CN114657275B - Primer pair combination, kit and detection method for detecting transgenic alfalfa - Google Patents

Abstract

Translated fromChinese

本申请涉及生物技术领域，尤其涉及一种检测转基因苜蓿的引物对组合、试剂盒及检测方法。所述引物对组合其核苷酸序列如SEQ ID NO.1至SEQ ID NO.18所示；通过本申请的引物组合，操作简单，经过一次样品前处理，单管PCR扩增，文库构建与测序就可以同步检测多样品或一个样品中多种转基因成分，具有平行分析和多重判断的特点，大大提升了转基因产品的检测效率，节约了检测成本。

The application relates to the field of biotechnology, in particular to a combination of primers, a kit and a detection method for detecting transgenic alfalfa. The nucleotide sequence of the primer pair combination is shown in SEQ ID NO.1 to SEQ ID NO.18; through the primer combination of the present application, the operation is simple, after a sample pretreatment, single-tube PCR amplification, library construction and Sequencing can simultaneously detect multiple samples or multiple genetically modified components in one sample, which has the characteristics of parallel analysis and multiple judgments, which greatly improves the detection efficiency of genetically modified products and saves detection costs.

Description

Translated fromChinese

一种检测转基因苜蓿的引物对组合、试剂盒及检测方法A primer pair combination, kit and detection method for detecting transgenic alfalfa

技术领域technical field

本申请涉及生物技术领域，尤其涉及一种检测转基因苜蓿的引物对组合、试剂盒及检测方法。The application relates to the field of biotechnology, in particular to a combination of primers, a kit and a detection method for detecting transgenic alfalfa.

背景技术Background technique

苜蓿是一种豆科多年生牧草，具备高营养、高产量、适应力强等特点。同时，苜蓿是一种高蛋白，因蛋白质含量高、含多种矿物元素、维生素和碳水化合物丰富以及能进行生物固氮等优点，在农牧业生产中发挥着重要的作用，具有很高的经济和生态价值。Alfalfa is a leguminous perennial forage with the characteristics of high nutrition, high yield and strong adaptability. At the same time, alfalfa is a kind of high protein, because of its high protein content, rich in various mineral elements, rich in vitamins and carbohydrates, and its ability to carry out biological nitrogen fixation, it plays an important role in agricultural and animal husbandry production and has a high economic value. and ecological value.

传统的栽培方法具有时间长，产量低，成本高的局限性，无法满足现今社会对于育种多元化的需求。随着转基因技术的不断发展，植物转基因技术在苜蓿的遗传改良中具有极高的应用价值，从根本上加大育种进程，提高生产产量以及质量。但是转基因苜蓿被直接或间接地制成食品以及国际社会对转基因产品安全性问题的日益关注，农产品中的转基因成分检测已纳入国内外检验检疫部门的检测项目并逐渐得到加强。因此，开发高效、便捷的转基因食品检测技术显得至关重要。Traditional cultivation methods have the limitations of long time, low yield, and high cost, and cannot meet the needs of today's society for diversified breeding. With the continuous development of transgenic technology, plant transgenic technology has extremely high application value in the genetic improvement of alfalfa, fundamentally increasing the breeding process and improving production yield and quality. However, genetically modified alfalfa is directly or indirectly made into food and the international community is increasingly concerned about the safety of genetically modified products. The detection of genetically modified ingredients in agricultural products has been included in the inspection items of domestic and foreign inspection and quarantine departments and has been gradually strengthened. Therefore, it is very important to develop efficient and convenient genetically modified food detection technology.

转基因产品的检测技术主要包括基于蛋白的检测方法和基于核酸的检测方法。目前基于核酸的PCR检测方法仍是目前最普遍、最准确的转基因检测技术，其主要包括普通定性PCR、巢式PCR、环介导等温扩增技术（LAMP)、荧光定量PCR多重PCR等方法。相对于普通定性PCR 方法，巢式PCR高了检测的灵敏度，容易造成假阳性。LAMP操作简单、特异性强，然而引物设计较为复杂，容易造成DNA 污染，影响后续的实验。荧光定量PCR 方法具有重复性好、灵敏度高、核酸交叉污染少的优点，但其成本高，并且需要特殊检测仪器。普通的多重PCR 方法可以在一个反应中同时检测多个基因，但一般不超过6重，否则引物之间干扰较大，影响检测效果。基因芯片和数字PCR技术也是常用的转基因产品检测技术，它们均具有高通量、灵敏度高、特异性强等优点，且可平行检测1 个转基因作物中多个基因或同时检测多种转基因作物；然而其成本高昂，需要专门的仪器设备，要求操作人员具有较高的专业素质，这些因素限制了该技术在检测中的广泛应用。The detection techniques of genetically modified products mainly include protein-based detection methods and nucleic acid-based detection methods. At present, the nucleic acid-based PCR detection method is still the most common and accurate transgenic detection technology, which mainly includes ordinary qualitative PCR, nested PCR, loop-mediated isothermal amplification technology (LAMP), fluorescent quantitative PCR multiplex PCR and other methods. Compared with ordinary qualitative PCR methods, nested PCR has higher detection sensitivity and is likely to cause false positives. LAMP is easy to operate and has strong specificity. However, the design of primers is relatively complicated, which may easily cause DNA contamination and affect subsequent experiments. The fluorescent quantitative PCR method has the advantages of good repeatability, high sensitivity, and less nucleic acid cross-contamination, but it is expensive and requires special detection instruments. Ordinary multiplex PCR method can simultaneously detect multiple genes in one reaction, but generally not more than 6 multiplexes, otherwise the interference between primers will be large, which will affect the detection effect. Gene chips and digital PCR technologies are also commonly used detection technologies for genetically modified products. They both have the advantages of high throughput, high sensitivity, and strong specificity, and can detect multiple genes in one genetically modified crop in parallel or detect multiple genetically modified crops at the same time; However, it is expensive, requires specialized equipment, and requires operators to have high professional quality. These factors limit the wide application of this technology in detection.

因此研发一种高效、灵敏和通量高的转基因产品检测方法，成为亟待解决的关键问题。Therefore, the development of an efficient, sensitive and high-throughput detection method for genetically modified products has become a key problem to be solved urgently.

发明内容Contents of the invention

本申请提供了一种检测转基因苜蓿的引物对组合、试剂盒及检测方法，以解决基于定量PCR技术检测转基因苜蓿通量低且检测成本高的技术问题。The application provides a combination of primers, a kit and a detection method for detecting transgenic alfalfa to solve the technical problems of low throughput and high detection cost of detecting transgenic alfalfa based on quantitative PCR technology.

第一方面，本申请提供了一种检测转基因苜蓿的引物对组合，所述引物对组合包括：In a first aspect, the application provides a primer pair combination for detecting transgenic alfalfa, the primer pair combination comprising:

特异性扩增p35S的引物对，其核苷酸序列如SEQ ID NO.1至SEQ ID NO.2所示；A pair of primers for specifically amplifying p35S, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2;

特异性扩增t35S的引物对，其核苷酸序列如SEQ ID NO.3至SEQ ID NO.4所示；A pair of primers for specifically amplifying t35S, the nucleotide sequences of which are shown in SEQ ID NO.3 to SEQ ID NO.4;

特异性扩增pNOS的引物对，其核苷酸序列如SEQ ID NO.5至SEQ ID NO.6所示；A pair of primers for specifically amplifying pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6;

特异性扩增pFMV35S的引物对，其核苷酸序列如SEQ ID NO.7至SEQ ID NO.8所示；A pair of primers for specifically amplifying pFMV35S, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8;

特异性扩增tNOS的引物对，其核苷酸序列如SEQ ID NO.9至SEQ ID NO.10所示；A pair of primers for specifically amplifying tNOS, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;

特异性扩增PAT的引物对，其核苷酸序列如SEQ ID NO.11至SEQ ID NO.12所示；A pair of primers for specifically amplifying PAT, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;

特异性扩增bar的引物对，其核苷酸序列如SEQ ID NO.13至SEQ ID NO.14所示；A pair of primers for specifically amplifying bar, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14;

特异性扩增NPtII的引物对，其核苷酸序列如SEQ ID NO.15至SEQ ID NO.16所示；A pair of primers for specifically amplifying NPtII, the nucleotide sequences of which are shown in SEQ ID NO.15 to SEQ ID NO.16;

和/或，特异性扩增cp4epsps的引物对，其核苷酸序列如SEQ ID NO.17至SEQ IDNO.18所示。And/or, a pair of primers for specifically amplifying cp4epsps, the nucleotide sequences of which are shown in SEQ ID NO.17 to SEQ ID NO.18.

可选的，所述引物对组合还包括特异性扩增选自下组的苜蓿转基因品系的引物对：Ms_J163和/或Ms_J101。Optionally, the combination of primer pairs further includes a pair of primers for specifically amplifying transgenic lines of alfalfa selected from the following group: Ms_J163 and/or Ms_J101.

可选的，所述引物对组合还包括：Optionally, the primer pair combination also includes:

特异性扩增Ms_J163的引物对，其核苷酸序列如SEQ ID NO.19至SEQ ID NO.20所示；A pair of primers for specifically amplifying Ms_J163, the nucleotide sequences of which are shown in SEQ ID NO.19 to SEQ ID NO.20;

和/或，特异性扩增Ms_J101的引物对，其核苷酸序列如SEQ ID NO.21至SEQ IDNO.22所示。And/or, a pair of primers for specifically amplifying Ms_J101, the nucleotide sequences of which are shown in SEQ ID NO.21 to SEQ ID NO.22.

可选的，所述特异性扩增Ms_Acc的引物对，其核苷酸序列如SEQ ID NO.23至SEQID NO.24所示。Optionally, the nucleotide sequences of the pair of primers for specifically amplifying Ms_Acc are shown in SEQ ID NO.23 to SEQ ID NO.24.

可选的，所述引物对组合还包括特异性扩增选自下组的苜蓿转基因元件的引物对：p35S、t35S、pNOS、tNOS、pFMV35S、PAT、bar、NPtII和cp4epsps。Optionally, the primer pair combination further includes a primer pair for specifically amplifying the alfalfa transgenic element selected from the group consisting of p35S, t35S, pNOS, tNOS, pFMV35S, PAT, bar, NPtII and cp4epsps.

第二方面，本申请提供了一种检测转基因苜蓿的引物对组合的试剂盒，所述试剂盒包括：第一方面所述的引物对组合。In a second aspect, the present application provides a kit for detecting a combination of primers in transgenic alfalfa, the kit comprising: the combination of primers in the first aspect.

可选的，所述试剂盒还包括多重PCR预混液。Optionally, the kit also includes a multiplex PCR master mix.

可选的，所述试剂盒还包括第一容器，所述第一容器内含有所述引物对组合。Optionally, the kit further includes a first container containing the combination of primer pairs.

第三方面，本申请提供了第一方面所述的引物对组合用于检测转基因苜蓿的方法，所述方法包括以下步骤：In a third aspect, the present application provides a method for detecting transgenic alfalfa using the combination of primers described in the first aspect, the method comprising the following steps:

得到待测苜蓿的DNA和所述引物对组合；Obtain the DNA of alfalfa to be tested and the primer pair combination;

以所述DNA为模板，将所述引物对组合加入反应体系中，进行扩增反应，得到扩增产物；Using the DNA as a template, adding the primer pair combination into the reaction system, performing an amplification reaction, and obtaining an amplification product;

将所述扩增产物进行高通量测序，得到高通量文库；performing high-throughput sequencing on the amplified product to obtain a high-throughput library;

将所述高通量文库中的基因序列进行分析，得到检测转基因苜蓿的结果。The gene sequence in the high-throughput library is analyzed to obtain the result of detecting the transgenic alfalfa.

第四方面，苜蓿引物对组合的用途，所述引物对组合用于制备并检测苜蓿转基因成分和转基因品系；In the fourth aspect, the use of alfalfa primer pair combinations, the primer pair combinations are used to prepare and detect alfalfa transgenic components and transgenic lines;

所述引物对组包括序列如SEQ ID NO.1至SEQ ID NO.24所示的引物。The primer pair set includes primers whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.24.

本申请实施例提供的上述技术方案与现有技术相比具有如下优点：Compared with the prior art, the above-mentioned technical solutions provided by the embodiments of the present application have the following advantages:

本申请实施例提供的检测转基因苜蓿，包括转基因成分和转基因品系的引物组合，所述引物对组合所述引物对组合其核苷酸序列如SEQ ID NO.1至SEQ ID NO.18所示；通过本申请的引物组合，操作简单，经过一次样品前处理，单管PCR 扩增，文库构建与测序就可以同步检测多样品或一个样品中多种转基因成分，具有平行分析和多重判断的特点，大大提升了转基因产品的检测效率，节约了检测成本。The detection of transgenic alfalfa provided in the examples of the present application includes a combination of primers for transgenic components and transgenic lines, and the nucleotide sequences of the primer pair combination are shown in SEQ ID NO.1 to SEQ ID NO.18; The primer combination of this application is easy to operate. After one sample pretreatment, single-tube PCR amplification, library construction and sequencing, multiple samples or multiple transgenic components in one sample can be detected simultaneously. It has the characteristics of parallel analysis and multiple judgments. The detection efficiency of genetically modified products is greatly improved, and the detection cost is saved.

附图说明Description of drawings

此处的附图被并入说明书中并构成本说明书的一部分，示出了符合本发明的实施例，并与说明书一起用于解释本发明的原理。The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description serve to explain the principles of the invention.

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单的介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, In other words, other drawings can also be obtained from these drawings without paying creative labor.

图1为本申请实施例提供的一种全面检测转基因苜蓿的试剂盒的检测方法的流程示意图。Fig. 1 is a schematic flowchart of a detection method of a kit for comprehensive detection of transgenic alfalfa provided by the embodiment of the present application.

具体实施方式Detailed ways

为使本申请实施例的目的、技术方案和优点更加清楚，下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请的一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本申请保护的范围。In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments It is a part of the embodiments of this application, but not all of them. Based on the embodiments in the present application, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present application.

影响多重PCR反应的因素有很多，比如：引物特异性，如果引物与体系中其他非目的基因片段结合力更强，那么目的基因结合引物的能力就会受到竞争，从而导致扩增效率下降；通过本申请设计的引物对组合，可以使PCR反应得到的扩增产物检测效率和准确率得到有效提升。There are many factors that affect the multiplex PCR reaction, such as: primer specificity, if the primer has a stronger binding force to other non-target gene fragments in the system, the ability of the target gene to bind the primer will be competed, resulting in a decrease in amplification efficiency; through The combination of primer pairs designed in this application can effectively improve the detection efficiency and accuracy of the amplification products obtained by PCR reaction.

第一方面，本申请提供了一种检测转基因苜蓿的引物对组合，所述引物对组合包括：In a first aspect, the application provides a primer pair combination for detecting transgenic alfalfa, the primer pair combination comprising:

特异性扩增p35S的引物对，其核苷酸序列如SEQ ID NO.1至SEQ ID NO.2所示；A pair of primers for specifically amplifying p35S, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2;

特异性扩增t35S的引物对，其核苷酸序列如SEQ ID NO.3至SEQ ID NO.4所示；A pair of primers for specifically amplifying t35S, the nucleotide sequences of which are shown in SEQ ID NO.3 to SEQ ID NO.4;

特异性扩增pNOS的引物对，其核苷酸序列如SEQ ID NO.5至SEQ ID NO.6所示；A pair of primers for specifically amplifying pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6;

特异性扩增pFMV35S的引物对，其核苷酸序列如SEQ ID NO.7至SEQ ID NO.8所示；A pair of primers for specifically amplifying pFMV35S, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8;

特异性扩增tNOS的引物对，其核苷酸序列如SEQ ID NO.9至SEQ ID NO.10所示；A pair of primers for specifically amplifying tNOS, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;

特异性扩增PAT的引物对，其核苷酸序列如SEQ ID NO.11至SEQ ID NO.12所示；A pair of primers for specifically amplifying PAT, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;

特异性扩增bar的引物对，其核苷酸序列如SEQ ID NO.13至SEQ ID NO.14所示；A pair of primers for specifically amplifying bar, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14;

特异性扩增NPtII的引物对，其核苷酸序列如SEQ ID NO.15至SEQ ID NO.16所示；A pair of primers for specifically amplifying NPtII, the nucleotide sequences of which are shown in SEQ ID NO.15 to SEQ ID NO.16;

和/或，特异性扩增cp4epsps的引物对，其核苷酸序列如SEQ ID NO.17至SEQ IDNO.18所示。And/or, a pair of primers for specifically amplifying cp4epsps, the nucleotide sequences of which are shown in SEQ ID NO.17 to SEQ ID NO.18.

上述引物的长度介于在18-30bp之间，引物间互不干扰；所述多重PCR引物的对数范围包括但不限于：1-12对，优选的为9-12对，其中，1-12对之间根据具体检测样品的情况适当调整。为了实现对转基因苜蓿的检测，收集了9个常用的苜蓿转基因元件和2种转基因品系，覆盖了市面上大多数转基因苜蓿品系的常用转基因元件，所述多重PCR引物的对数范围为：1-12对，相比常规的8对特异性的多重PCR，具有检测通量和灵敏度高的优势。The length of the above primers is between 18-30bp, and the primers do not interfere with each other; the logarithmic range of the multiple PCR primers includes but is not limited to: 1-12 pairs, preferably 9-12 pairs, wherein, 1- The 12 pairs are properly adjusted according to the situation of the specific test samples. In order to realize the detection of transgenic alfalfa, 9 commonly used alfalfa transgenic elements and 2 transgenic lines were collected, covering the commonly used transgenic elements of most transgenic alfalfa lines on the market. The logarithmic range of the multiplex PCR primers is: 1- 12 pairs, compared with the conventional multiplex PCR with 8 pairs of specificity, it has the advantages of high detection throughput and sensitivity.

在一些实施方式中，所述引物对组合还包括特异性扩增选自下组的苜蓿转基因品系内参引的引物对：Ms_J163和/或Ms_J101。In some embodiments, the primer pair combination further includes a primer pair for specifically amplifying internal references in alfalfa transgenic lines selected from the group consisting of Ms_J163 and/or Ms_J101.

本申请对目标苜蓿转基因品系或转基因元件的设计PCR扩增引物对，通过高通量测序分析，可以迅速和全面的得到待检测基因的多角度信息，省时省力；为了实现多组引物在一个实验流程中可以进行同时添加，而不是进行多次独立的平行实验，多重PCR引物的扩增产物进行高通量测序，可以达到节省人工和成本的目的。This application designs PCR amplification primer pairs for target alfalfa transgenic lines or transgenic elements. Through high-throughput sequencing analysis, the multi-angle information of the gene to be detected can be obtained quickly and comprehensively, saving time and effort; in order to realize multiple sets of primers in one In the experimental process, simultaneous addition can be performed instead of multiple independent parallel experiments. The amplification products of multiple PCR primers can be sequenced through high-throughput, which can save labor and cost.

另外，所述引物组合的扩增产物可以进行一次高通量测序和分析，得到多个检测结果，包括转基因成分、判断待测样品中是否含有靶标分子、确定待测样品中内参基因与靶标分子的拷贝数目，以确定外源基因的含量；避免了传统Real-time PCR技术一次只能实现一个目的，需要进行多次扩增和检测才能覆盖样本中的多个靶标转基因成分，而本申请的引物在进行检测时，可以通过一次高通量测序和分析，得到转基因作物中多个基因或同时检测多种转基因作物检测结果，大大提高了检测的效率和灵敏度，节省了成本，便于应用推广。In addition, the amplified product of the primer combination can be subjected to high-throughput sequencing and analysis to obtain multiple detection results, including transgenic components, judging whether the sample to be tested contains target molecules, and determining the internal reference gene and target molecule in the sample to be tested. to determine the content of exogenous genes; avoiding the fact that traditional Real-time PCR technology can only achieve one purpose at a time, and requires multiple amplifications and detections to cover multiple target transgenic components in the sample, while the present application When the primers are tested, multiple genes in transgenic crops can be obtained through one high-throughput sequencing and analysis, or the detection results of multiple genetically modified crops can be detected at the same time, which greatly improves the efficiency and sensitivity of detection, saves costs, and facilitates application and promotion.

在一些实施方式中，所述引物对组合中：In some embodiments, in the primer pair combination:

特异性扩增Ms_J163的引物对，其核苷酸序列如SEQ ID NO.19至SEQ ID NO.20所示；A pair of primers for specifically amplifying Ms_J163, the nucleotide sequences of which are shown in SEQ ID NO.19 to SEQ ID NO.20;

和/或，特异性扩增Ms_J101的引物对，其核苷酸序列如SEQ ID NO.21至SEQ IDNO.22所示。And/or, a pair of primers for specifically amplifying Ms_J101, the nucleotide sequences of which are shown in SEQ ID NO.21 to SEQ ID NO.22.

综上，为了增强所述引物对的适用性和灵敏性，所设计引物长度介于在18-30bp之间，引物间互不干扰，且所有引物可以组合成引物池进行多重PCR扩增，即所有设计的引物可以在一个扩增反应中均正常扩增，经使用证实，灵敏度高和适用性强；同时，PCR扩增引物对组由正向引物和反向引物组成，一个为上游引物，一个为下游引物。In summary, in order to enhance the applicability and sensitivity of the primer pair, the length of the designed primers is between 18-30bp, the primers do not interfere with each other, and all primers can be combined into a primer pool for multiplex PCR amplification, namely All the designed primers can be amplified normally in one amplification reaction. It has been proved by use that they have high sensitivity and strong applicability; at the same time, the PCR amplification primer pair set is composed of forward primer and reverse primer, one is the upstream primer, One is the downstream primer.

在一些实施方式中，所述特异性扩增Ms_Acc的引物对，其核苷酸序列如SEQ IDNO.23至SEQ ID NO.24所示。In some embodiments, the nucleotide sequences of the pair of primers for specifically amplifying Ms_Acc are shown in SEQ ID NO.23 to SEQ ID NO.24.

为了实现检测苜蓿转基因成分和转基因品系的目的，选用了苜蓿内参基因，可以实现对样品中转基因成分含量进行定量的作用。In order to achieve the purpose of detecting the transgenic components and transgenic lines of alfalfa, the internal reference gene of alfalfa was selected, which can realize the quantification of the content of the transgenic components in the sample.

优选的，引物对组合包括扩增9种常用苜蓿转基因元件、2种转基因品系特异性序列以及1种苜蓿内参基因Ms_Acc的引物对。Preferably, the primer pair combination includes primer pairs for amplifying 9 common alfalfa transgenic elements, 2 transgenic line-specific sequences, and 1 alfalfa internal reference gene Ms_Acc.

在一些实施方式中，所述引物对组合还包括特异性扩增选自下组的苜蓿转基因元件的引物对：p35S、t35S、pNOS、tNOS、pFMV35S、PAT、bar、NPtII和cp4epsps。In some embodiments, the primer pair combination further comprises a primer pair that specifically amplifies an alfalfa transgenic element selected from the group consisting of p35S, t35S, pNOS, tNOS, pFMV35S, PAT, bar, NPtII, and cp4epsps.

在现有引物对组合的基础上，还可以包括特异性扩增相同的苜蓿转基因元件的其他引物对引物组合，还可以包括后期可根据新收集的转基因元件进行定期增加多重PCR引物的对数组，经验证可达3000对，扩增效果依然很好。On the basis of the existing primer pair combination, other primer pairs and primer combinations that specifically amplify the same alfalfa transgenic element can also be included, and the pair group of multiplex PCR primers that can be periodically increased according to the newly collected transgenic element in the later stage can also be included, It has been verified that it can reach 3000 pairs, and the amplification effect is still very good.

上述引物与其扩增的上述苜蓿转基因元件及品系的特异性核苷酸序列、相应引物对的编号及引物对的核苷酸序列如表1所示。The above primers and the specific nucleotide sequences of the alfalfa transgenic elements and strains amplified by the above primers, the numbers of the corresponding primer pairs and the nucleotide sequences of the primer pairs are shown in Table 1.

第二方面，本申请提供了一种检测转基因苜蓿的引物对组合的试剂盒，所述试剂盒包括：第一方面所述的引物对组合。In a second aspect, the present application provides a kit for detecting a combination of primers in transgenic alfalfa, the kit comprising: the combination of primers in the first aspect.

在一些实施方式中，所述试剂盒还包括多重PCR预混液。In some embodiments, the kit further includes a multiplex PCR master mix.

具体的，多重PCR预混液的组分包括所述油菜的转基因元件和内参基因的各引物组合。的。组合时，每条引物按照1:1的比例进行预混，根据不同的实验目的进行各引物的混合，具体实施实例中，每条引物浓度是2 nM。Specifically, the components of the multiplex PCR premix include the primer combinations of the rapeseed transgenic element and the internal reference gene. of. When combining, each primer is premixed according to a ratio of 1:1, and each primer is mixed according to different experimental purposes. In a specific implementation example, the concentration of each primer is 2 nM.

在一些实施方式中，所述试剂盒还包括第一容器，所述第一容器内含有所述引物对组合。In some embodiments, the kit further includes a first container containing the combination of primer pairs.

第三方面，本申请提供了第一方面所述的引物对组合的检测方法，如图1所示，所述方法包括以下步骤：In a third aspect, the present application provides the detection method of the primer pair combination described in the first aspect, as shown in Figure 1, the method comprises the following steps:

S1.得到待测苜蓿的DNA和所述引物对组合；S1. Obtain the DNA of alfalfa to be tested and the primer pair combination;

S2.以所述DNA为模板，将所述引物对组合加入反应体系中，进行扩增反应，得到扩增产物；S2. Using the DNA as a template, adding the primer pair combination into the reaction system, performing an amplification reaction, and obtaining an amplification product;

S3.将所述扩增产物进行高通量测序，得到高通量文库；S3. performing high-throughput sequencing on the amplified product to obtain a high-throughput library;

S4.将所述高通量文库中的基因序列进行分析，得到检测转基因苜蓿的结果。S4. Analyzing the gene sequence in the high-throughput library to obtain the result of detecting the transgenic alfalfa.

第四方面，苜蓿引物对组合的用途，所述引物对组合用于制备并检测苜蓿转基因成分和转基因品系；In the fourth aspect, the use of alfalfa primer pair combinations, the primer pair combinations are used to prepare and detect alfalfa transgenic components and transgenic lines;

所述引物对组包括序列如SEQ ID NO.1至SEQ ID NO.24所示的引物。The primer pair set includes primers whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.24.

具体地，高通量测序可以是二代测序，也可以是3代测序，得到的高通量文库可以从多个维度分析转基因的成分，包括但不限于实施案例中的转基因元件。应用于苜蓿及其制品的转基因成分和转基因品系的定性与定量检测。Specifically, high-throughput sequencing can be next-generation sequencing or third-generation sequencing, and the obtained high-throughput library can analyze transgenic components from multiple dimensions, including but not limited to the transgenic elements in the examples. Applied to the qualitative and quantitative detection of genetically modified ingredients and genetically modified lines of alfalfa and its products.

使用上述方法，可一次性对多样本的所有转基因成分及转基因品系进行检测，具有高通量、高灵敏、准确和快速等优势，可应用于苜蓿及其制品的转基因成分的定性与定量检测。Using the above method, all genetically modified ingredients and genetically modified lines of multiple samples can be detected at one time, which has the advantages of high throughput, high sensitivity, accuracy and speed, and can be applied to the qualitative and quantitative detection of genetically modified ingredients in alfalfa and its products.

本申请实施例的方法中，所述扩增反应包括普通PCR扩增反应或实时荧光PCR扩增反应；所述扩增反应的环境/程序包括：所述扩增反应的环境/程序包括：94℃预变性5分钟；第一步扩增反应，94℃变性15s，62℃～56℃退火30s，12个Touch Down cycle，（每个循环退火及延伸的温度降低0 .5℃）；第二步扩增反应，94℃变性15s，57℃退火30S，22个cycle。In the method of the embodiment of the present application, the amplification reaction includes an ordinary PCR amplification reaction or a real-time fluorescent PCR amplification reaction; the environment/program of the amplification reaction includes: the environment/program of the amplification reaction includes: 94 Pre-denaturation at ℃ for 5 minutes; the first step of amplification reaction, denaturation at 94°C for 15s, annealing at 62°C to 56°C for 30s, 12 Touch Down cycles (annealing and extension temperature decreased by 0.5°C for each cycle); the second Step amplification reaction, denaturation at 94°C for 15s, annealing at 57°C for 30s, 22 cycles.

另外，所述反应体系包括但不限于：反应体系包括：总体系40μl，引物预混液2.5μl、2×buffer：20μl，多重PCR扩增酶：0.5μl；其余的用水补充。In addition, the reaction system includes but is not limited to: the reaction system includes: 40 μl of total system, 2.5 μl of primer premix, 2×buffer: 20 μl, multiplex PCR amplification enzyme: 0.5 μl; the rest is supplemented with water.

优选地，所述的方法的扩增反应的环境/程序包括：94℃预变性15分钟；第一步扩增反应，94℃变性20秒，65℃～57℃退火并延伸60秒，10个Touch Down循环，（每个循环退火及延伸的温度降低0 .8℃）；第二步扩增反应，94℃变性20秒，57℃退火并延伸60秒，26个循环。Preferably, the environment/program of the amplification reaction of the method includes: pre-denaturation at 94°C for 15 minutes; the first step of amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 65°C to 57°C for 60 seconds, 10 Touch Down cycle, (annealing and extension temperature decreased by 0.8°C for each cycle); the second step amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, 26 cycles.

再优选地，所述的方法的反应体系包括：总体系30μl，引物对：2μl、2×buffer：15ul，多重扩增酶：0.5μl；剩余的用水补充。More preferably, the reaction system of the method includes: total system 30 μl, primer pair: 2 μl, 2×buffer: 15 μl, multiplex amplification enzyme: 0.5 μl; the rest is supplemented with water.

在一些实施方式中，所述高通量文库的浓度≥2ng/ul。In some embodiments, the concentration of the high-throughput library is > 2 ng/ul.

控制高通量文库的浓度，避免了克隆而引起的系统偏差，简化了实验操作，提高了测序效率。Controlling the concentration of the high-throughput library avoids systematic deviation caused by cloning, simplifies experimental operations, and improves sequencing efficiency.

PCR扩增引物对组的用途，用于制备检测试剂盒，所述检测试剂盒用于检测转基因苜蓿和转基因品系；The purpose of PCR amplification primer pair group is used for preparing detection kit, and described detection kit is used for detecting transgenic alfalfa and transgenic strain;

所述引物对组包括序列如SEQ ID NO.1至SEQ ID NO.26所示的引物。The primer pair set includes primers whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.26.

本发明所提供的试剂盒能灵敏的检测样品中转基因含量为0.05％的苜蓿转基因产品。The kit provided by the invention can sensitively detect alfalfa transgene products with a transgene content of 0.05% in samples.

本发明的重现性试验中每个样品不同文库间、不同建库批次间检测结果重现率r＝100％，准确率a=98.7％。In the reproducibility test of the present invention, the reproducibility rate of detection results between different libraries and different database building batches of each sample is r=100%, and the accuracy rate a=98.7%.

本发明的所述试剂盒在复杂模板中检测多种苜蓿转基因品系，具有高度特异性。The kit of the invention can detect multiple alfalfa transgenic lines in complex templates, and has high specificity.

综上，本申请克服了现有技术费时费力成本高的缺陷，提供的苜蓿转基因品系检测试剂盒操作简单，快速灵敏，检测通量大，检测结果重复性好，多样本多靶标序列检测成本低廉，对种子站、农科院所及海关进出口岸的转基因制品检测具有重要应用。In summary, this application overcomes the time-consuming, labor-intensive and high-cost defects of the prior art, and provides a detection kit for alfalfa transgenic strains that is simple to operate, fast and sensitive, with large detection throughput, good repeatability of detection results, and low cost for multi-sample multi-target sequence detection , It has an important application for the detection of genetically modified products in seed stations, agricultural science institutes and customs import and export ports.

下面将结合实施例、对比例及实验数据对本发明的方法进行详细说明。The method of the present invention will be described in detail below in conjunction with examples, comparative examples and experimental data.

本申请实施例的技术方案为解决上述技术问题，总体思路如下：The technical solution of the embodiment of the present application is to solve the above-mentioned technical problems, and the general idea is as follows:

筛选常用的苜蓿转基因产品检测元件及内参基因作为检测目标。包括13种检测常用的转基因元件：p35S、t35S、pNOS、tNOS、tPINⅡ、pZmUBI、pRBCS4、tE9、Bar、PAT、HPT、GUS、Cry1Ab-Ac；包括苜蓿内参基因的序列：SPS。Commonly used alfalfa transgenic product detection elements and internal reference genes were screened as detection targets. Including 13 commonly used transgenic elements for detection: p35S, t35S, pNOS, tNOS, tPINⅡ, pZmUBI, pRBCS4, tE9, Bar, PAT, HPT, GUS, Cry1Ab-Ac; including the sequence of the alfalfa reference gene: SPS.

接着，本发明开发了用于检测所述的转基因元件和苜蓿内参基因的多重PCR引物组合物，其中13对针对13种转基因元件，2对针对两个内参基因。所述引物互相间不冲突，可以通过多重PCR进行高效的扩增。Next, the present invention developed a multiplex PCR primer composition for detecting the transgenic elements and alfalfa internal reference genes, wherein 13 pairs were aimed at 13 kinds of transgenic elements, and 2 pairs were aimed at two internal reference genes. The primers do not conflict with each other and can be efficiently amplified by multiplex PCR.

所述多重PCR引物组合物可以用于开发转基因元件检测试剂盒。The multiplex PCR primer composition can be used to develop a transgenic element detection kit.

本发明所提供的试剂盒能灵敏的检测样品中含量为0.05％的转基因成分。The kit provided by the invention can sensitively detect the genetically modified component with a content of 0.05% in the sample.

本发明的重现性试验中每个样品不同文库间、不同建库批次间检测结果重现率r＝100％，准确率a=98.7％。In the reproducibility test of the present invention, the reproducibility rate of detection results between different libraries and different database building batches of each sample is r=100%, and the accuracy rate a=98.7%.

本发明的所述试剂盒在复杂模板中检测多种转基因成分具有高度特异性。The kit of the present invention has high specificity in detecting multiple transgenic components in complex templates.

下面将结合实施例、对比例及实验数据对本申请的一种全面检测苜蓿转基因成分试剂盒及其应用进行详细说明。A comprehensive detection kit for genetically modified alfalfa components of the present application and its application will be described in detail below in conjunction with examples, comparative examples and experimental data.

实施例1 靶标转基因元件、品系的筛选和多重PCR扩增引物的设计Example 1 Target transgenic elements, screening of strains and design of multiplex PCR amplification primers

本申请实施例中，靶标转基因成分主要指的是转基因元件和品系，他们及实施例中使用的苜蓿内参基因，主要收集于常用的转基因数据库、国家标准、行业标准或者现有的文献中，尽量收集的全，以保证检测的特异性和准确性。其中筛选的转基因元件、品系和内参基因的名称如表1所示。In the examples of this application, the target transgenic components mainly refer to transgenic elements and strains. They and the alfalfa internal reference genes used in the examples are mainly collected from commonly used transgenic databases, national standards, industry standards or existing literature, and try to Collect all to ensure the specificity and accuracy of detection. The names of the screened transgenic elements, strains and internal reference genes are shown in Table 1.

表1 本发明所筛选的靶标分子及其引物序列。Table 1 Target molecules screened in the present invention and their primer sequences.

本申请实施例中，利用Primer3Plus设计多重PCR引物，所设引物长度介于在18-30bp之间，引物间互不干扰，这个主要评估引物之间的二聚体，或者引物内部的发夹结构，以及非靶标序列的非特异扩增，评估后的所有引物可以组合成引物池进行多重PCR扩增，即所有设计的引物可以在一个扩增反应中均正常扩增。具体的引物序列包括：SEQ ID NO.1～SEQ ID NO.24所示。In the examples of this application, Primer3Plus is used to design multiple PCR primers. The length of the primers is between 18-30bp, and the primers do not interfere with each other. This mainly evaluates the dimer between the primers, or the hairpin structure inside the primer. , and non-specific amplification of non-target sequences, all evaluated primers can be combined into a primer pool for multiple PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction. The specific primer sequences include: SEQ ID NO.1-SEQ ID NO.24.

实施例2检测苜蓿样品是否含有转基因成分Example 2 Detecting whether alfalfa samples contain genetically modified ingredients

1. 实验材料：转基因材料Ms_J101。实验材料转入了pFMV35S、cp4epsps，其转基因含量为10%，用其作为研究材料。1. Experimental material: transgenic material Ms_J101. The experimental materials were transferred into pFMV35S and cp4epsps, and the transgene content was 10%, which were used as research materials.

2. DNA模板的准备：植物基因组的提取采用的是CTAB或天根生化科技（北京）有限公司的高效植物基因组DNA 提取试剂盒（DP350）。本实施例是用天根DNA提取试剂盒提取的待测样品DNA，每个样品做了三个生物重复。2. Preparation of DNA template: Plant genome was extracted using CTAB or Tiangen Biochemical Technology (Beijing) Co., Ltd. High-efficiency Plant Genome DNA Extraction Kit (DP350). In this example, the DNA of the sample to be tested was extracted with the Tiangen DNA Extraction Kit, and three biological repetitions were made for each sample.

3．PCR扩增、文库构建与测序3. PCR amplification, library construction and sequencing

利用12对多重PCR扩增引物扩增样本的基因组DNA；将每个样本的扩增产物连接测序接头和特异样本DNA条形码后混合，成为高通量测序文库；利用高通量测序平台检测高通量测序文库并对高通量测序数据进行质量控制。本步骤需要根据检测的准确性、灵敏性等要求，研究调整扩增循环数、测序深度等关键参数；本步骤也可连接三代测序，以实现二代测序与三代测序间优势互补。Use 12 pairs of multiplex PCR amplification primers to amplify the genomic DNA of the sample; connect the amplified product of each sample to the sequencing adapter and the DNA barcode of the specific sample and mix to form a high-throughput sequencing library; use a high-throughput sequencing platform to detect the high-throughput Quantitative sequencing libraries and quality control of high-throughput sequencing data. This step needs to study and adjust key parameters such as the number of amplification cycles and sequencing depth according to the requirements of detection accuracy and sensitivity; this step can also be connected with third-generation sequencing to realize the complementary advantages between second-generation sequencing and third-generation sequencing.

4.结果的判定4. Judgment of results

1）据测试样品中的转基因品系的信号指数S和空白对照中转基因品系的信号指数P判定污染是否可接受，其中：1) According to the signal index S of the transgenic strain in the test sample and the signal index P of the transgenic strain in the blank control, determine whether the contamination is acceptable, where:

空白对照噪音指数P=nc/Nc，其中nc和Nc分别代表空白对照中，转基因品系的测序片段的数量和总测序片段数量。Blank control noise index P=nc/Nc, where nc and Nc represent the number of sequenced fragments and the total number of sequenced fragments of the transgenic line in the blank control, respectively.

测试样品的信号指数S=nt/Nt，其中，nt和Nt分别代表测试样品中，转基因品系的测序片段的数量和总测序片段数量。The signal index of the test sample S=nt/Nt, wherein, nt and Nt respectively represent the number of sequenced fragments and the total number of sequenced fragments of the transgenic line in the test sample.

信噪比=S/PSNR=S/P

2）转基因结果的判断2) Judgment of genetically modified results

利用待测样本DNA条形码和同源比对，将每个测序片段分配到每个目标物种的每个靶标位置上，所述的靶标包括转基因元件、品系和内参基因。根据每个靶标位置上的测序序列数目，以实现转基因元件或品系的绝对定量。当比对内参基因和转基因元件上的测序序列超过指定阈值时，定性判定样品中含有转基因成分；当样品中含有转基因成分时，根据转基因元件和品系与内参基因的测序序列的比值，定量判定样品中的外源基因的含量。本实施例中转基因含量的计算公式如（A）所示：Using the DNA barcode of the sample to be tested and the homologous comparison, each sequencing fragment is assigned to each target position of each target species, and the target includes transgenic elements, strains and internal reference genes. Based on the number of reads per target position for absolute quantification of transgenic elements or lines. When the sequence sequence on the comparison of the internal reference gene and the transgenic element exceeds the specified threshold, it is qualitatively determined that the sample contains a genetically modified component; The content of exogenous genes in. The formula for calculating the transgene content in this example is shown in (A):

CtestDNACtestDNA—— 测试样品的转基因含量- Test samples for GMO contenttTitT——测试样品中的每种转基因元件和品系的测序序列数目- the number of sequenced reads for each transgenic element and line in the test sampletRitR—— 测试样品中检出的每种内参基因片段的测序序列目——The sequencing sequence list of each internal reference gene fragment detected in the test samplemm—— 测试样品中检出的内参基因片段的总数——The total number of internal reference gene fragments detected in the test samplenno—— 标准品中检出的转基因元件和品系片段的总数- the total number of transgenic elements and line fragments detected in the standards

根据本实例，共检测了2个样品，1个转基因品系Ms_J101和一个阴性样品，每个样品做了三个生物重复，结果如表2所示：阴性样品中常用的启动子和终止子在阴性苜蓿中也会检测出几个序列，要求测序reads数目小于5条的序列过滤掉。本发明规定当信噪比大于10倍时，可判定检测体系中的污染是可接受的。当样品中转基因品系的信噪比大于10，判定样本中检出转基因品系的核酸。According to this example, a total of 2 samples were tested, 1 transgenic line Ms_J101 and 1 negative sample. Three biological replicates were done for each sample, and the results are shown in Table 2: the commonly used promoters and terminators in negative Several sequences are also detected in alfalfa, and sequences requiring less than 5 sequencing reads are filtered out. The present invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be judged that the contamination in the detection system is acceptable. When the signal-to-noise ratio of the transgenic strain in the sample is greater than 10, it is determined that the nucleic acid of the transgenic strain is detected in the sample.

表2 本实施例2待测样品的转基因检测结果Table 2 Transgenic detection results of samples to be tested in Example 2

由表可知，Ms_J101中的所对应的各转基因元件在三个重复实验中均被有效检出，而且含量接近于其含量；从该表说明苜蓿转基因试剂盒可以用来检测转基因产品。It can be seen from the table that the corresponding transgenic elements in Ms_J101 were effectively detected in three repeated experiments, and the content was close to its content; the table shows that the alfalfa transgenic kit can be used to detect genetically modified products.

实施例3 准确性、特异性与灵敏度评估Example 3 Evaluation of accuracy, specificity and sensitivity

转基因苜蓿品种Ms_J101和Ms_J163转基因标准品制备不同质量百分比的转基因样本来评估所开发技术的准确度、特异性和灵敏度。具体地，各样本的转基因含量采用质量百分进行稀释，具体地把转基因苜蓿Ms_J101和Ms_J163用阴性苜蓿分别稀释成10%，1%，0.1%，0.05%，0.025%和0.01%的样本，分别对应于转基因品系Ms_J101的稀释样本编号（A1，A2，A3，A4，A5，A6）和转基因品系Ms_J163的稀释样本编号（B1，B2，B3，B4，B5，B6）。定性检测的准确度指真阳性与真阴性所占的比例，定量准确度是指多次测定的平均值与真值相符合的程度，以误差来表示。特异性又称真阴性率，多次检测检出的真阴性占所有阴性的百分比。灵敏度指在95%置信度下，能够检出的转基因品系的最低含量，即检测下限。按照实施例2的方法进行检测，每个样本三个生物重复，结果如表3所示。The transgenic standards of transgenic alfalfa varieties Ms_J101 and Ms_J163 were prepared with different mass percentages of transgenic samples to evaluate the accuracy, specificity and sensitivity of the developed technology. Specifically, the transgene content of each sample was diluted by mass percentage, specifically, the transgenic alfalfa Ms_J101 and Ms_J163 were diluted with negative alfalfa to 10%, 1%, 0.1%, 0.05%, 0.025% and 0.01% samples, respectively. Corresponding to the dilution sample numbers (A1, A2, A3, A4, A5, A6) of the transgenic line Ms_J101 and the dilution sample numbers (B1, B2, B3, B4, B5, B6) of the transgenic line Ms_J163. The accuracy of qualitative detection refers to the proportion of true positives and true negatives, and the quantitative accuracy refers to the degree to which the average value of multiple measurements coincides with the true value, expressed as an error. Specificity, also known as the true negative rate, is the percentage of true negatives detected by multiple tests to all negatives. Sensitivity refers to the lowest content of transgenic strains that can be detected at a 95% confidence level, that is, the lower limit of detection. The detection was carried out according to the method of Example 2, and each sample was replicated three times, and the results are shown in Table 3.

表3本发明方法的准确性与灵敏度评估Accuracy and sensitivity evaluation of the method of the present invention in table 3

注：+代表检出，－代表未检出，A1和B1代表转基因含量为10%，A2和B2代表转基因含量为1%，A3和B3代表转基因含量为0.1%，A4和B4代表转基因含量为0.05%，A5和B5代表转基因含量为0.025%，A6和B6代表转基因含量为0.01%。Note: + means detected, - means not detected, A1 and B1 represent GM content of 10%, A2 and B2 represent GM content of 1%, A3 and B3 represent GM content of 0.1%, A4 and B4 represent GM content of 0.1%. 0.05%, A5 and B5 represent 0.025% GMO content, A6 and B6 represent 0.01% GMO content.

由表可知，所述试剂盒能在转基因含量为0.05%的样本中稳定地检出各转基因元件，而在阴性的样本中检出最多检出1个转基因成分，说明所述试剂盒特异性强，说明所述试剂盒特异性强，能够明显区分转基因含量为0.05%的样本和阴性样本，具有技术稳定性和转基因含量为0.05%的检测灵敏度。It can be seen from the table that the kit can stably detect each transgenic element in a sample with a transgene content of 0.05%, and detect at most one transgenic element in a negative sample, indicating that the kit has strong specificity , indicating that the kit has strong specificity, can clearly distinguish samples with a GMO content of 0.05% from negative samples, and has technical stability and detection sensitivity with a GMO content of 0.05%.

实施例Example

为了验证本发明的准确性及批量样品转基因检测中的作用，实验室选取了某公司79份未知基因型的苜蓿叶片样本进行检测，采用实施例2的检测方法，检测结果并与该公司的保存类型进行比较，并统计结果的一致性。分析结果表明在79份测试样品中，只有1份样品的结果不一致，检测结果的一致性高达98.7％，因此较好地证明了方法的准确性。In order to verify the accuracy of the present invention and the effect of batch sample transgenic detection, the laboratory selected 79 alfalfa leaf samples of unknown genotypes from a certain company for detection, using the detection method of Example 2, and the detection results were compared with the company's preservation Types are compared, and the consistency of the results is counted. The analysis results showed that among the 79 test samples, only one sample had inconsistent results, and the consistency of the test results was as high as 98.7%, thus better proving the accuracy of the method.

本发明又一优势包括：Yet another advantage of the present invention includes:

1）操作简单，经过一次样品前处理，单管PCR 扩增，文库构建与测序就可以同步检测多样品或一个样品中多种转基因成分，具有平行分析和多重判断的特点，大大提升了转基因产品的检测效率；1) The operation is simple. After one sample pretreatment, single-tube PCR amplification, library construction and sequencing, multiple samples or multiple transgenic components in one sample can be detected simultaneously. It has the characteristics of parallel analysis and multiple judgments, which greatly improves the quality of transgenic products. detection efficiency;

2）检验对象全，包含了苜蓿当前常见的9种转基因元件和2种转基因品系，并且可以很方便地加入新的检测靶标序列，避免单个靶标扩增失败，提高的了检测的特异性、准确度和灵敏性；2) The test object is complete, including 9 common transgenic elements and 2 transgenic lines of alfalfa, and can easily add new detection target sequences to avoid the failure of single target amplification and improve the specificity and accuracy of detection speed and sensitivity;

3）试剂盒融合二代测序平台进行扩增产物的测序，提高了检测系统的通量和可重复性，并且检测结果可以直接数字化，适用于转基因苜蓿及其制品的大规模检测。3) The kit is integrated with a next-generation sequencing platform to sequence the amplified products, which improves the throughput and repeatability of the detection system, and the detection results can be directly digitized, which is suitable for large-scale detection of genetically modified alfalfa and its products.

需要说明的是，在本文中，诸如“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者任何其他变体意在涵盖非排他性地包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。It should be noted that in this article, relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply these No such actual relationship or order exists between entities or operations. Furthermore, the term "comprises", "comprising", or any other variation is intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus comprising a set of elements includes not only those elements, but also items not expressly listed. other elements, or also include elements inherent in such a process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article or apparatus comprising said element.

以上所述仅是本发明的具体实施方式，使本领域技术人员能够理解或实现本发明。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其他实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所申请的原理和新颖特点相一致的最宽的范围。The above descriptions are only specific embodiments of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Accordingly, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

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Claims (8)

Translated fromChinese

1.一种检测转基因苜蓿的引物对组合物，其特征在于，所述引物对组合物包括：1. a kind of primer pair composition that detects transgenic alfalfa, it is characterized in that, described primer pair composition comprises:特异性扩增p35S的引物对，其核苷酸序列如SEQ ID NO.1至SEQ ID NO.2所示；A pair of primers for specifically amplifying p35S, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2;特异性扩增t35S的引物对，其核苷酸序列如SEQ ID NO.3至SEQ ID NO.4所示；A pair of primers for specifically amplifying t35S, the nucleotide sequences of which are shown in SEQ ID NO.3 to SEQ ID NO.4;特异性扩增pNOS的引物对，其核苷酸序列如SEQ ID NO.5至SEQ ID NO.6所示；A pair of primers for specifically amplifying pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6;特异性扩增pFMV35S的引物对，其核苷酸序列如SEQ ID NO.7至SEQ ID NO.8所示；A pair of primers for specifically amplifying pFMV35S, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8;特异性扩增tNOS的引物对，其核苷酸序列如SEQID NO.9至SEQ ID NO.10所示；A pair of primers for specifically amplifying tNOS, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;特异性扩增PAT的引物对，其核苷酸序列如SEQ ID NO.11至SEQ ID NO.12所示；A pair of primers for specifically amplifying PAT, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;特异性扩增bar的引物对，其核苷酸序列如SEQ ID NO.13至SEQ ID NO.14所示；A pair of primers for specifically amplifying bar, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14;特异性扩增NPtII的引物对，其核苷酸序列如SEQ ID NO.15至SEQ ID NO.16所示；A pair of primers for specifically amplifying NPtII, the nucleotide sequences of which are shown in SEQ ID NO.15 to SEQ ID NO.16;和，特异性扩增cp4epsps的引物对，其核苷酸序列如SEQ ID NO.17至SEQ ID NO.18所示。And, a pair of primers for specifically amplifying cp4epsps, the nucleotide sequences of which are shown in SEQ ID NO.17 to SEQ ID NO.18.2.根据权利要求1所述的引物对组合物，其特征在于，所述引物对组合物还包括：2. primer pair composition according to claim 1, is characterized in that, described primer pair composition also comprises:特异性扩增Ms_J163的引物对，其核苷酸序列如SEQ ID NO.19至SEQ ID NO.20所示；A pair of primers for specifically amplifying Ms_J163, the nucleotide sequences of which are shown in SEQ ID NO.19 to SEQ ID NO.20;和/或，特异性扩增Ms_J101的引物对，其核苷酸序列如SEQ ID NO.21至SEQ ID NO.22所示。And/or, a pair of primers for specifically amplifying Ms_J101, the nucleotide sequences of which are shown in SEQ ID NO.21 to SEQ ID NO.22.3.根据权利要求1所述的引物对组合物，其特征在于，还包括特异性扩增Ms_Acc的引物对，其核苷酸序列如SEQ ID NO.23至SEQ ID NO.24所示。3. The primer pair composition according to claim 1, further comprising a primer pair for specifically amplifying Ms_Acc, the nucleotide sequences of which are shown in SEQ ID NO.23 to SEQ ID NO.24.4.一种检测转基因苜蓿的引物对组合物的试剂盒，其特征在于，所述试剂盒包括：权利要求1-3任意一项所述的引物对组合物。4. A kit for detecting a primer pair composition for transgenic alfalfa, characterized in that the kit comprises: the primer pair composition according to any one of claims 1-3.5.根据权利要求4所述的试剂盒，其特征在于，所述试剂盒还包括多重PCR预混液。5. The kit according to claim 4, characterized in that, the kit also includes a multiplex PCR master mix.6.根据权利要求4所述的试剂盒，其特征在于，所述试剂盒还包括第一容器，所述第一容器内含有所述引物对组合物。6. The kit according to claim 4, further comprising a first container containing the primer pair composition.7.一种如权利要求1-3任意一项所述的引物对组合物用于检测转基因苜蓿的方法，其特征在于，所述方法包括以下步骤：7. A primer pair composition as described in any one of claims 1-3 is used to detect the method for transgenic alfalfa, it is characterized in that, described method comprises the following steps:得到待测苜蓿的DNA和所述引物对组合物；Obtain the DNA of alfalfa to be tested and the primer pair composition;以所述DNA为模板，将所述引物对组合物加入反应体系中，进行扩增反应，得到扩增产物；Using the DNA as a template, adding the primer pair composition into the reaction system, performing an amplification reaction, and obtaining an amplification product;将所述扩增产物进行高通量测序，得到高通量文库；performing high-throughput sequencing on the amplified product to obtain a high-throughput library;将所述高通量文库中的基因序列进行分析，得到检测转基因苜蓿的结果。The gene sequence in the high-throughput library is analyzed to obtain the result of detecting the transgenic alfalfa.8.苜蓿引物对组合物的用途，其特征在于，所述引物对组合物用于检测苜蓿转基因成分和转基因品系；8. the purposes of alfalfa primer pair composition, it is characterized in that, described primer pair composition is used for detecting alfalfa transgenic composition and transgenic line;所述引物对组合物包括序列如SEQ ID NO.1至SEQ ID NO.24所示的引物。The primer pair composition includes primers whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.24.

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