a driving force, and a capillary is used as a separation channel to separate the enthalpy and distribution behavior between components in the sample. Phase separation technology. The combination of the two is a new trend of GM0 detection, but many experiments have found that more than 10 samples to be tested are prone to false positive results (Chaouachi M, et al, 2008, Agric. Food Chem., 56, 11596-11606), The method needs to be further improved.
液相芯片技术， 结合了流式细胞技术和酶标检测技术， 通过调配 2种荧光染料的比例 获得 100多种荧光组合的小球， 每个荧光组合小球都标记有一种和靶 DNA序列进行结合的 探针 (Fantozzi A, et al , 2008 , Food Anal. Methods, 1， 10-17 )， 在探针与样品结合 后检测微球的荧光信号和样品信号即可得到检测结果。 理论上微球可以结合任何引物 /探 针组合来更好地适应高通量检测的需求， 但液相芯片的缺点是还在进一步的研究中， 且需 要购买昂贵的液相芯片仪器。  The liquid phase chip technology combines flow cytometry and enzyme label detection technology to obtain more than 100 kinds of fluorescent combined spheres by blending the ratio of the two fluorescent dyes. Each fluorescent combination sphere is labeled with a target DNA sequence. The bound probe (Fantozzi A, et al, 2008, Food Anal. Methods, 1, 10-17), the detection result can be obtained by detecting the fluorescence signal and the sample signal of the microsphere after the probe is combined with the sample. In theory, microspheres can be combined with any primer/probe combination to better accommodate high-throughput detection, but the shortcomings of liquid-phase chips are still in further research and the need to purchase expensive liquid-phase chip instruments.
固相芯片技术， 是一种高度集成的 DNA检测技术。 采用专用自动化设备将已知 DNA寡 核苷酸探针排列在玻片或硅片上， 然后将待测样品即 DNA与芯片探针杂交， 有相对应靶标 的 DNA就会出现相应的杂交信号， 这样就可以一次实验检测多个靶标 DNA的目的， 具有高 通量、 灵敏性高、 自动化程度高等特点。 本发明为固相 DNA芯片技术， 相比较上述几种方法， 有其特有的优点如， 实验原理、 操作简单， 所需时间短、 成本低廉， 对昂贵设备依赖性小； 随着转基因产品的不断增加， 可以有效地解决了大量转基因靶标同时检测的问题。 Solid phase chip technology is a highly integrated DNA detection technology. The known DNA oligonucleotide probe is arranged on a slide or a silicon wafer by using a special automatic device, and then the DNA to be tested is hybridized with the chip probe, and the corresponding hybridization signal is generated by the DNA corresponding to the target. This allows the purpose of detecting multiple target DNAs in one experiment, with high throughput, high sensitivity, and high degree of automation. The invention is a solid phase DNA chip technology, and has the unique advantages such as the experimental principle, the simple operation, the short time required, the low cost, and the low dependence on expensive equipment; compared with the above several methods; The increase can effectively solve the problem of simultaneous detection of a large number of transgenic targets.
发明内容 Summary of the invention
本发明的目的在于提供一种提高检测效率和检测准确性， 降低检测成本， 缩短检测时 间的适用于转基因产品高通量检测的 DNA芯片。 该 DNA芯片适用于全球转基因产品高通量 检测， 可以检测目前涵盖约 97%的转基因靶标序列。  SUMMARY OF THE INVENTION An object of the present invention is to provide a DNA chip suitable for high-throughput detection of transgenic products, which improves detection efficiency and detection accuracy, reduces detection cost, and shortens detection time. The DNA chip is suitable for high-throughput detection of global transgenic products and can detect about 97% of transgenic target sequences currently covered.
本发明的目的是通过以下技术方案来实现的：  The object of the present invention is achieved by the following technical solutions:
本发明涉及一种适用于转基因产品高通量检测的 DNA芯片，所述 DNA芯片的载体 上固 定有可检测 97个转基因靶标的特异性碰探针，所述 DNA探针为 SEQ ID NO. 1〜SEQ ID NO. 97 所示的核酸序列。  The invention relates to a DNA chip suitable for high-throughput detection of a transgenic product, wherein the DNA chip has a specific touch probe capable of detecting 97 transgenic targets, and the DNA probe is SEQ ID NO. ~ The nucleic acid sequence shown in SEQ ID NO.
优选地， 在所述 DNA探针核心碱基的 5 ' 端连接了 10〜30个 T碱基的氨基连接臂。 这 样连接目的是为了有效地将探针连接和固定在载体上，  Preferably, an amino linker of 10 to 30 T bases is attached to the 5' end of the DNA probe core base. The purpose of such a connection is to effectively connect and fix the probe to the carrier.
进一步优选地， 在所述 DNA探针核心碱基的 5 ' 端连接了 15个 T碱基的氨基连接臂。 优选地， 所述 DNA探针可与含有转基因成分的 PCR扩增产物特异性互补结合。  Further preferably, 15 T base amino link arms are attached to the 5' end of the DNA probe core base. Preferably, the DNA probe is specifically complementary to a PCR amplification product containing a transgenic component.
优选地， 所述载体为载玻片、 硅片、 硝酸纤维素膜、 尼龙膜或聚苯乙烯。  Preferably, the carrier is a slide, a silicon wafer, a nitrocellulose membrane, a nylon membrane or polystyrene.
本发明还涉及一种用于高通量检测转基因农产品的试剂盒，所述试剂盒包括上述的 DNA 心片。  The invention also relates to a kit for high throughput detection of transgenic agricultural products, the kit comprising the DNA core described above.
本发明具有的有益效果为： 可提高检测效率和检测准确性， 降低检测成本， 缩短检测 时间， 能与多种转基因靶标序列的扩增产物进行特异性杂交， 本发明可同时检测 97种转基 因靶标序列， 基本涵盖了约 97%的转基因产品的靶标序列， 可适用于各种转基因产品的检 测和标识， 以及作为转基因成分定量实验、 制定转基因产品安全管理的重要技术支持。 附图说明  The invention has the beneficial effects of: improving detection efficiency and detection accuracy, reducing detection cost, shortening detection time, and specifically hybridizing with amplification products of various transgenic target sequences, and the invention can simultaneously detect 97 transgenic targets The sequence, which covers approximately 97% of the target sequences of transgenic products, can be applied to the detection and labeling of various transgenic products, and as an important technical support for the quantitative experiments of genetically modified components and the safety management of genetically modified products. DRAWINGS
通过阅读参照以下附图对非限制性实施例所作的详细描述， 本发明的其它特征、 目的和优点将会变得更明显：  Other features, objects, and advantages of the present invention will become apparent from the Detailed Description of Description
图 1为 97种转基因靶标检测碰芯片的探针分布模式图；  Figure 1 is a schematic diagram of probe distribution patterns of 97 transgenic targets for detecting a touch chip;
图 2 为实验室自配的 4种转基因大豆混合样品的杂交结果示意图；  Figure 2 is a schematic diagram showing the results of hybridization of four kinds of transgenic soybean mixed samples in the laboratory;
图 3 为实验室自配的 8种转基因水稻混合样品的杂交结果示意图。 具体实施方式 Figure 3 is a schematic diagram showing the results of hybridization of 8 kinds of transgenic rice mixed samples in the laboratory. detailed description
下面结合附图和具体实施例对本发明进行详细说明。 以下实施例将有助于本领域的技 术人员进一步理解本发明， 但不以任何形式限制本发明。 应当指出的是， 对本领域的普通 技术人员来说， 在不脱离本发明构思的前提下， 还可以做出若干调整和改进。 这些都属于 本发明的保护范围。  The invention will be described in detail below with reference to the drawings and specific embodiments. The following examples are intended to further understand the invention, but are not intended to limit the invention in any way. It should be noted that a number of adjustments and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of protection of the present invention.
本发明利用生物芯片技术在一种载体， 如载玻片、 硅片、 膜或高分子材料上固定可检 测 97个转基因靶标的特异性 DNA探针， 形成较大密度 （200/microscope slide ) 的 DNA探 针阵列。 用载体表面的特异性碰探针杂交方法来检测转基因产品中可能存在的绝大多数 外源***片段。 具体步骤如下：  The invention utilizes biochip technology to fix a specific DNA probe capable of detecting 97 transgenic targets on a carrier such as a slide, a silicon wafer, a membrane or a polymer material to form a larger density (200/microscope slide). DNA probe array. A specific probe hybridization method on the surface of the vector is used to detect the vast majority of exogenous inserts that may be present in the transgenic product. Specific steps are as follows:
1、 常用转基因靶标序列的分析整理  1. Analysis and sorting of commonly used transgenic target sequences
通过 GMDD (GMO detection database , GMDD) , GM Crop Database和国内外的专利数 据库等， 分析整理出了目前广泛存在于全球种植的转基因事件和其常用的转基因元件共计 97个靶标序列， 其中事件 55种， 元件 44种， 内源基因 8种， 并整理出 55个事件靶标序 列中含有的上述 44种元件的详细信息， 用于后续探针杂交结果的分析验证。  Through GMDD (GMO detection database, GMDD), GM Crop Database and patent databases at home and abroad, a total of 97 target sequences of transgenic events and their commonly used transgenic components, which are widely distributed in the world, are analyzed and analyzed, among which 55 species are involved. 44 kinds of components, 8 endogenous genes, and compiled detailed information of the above 44 kinds of elements contained in 55 event target sequences, for analysis and verification of subsequent probe hybridization results.
1、 DNA芯片的设计和制备  1. Design and preparation of DNA chips
( 1 ) 探针设计、 筛选和制备  (1) Probe design, screening and preparation
针对上述 97个靶标序列的扩增产物， 设计特异性的寡核苷酸探针， 其中， 事件序列的 产物由于是品系特异性的引物扩增产物， 只设计了一种探针， 对于元件和内源基因的探针， 分别设计了 2-4对， 探针片段大小为 （25 ± 5 ) bp, 探针合成时在核心碱基的 5 ' 端连接了 15个 T碱基的氨基连接臂， 即 5 ' -NH2- (T ) 15 -核心碱基。 合成后用反向柱纯化， 紫外定 探针筛选： 利用靶标序列的基因组模板进行一轮常规 PCR， 扩增后将产物稀释 1000倍 再利用通用引物进行二轮 PCR， 扩增产物与所设计的寡核苷酸探针进行杂交， 筛选出信号 与特异性强的探针。 Designing specific oligonucleotide probes for the amplification products of the above 97 target sequences, wherein the product of the event sequence is a strain-specific primer amplification product, only one probe is designed for the component and The probes of the endogenous gene were designed with 2-4 pairs, and the probe fragment size was (25 ± 5 ) bp. The probe was synthesized with 15 T base amino linkers at the 5 ' end of the core base. , ie 5 ' -NH 2 - (T ) 15 -core base. After synthesis, it is purified by reverse column, and UV-targeted probe: a routine PCR is performed using the genomic template of the target sequence, and the product is diluted 1000 times after amplification, and then the universal primer is used for two-round PCR, and the amplified product and the designed product are designed. Oligonucleotide probes are hybridized to screen for probes with strong signal and specificity.
探针制备： 寡核苷酸探针用 50%的 DMS0点样液溶解到 lOumol/L, 用基因芯片点样仪将 寡核苷酸探针点到经醛基化处理的载玻片上， 每个探针重复点样 4次， 以验证检测结果的 可重复性， 点样方式见附图 1， 图中， 玻片上有 97 X 2 X 2 (每个点重复 4次， 每一排的两 个点为同一个探针） 靶标序列探针， 上半部分， 从左到右， 按顺序排列； 下半部分， 为从 右到左按顺序排列。 白点即为探针， 灰点带圈为空白， ***灰点为 landmark Probe preparation: The oligonucleotide probe was dissolved in lOumol/L with 50% DMS0 spotting solution, and the oligonucleotide probe was spotted onto the aldehyde-treated glass slide using a gene chip spotter. The probes were repeatedly sampled 4 times to verify the repeatability of the test results. See Figure 1 for the spotting method. In the figure, there are 97 X 2 X 2 on the slide (4 times for each point, 2 for each row) The points are the same probe) the target sequence probe, the upper part, from left to right, in order; the lower part, for Right to left in order. The white point is the probe, the gray dot is blank, and the outer gray dot is landmark.
( 2 ) 探针芯片前处理  (2) Probe chip pre-processing
制备好的探针芯片在 37°C湿盒中过夜固定， 固定后用 0. 2%SDS洗 3min， 去离子水清洗 后用封闭液（新配置的 0. 3%NaBH4溶液 +1 X PBS+25%无水乙醇）封闭 5min， 以去除自由醛基， 再用水充分冲洗， 最后离心干燥或氮气吹干。 制备好的 DNA芯片位于常温、 干燥处保存。 The prepared probe chip was fixed in a wet box at 37 ° C overnight, fixed after washing with 0.2% SDS for 3 min, washed with deionized water and then blocked solution (new configuration of 0.3% NaBH 4 solution + 1 X PBS) +25% absolute ethanol) was blocked for 5 min to remove free aldehyde groups, rinsed thoroughly with water, and finally centrifuged or blown dry with nitrogen. The prepared DNA chip is stored at a normal temperature and in a dry place.
( 3 ) 杂交与杂交后处理  (3) Hybridization and post-hybridization
30uL杂交反应体系中加入 3uL荧光标记的 PCR产物。杂交缓冲液的组分包括： 20 x SSC, 50 X Denhardt ' s reagent (生工，上海）和 0. 2%SDS (w/v)。将杂交液于 95 °C变性处理 5 min, 然后即刻置于冰上骤冷， 冰浴 3 mir！。使用晶芯多样品芯片盖片（北京博奧生物有限公司）， 并通过加样孔将 30uL处理好的样品加入到 DNA微阵列， 然后置于湿盒， 55 °C反应 3h。  3 uL of the fluorescently labeled PCR product was added to the 30 uL hybridization reaction system. The components of the hybridization buffer include: 20 x SSC, 50 X Denhardt's reagent (production, Shanghai) and 0.2% SDS (w/v). The hybridization solution was denatured at 95 °C for 5 min, then immediately placed on ice and quenched, ice bath 3 mir! . A crystal core multi-sample chip cover slip (Beijing Boao Biotechnology Co., Ltd.) was used, and 30 uL of the treated sample was added to the DNA microarray through the sample well, and then placed in a wet box and reacted at 55 ° C for 3 h.
反应完毕， 将芯片分别使用清洗液 I和清洗液 I I进行清洗， 每次 5 min， 缓慢晃 动。 其中， 清洗液 I为 20 X SSC, 2%SDS, 42°C预热。 清洗液 II为 0. 2%SDS。 然后用去离子 清洗， 再离心干燥或氮气吹干。  After the reaction was completed, the chips were washed with the cleaning solution I and the cleaning solution I I, and slowly shaken for 5 minutes each time. Among them, the cleaning solution I was 20 X SSC, 2% SDS, and preheated at 42 °C. The cleaning solution II is 0.2% SDS. It is then rinsed with deionized water, dried by centrifugation or blown dry with nitrogen.
( 4) 扫描和结果分析  (4) Scan and result analysis
使用芯片扫描仪 GenePixTM4200A Scanner (Molecular Devices, USA)获得图像和数据， 扫描通道使用 635 nm和 532 nm, 扫描分辨率为 5um， PMT分别为 350和 300， power均为 100%， 扫描 1-2次。 将芯片扫描结果与先前整理的事件、 元件包含详细信息进行比较， 判 断芯片检测。 Chip scanner using GenePix TM 4200A Scanner (Molecular Devices, USA) and obtain image data, and scans channels using the 635 nm 532 nm, scanning resolution 5um, PMT, respectively 350 and 300, power is 100%, the scanning 1- 2 times. The chip scan result is compared with the previously collated event and the component contains detailed information to judge the chip detection.
2、 结合实施例对本发明的探针芯片进行进一步说明：  2. The probe chip of the present invention is further described in conjunction with the embodiments:
实施例 1  Example 1
设计合适的引物和探针是准确进行 GM0检测的关键。 针对目前转基因产品中主要含有 的 97种靶标序列（41种常用***元件， 48种常见事件和 8种内源基因）， 设计特异性的引 物和探针， 设计的引物用常规 PCR进行特异性验证， 设计的多个探针用基因组模板进行筛 选， 最终确定了最为准确的引物和探针。  Designing the appropriate primers and probes is the key to accurate GM0 detection. Designed specific primers and probes for the 97 target sequences (41 commonly used inserts, 48 common events and 8 endogenous genes) contained in the current transgenic products. The designed primers were specifically verified by conventional PCR. Multiple probes were designed to be screened using genomic templates to determine the most accurate primers and probes.
将实验室已有的 4种转基因大豆事件（A2704-12 , A5547-127, GTS40-3-2和 M0N89788 ) 的种子粉末， 利用商业化的 DNA提取试剂盒进行 DNA的提取和纯化， 再利用 NanoDrop 1000 测出碰的浓度， 并统一稀释到 10ng/ul， 4种模板各取 lOul充分混合作为待测样品。  The seed powders of the four transgenic soybean events (A2704-12, A5547-127, GTS40-3-2 and M0N89788) in the laboratory were extracted and purified using a commercial DNA extraction kit, and then NanoDrop was used. 1000 The measured concentration of the collision was uniformly diluted to 10 ng/ul, and each of the four templates was thoroughly mixed as a sample to be tested.
利用亲疏水微孔芯片技术进行芯片 PCR反应。 即事先用基因芯片点样仪将设计好的 97 对引物点入芯片微孔中， 引流法加入 PCR反应体系， 从而进行第一轮的芯片 PCR反应。 待 反应结束后通过离心方式收集微孔中扩增产物 （离心前加入 50ul 去离子水便于取出部分 PCR产物）， 取出 2ul加入到第二轮的普通 PCR体系， 该轮扩增的引物为通用引物， 目的是 对各目标片段进行平行地再放大， 以便于检测和分析。 The chip PCR reaction was carried out using a hydrophobic microporous chip technique. That is, the gene chip spotter will be designed in advance. The primers were inserted into the micropores of the chip, and the PCR reaction system was introduced by the drainage method, thereby performing the first round of chip PCR reaction. After the reaction is completed, the amplification products in the micropores are collected by centrifugation (50 ul of deionized water is added before centrifugation to facilitate the removal of part of the PCR product), and 2 ul is taken to the second round of the common PCR system, and the amplified primers are universal primers. The purpose is to re-amplify each target segment in parallel for detection and analysis.
探针芯片检测， 利用芯片上寡核苷酸探针与扩增产物进行杂交检测。 配置杂交体系， 包括杂交缓冲液、 扩增产物等， 杂交液于 95 °C变性处理 5 min, 然后即刻置于冰上骤冷， 冰浴 3 mir！。 使用晶芯多样品芯片盖片 （北京博奧生物有限公司）， 并通过加样孔将 20uL 处理好的杂交样品加入到 DNA微阵列， 然后置于湿盒， 55 °C反应 3h。 反应完毕， 将芯片分 别使用清洗液 I和清洗液 II进行清洗，每次 5 min,缓慢晃动。其中，清洗液 I为 20 X SSC, 2%SDS, 42°C预热。 清洗液 II为 0. 2%SDS。 然后用去离子清洗， 再离心干燥或氮气吹干。  Probe chip detection, hybridization detection using an on-chip oligonucleotide probe and an amplification product. The hybridization system is configured, including hybridization buffer, amplification product, etc., and the hybridization solution is denatured at 95 °C for 5 min, then immediately placed on ice and quenched, ice bath 3 mir! . A core multi-sample chip cover slip (Beijing Boao Biotechnology Co., Ltd.) was used, and 20 uL of the treated hybrid sample was added to the DNA microarray through the sample well, and then placed in a wet box and reacted at 55 ° C for 3 h. After the reaction is completed, the chips are washed with the cleaning solution I and the cleaning solution II, respectively, and shaken slowly for 5 minutes each time. Among them, the cleaning solution I was 20 X SSC, 2% SDS, and preheated at 42 °C. The cleaning solution II is 0.2% SDS. It is then rinsed with deionized water, dried by centrifugation or blown dry with nitrogen.
扫描和结果分析， 使用芯片扫描仪 GenePixTM4200A Scanner (Molecular Devices, USA) 获得图像和数据， 扫描通道使用 635 nm和 532 nm, 扫描分辨率为 5um， PMT分别为 350 和 300， power均为 100%， 扫描 1_2次。 将芯片扫描结果如附图 2。 由图 2， 并结合模式图 1可知， 杂交图中有探针信号的分别是序号： 1、 3、 6、 8、 11、 12、 29、 30、 34、 74、 75、 76、 77、 78和 92， 其对应的靶标名称分别是： P- CaMV35s、 bla、 T- nos、 Pat、 CP4 epsps, CTP2 、 CMoVb、 E9、 P-FMV/TSFU A2704-12 , A5547-127, GTS40-3-2的 5 ' 和 3 ' 端、 M0N89788 以及大豆内源基因 ZeCi /7。 根据已知的 4种转基因大豆事件， 并査找相关数据库和文献得 出， 理论上的 4种事件和大豆内源信号完全检测出， 所含元件信号也完全符合， 除信号 3、 29和 30杂交信号较弱， 探针图调整为灰度图后显示不明显外。 对于一个样品的两次重复， 杂交结果高度吻合， 且与理论值比较， 所含事件和内源信息 100%准确， 所含元件信息也有 极高的特异性和准确性。 Scanning and analysis of results using a chip scanner GenePix TM 4200A Scanner (Molecular Devices, USA) and obtain image data, and scans channels using the 635 nm 532 nm, scanning resolution 5um, PMT, respectively 350 and 300, power is 100 %, scan 1_2 times. The chip scan results are shown in Figure 2. 2, and in conjunction with the mode diagram 1, it can be seen that the probe signals in the hybridization diagram are respectively number: 1, 3, 6, 8, 11, 12, 29, 30, 34, 74, 75, 76, 77, 78 And 92, the corresponding target names are: P-CaMV35s, bla, T-nos, Pat, CP4 epsps, CTP2, CMoVb, E9, P-FMV/TSFU A2704-12, A5547-127, GTS40-3-2 The 5' and 3' ends, M0N89788 and the soybean endogenous gene Z eC i /7. According to the known four genetically modified soybean events, and looking up the relevant database and literature, the theoretical four events and the soybean endogenous signal were completely detected, and the contained component signals were also completely consistent, except for the hybridization of signals 3, 29 and 30. The signal is weak, and the probe map is adjusted to be grayscale and the display is not obvious. For two replicates of a sample, the hybridization results are highly consistent, and compared with the theoretical values, the contained events and endogenous information are 100% accurate, and the contained component information is also highly specific and accurate.
实施例 2  Example 2
利用上述实验过程对 8种转基因水稻（TT51-1、 Kefeng NO. 6、 Kefeng NO. 8、 T2A- 1、 KMD1、 Bar68_l、 tlc_19和 RJ5)混合样进行检测， 芯片扫描结果如附图 3; 由图 3并结合 模式图 1可知， 杂交图中有探针信号的分别是： 1、 2、 3、 6、 8、 15、 19、 20、 21、 24、 26、 28、 29、 33、 39、 81、 82、 83、 84、 85、 86、 87、 88和 95，对应的靶标名称分别是： P- CaMV35s、 bar、 bla、 T_nos、 Pat 、 nptl l、 Pact、 P_Ubi、 P0rbcs、 crylAb/c、 CpTI、 crylC、 CMoVb 、 uidA、 Gus、 TT51-1、 Kefeng NO. 6、 Kefeng NO. 8、 T2A_1、 KMD1、 Bar68_l、 tlc_19、 RJ5 和水稻内源基因 SPs。 根据已知的 8种转基因大豆事件， 并査找相关数据库和文献得出， 理论上的 8个事件和水稻内源信号完全检测出， 元件信号除理论信号完全符合外只多出 8 和 29两个元件信号， 故可以判定该基因芯片具有极高的特异性和准确性。 Using the above experimental procedure, a mixture of 8 transgenic rice (TT51-1, Kefeng NO. 6, Kefeng NO. 8, T2A-1, KMD1, Bar68_l, tlc_19 and RJ5) was tested. The chip scan results are shown in Figure 3; Figure 3 and in conjunction with the mode of Figure 1, the hybrid signal in the probe signal are: 1, 2, 3, 6, 8, 15, 19, 20, 21, 24, 26, 28, 29, 33, 39, 81, 82, 83, 84, 85, 86, 87, 88 and 95, the corresponding target names are: P-CaMV35s, bar, bla, T_nos, Pat, nptl l, Pact, P_Ubi, P0rbcs, crylAb/c, CpTI, crylC, CMoVb, uidA, Gus, TT51-1, Kefeng NO. 6, Kefeng NO. 8, T2A_1, KMD1, Bar68_l, tlc_19, RJ5 And rice endogenous gene S P s. According to the known 8 genetically modified soybean events, and looking up the relevant database and literature, the theoretical 8 events and the rice endogenous signal are completely detected, and the component signals are only 8 and 29 more than the theoretical signals. The component signal can be determined to have extremely high specificity and accuracy.
以上对本发明的具体实施例进行了描述。 需要理解的是， 本发明并不局限于上述特定 实施方式， 本领域技术人员可以在权利要求的范围内做出各种变形或修改， 这并不影响本 发明的实质内容。  The specific embodiments of the present invention have been described above. It is to be understood that the invention is not limited to the specific embodiments described above, and various modifications or changes may be made by those skilled in the art without departing from the scope of the invention.

Claims

权 利 要 求 书 claims
1、 一种适用于转基因产品高通量检测的 DNA芯片， 其特征在于， 所述 DNA芯片的载体 上固定有可检测 97个转基因靶标的特异性 DNA探针，所述 DNA探针为 SEQ ID NO. 1〜SEQ ID NO. 97所示的核酸序列。 1. A DNA chip suitable for high-throughput detection of genetically modified products, characterized in that a specific DNA probe that can detect 97 genetically modified targets is fixed on the carrier of the DNA chip, and the DNA probe is SEQ ID Nucleic acid sequences shown in NO. 1 to SEQ ID NO. 97.
2、 如权利要求 1所述的适用于转基因产品高通量检测的 DNA芯片， 其特征在于， 在所 述 DNA探针核心碱基的 5 ' 端连接了 10〜30个 T碱基的氨基连接臂。 2. The DNA chip suitable for high-throughput detection of genetically modified products as claimed in claim 1, characterized in that an amino linkage of 10 to 30 T bases is connected to the 5' end of the core base of the DNA probe. arm.
3、 如权利要求 2所述的适用于转基因产品高通量检测的 DNA芯片， 其特征在于， 在所 述 DNA探针核心碱基的 5 ' 端连接了 15个 T碱基的氨基连接臂。 3. The DNA chip suitable for high-throughput detection of genetically modified products as claimed in claim 2, characterized in that an amino connecting arm of 15 T bases is connected to the 5' end of the core base of the DNA probe.
4、 如权利要求 1所述的适用于转基因产品高通量检测的 DNA芯片， 其特征在于， 所述 DNA探针可与含有转基因成分的 PCR扩增产物特异性互补结合。 4. The DNA chip suitable for high-throughput detection of genetically modified products as claimed in claim 1, characterized in that the DNA probe can be specifically complementary to the PCR amplification product containing genetically modified components.
5、 如权利要求 1所述的适用于转基因产品高通量检测的 DNA芯片， 其特征在于， 所述 载体为载玻片、 硅片、 硝酸纤维素膜、 尼龙膜或聚苯乙烯。 5. The DNA chip suitable for high-throughput detection of genetically modified products as claimed in claim 1, wherein the carrier is a glass slide, silicon wafer, nitrocellulose membrane, nylon membrane or polystyrene.
6、 一种用于高通量检测转基因农产品的试剂盒， 其特征在于， 所述试剂盒包括权利要 求 1所述的 DNA芯片。 6. A kit for high-throughput detection of genetically modified agricultural products, characterized in that the kit includes the DNA chip of claim 1.
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