技术领域Technical Field
本发明属于生物技术领域，特别涉及一种检测转基因番木瓜的引物对组合、试剂盒及检测方法。The invention belongs to the field of biotechnology, and particularly relates to a primer pair combination, a kit and a detection method for detecting transgenic papaya.
背景技术Background Art
番木瓜是全球普遍种植的作物，含有丰富的植物蛋白和食用油脂，是重要的食用和饲用作物。在当前难以大幅增加番木瓜种植面积的背景下，利用现代生物技术培育高产优质转基因番木瓜新品种，对于促进番木瓜产业振兴具有十分重要的意义。但随着大量的转基因番木瓜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。Papaya is a crop commonly grown around the world. It is rich in plant protein and edible oils and is an important food and feed crop. In the current context where it is difficult to significantly increase the papaya planting area, the use of modern biotechnology to cultivate high-yield and high-quality transgenic papaya new varieties is of great significance for promoting the revitalization of the papaya industry. However, as a large number of transgenic papayas are directly or indirectly made into food and the international community is paying increasing attention to the safety of transgenic products, the detection of transgenic ingredients in agricultural products has been included in the detection projects of domestic and foreign inspection and quarantine departments and has been gradually strengthened. Therefore, it is crucial to develop efficient and convenient transgenic 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，但一般不超过六重，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 technology of genetically modified products mainly includes 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 genetically modified detection technology, which mainly includes ordinary qualitative PCR, nested PCR, loop-mediated isothermal amplification technology (LAMP), fluorescent quantitative PCR multiple PCR and other methods. Compared with the ordinary qualitative PCR method, nested PCR has high detection sensitivity and is prone to false positives. LAMP is simple to operate and has strong specificity, but the primer design is relatively complex and is prone to DNA contamination, affecting 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. The ordinary multiple PCR method can detect multiple genes simultaneously in one reaction, but generally not more than six times, otherwise the interference between primers is large, affecting the detection effect. Gene chips and digital PCR technology are also commonly used technologies for detecting 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, they are expensive, require specialized instruments and equipment, and require operators to have high professional qualities. These factors limit the widespread 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, developing an efficient, sensitive and high-throughput method for detecting genetically modified products has become a key issue that needs to be urgently addressed.
发明内容Summary of the invention
本申请提供了一种检测番木瓜转的引物对组合、试剂盒及检测方法，以解决如何高效检测转基因番木瓜和品系的技术问题。The present application provides a primer pair combination, a kit and a detection method for detecting transgenic papaya, so as to solve the technical problem of how to efficiently detect transgenic papaya and strains.
第一方面，本申请提供了一种检测转基因番木瓜的引物对组合，所述引物对组合包括:In a first aspect, the present application provides a primer pair combination for detecting transgenic papaya, 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 primer pair for specific amplification of 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 primer pair for specific amplification of 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 primer pair for specific amplification of pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6;
特异性扩增tNOS的引物对，其核苷酸序列如SEQ ID NO.7至SEQ ID NO.8所示；A primer pair for specific amplification of tNOS, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8;
特异性扩增NPtII的引物对，其核苷酸序列如SEQ ID NO.9至SEQ ID NO.10所示；A primer pair for specific amplification of NPtII, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;
特异性扩增PRSV_CP的引物对，其核苷酸序列如SEQ ID NO.11至SEQ ID NO.12所示；A primer pair for specific amplification of PRSV_CP, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;
特异性扩增replicase的引物对，其核苷酸序列如SEQ ID NO.13至SEQ ID NO.14所示；A primer pair for specifically amplifying replicase, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14;
和/或，特异性扩增GUS的引物对，其核苷酸序列如SEQ ID NO.15至SEQ ID NO.16所示。And/or, a primer pair for specific amplification of GUS, whose nucleotide sequences are shown in SEQ ID NO.15 to SEQ ID NO.16.
可选的，所述引物对组合包括：Optionally, the primer pair combination includes:
特异性扩增Rainbow-left的引物对，其核苷酸序列如SEQ ID NO.17至SEQ IDNO.18所示；A primer pair for specific amplification of Rainbow-left, the nucleotide sequences of which are shown in SEQ ID NO.17 to SEQ ID NO.18;
特异性扩增Rainbow-right的引物对，其核苷酸序列如SEQ ID NO.19至SEQ IDNO.20所示；A primer pair for specific amplification of Rainbow-right, the nucleotide sequences of which are shown in SEQ ID NO.19 to SEQ ID NO.20;
和/或，特异性扩增华农1号的引物对，其核苷酸序列如SEQ ID NO.21至SEQ IDNO.22所示。And/or, a primer pair for specific amplification of Huanong No. 1, whose nucleotide sequence is shown in SEQ ID NO.21 to SEQ ID NO.22.
可选的，所述引物对组合还包括用于扩增番木瓜内参基因CaP_papain的引物对。Optionally, the primer pair combination also includes a primer pair for amplifying the papaya internal reference gene CaP_papain.
可选的，扩增番木瓜内参基因CaP_papain的引物对，其核苷酸序列如SEQ IDNO.23至SEQ ID NO.24所示。Optionally, the primer pair for amplifying the papaya internal reference gene CaP_papain has a nucleotide sequence as shown in SEQ ID NO.23 to SEQ ID NO.24.
可选的，所述引物对组合包括：Optionally, the primer pair combination includes:
特异性扩增选自下组的番木瓜转基因元件的引物对：p35S、t35S、pNOS、tNOS、NPtII、PRSV_CP、replicase和GUS；所述引物对组合还包括特异性扩增选自下组的番木瓜转基因品系特异性序列的引物对：Rainbow-left、Rainbow-right和华农1号。A primer pair that specifically amplifies papaya transgenic elements selected from the following group: p35S, t35S, pNOS, tNOS, NPtII, PRSV_CP, replicase and GUS; the primer pair combination also includes a primer pair that specifically amplifies papaya transgenic line-specific sequences selected from the following group: Rainbow-left, Rainbow-right and Huanong No. 1.
第二方面，本申请提供了一种检测番木瓜转基因的试剂盒，所述试剂盒包括第一方面所述的检测转基因番木瓜的所述引物对组合。In a second aspect, the present application provides a kit for detecting transgenic papaya, the kit comprising the primer pair combination for detecting transgenic papaya described in the first aspect.
可选的，所述试剂盒包括第一容器，所述第一容器内含有所述引物对组合。Optionally, the kit includes a first container containing the primer pair combination.
可选的，所述试剂盒还包括多重PCR预混液。Optionally, the kit further comprises a multiplex PCR premix.
第三方面，本申请提供了第一方面所述的引物对组合、第二方面的所述的检测试剂盒在检测转基因番木瓜及其相关产品中的应用。In a third aspect, the present application provides the use of the primer pair combination described in the first aspect and the detection kit described in the second aspect in detecting transgenic papaya and related products thereof.
第四方面，本申请提供了一种检测转基因番木瓜的方法，所述方法包括以下步骤：In a fourth aspect, the present application provides a method for detecting transgenic papaya, the method comprising the following steps:
得到待测番木瓜的DNA和第一方面所述的引物对组合；Obtaining DNA of papaya to be tested and the primer pair combination described in the first aspect;
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 a reaction system, performing an amplification reaction, and obtaining an amplified 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，得到检测转基因番木瓜的结果。The gene sequence in the high-throughput library is analyzed to obtain the result of detecting the transgenic papaya.
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：The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:
本申请实施例提供的所述引物对组合，其核苷酸序列如SEQ ID NO.1至SEQ IDNO.16所示；通过一次高通量检测，实现多种转基因成分的定性与定量检测，避免了现有Real-time技术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，同时避免了该技术出现的假阳性或假阴性结果，因此本申请的引物对组合具有高效、准确和灵敏的优势，应用前景较好。The primer pair combination provided in the embodiment of the present application has a nucleotide sequence as shown in SEQ ID NO.1 to SEQ ID NO.16; through a single high-throughput detection, qualitative and quantitative detection of multiple genetically modified components is achieved, avoiding the need for multiple amplifications and detections in the existing Real-time technology to cover multiple target genetically modified components in the sample, while avoiding the false positive or false negative results of the technology. Therefore, the primer pair combination of the present application has the advantages of high efficiency, accuracy and sensitivity, and has a good application prospect.
附图说明BRIEF DESCRIPTION OF THE 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 embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.
图1为本申请实施例提供的一种检测转基因番木瓜的方法的流程示意图。FIG1 is a schematic diagram of a process for detecting transgenic papaya provided in an embodiment of the present application.
具体实施方式DETAILED DESCRIPTION
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 purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution 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 are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。本文中所使用的专业术语只是为了描述具体实施例的目的，并不是旨在限制本发明的保护范围。例如，室温可以是指10～35℃区间内的温度。Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. If there is a conflict, the present specification takes precedence. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. For example, room temperature can refer to a temperature within the range of 10 to 35°C.
除非另有特别说明，本发明中用到的各种原材料、试剂、仪器和设备等，均可通过市场购买得到或者可通过现有方法制备得到。Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.
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 technical problems, and the overall idea is as follows:
根据本发明一种典型的实施方式，提供了一种检测转基因番木瓜的引物对组合，所述引物对组合包括：特异性扩增p35S的引物对，其核苷酸序列如SEQ ID NO.1至SEQ IDNO.2所示；According to a typical embodiment of the present invention, a primer pair combination for detecting transgenic papaya is provided, the primer pair combination comprising: a primer pair for specific amplification of 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 primer pair for specific amplification of 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 primer pair for specific amplification of pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6;
特异性扩增tNOS的引物对，其核苷酸序列如SEQ ID NO.7至SEQ ID NO.8所示；A primer pair for specific amplification of tNOS, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8;
特异性扩增NPtII的引物对，其核苷酸序列如SEQ ID NO.9至SEQ ID NO.10所示；A primer pair for specific amplification of NPtII, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;
特异性扩增PRSV_CP的引物对，其核苷酸序列如SEQ ID NO.11至SEQ ID NO.12所示；A primer pair for specific amplification of PRSV_CP, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;
特异性扩增replicase的引物对，其核苷酸序列如SEQ ID NO.13至SEQ ID NO.14所示；A primer pair for specifically amplifying replicase, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14;
和/或，特异性扩增GUS的引物对，其核苷酸序列如SEQ ID NO.15至SEQ ID NO.16所示。And/or, a primer pair for specific amplification of GUS, whose nucleotide sequences are shown in SEQ ID NO.15 to SEQ ID NO.16.
具体地，转基因番木瓜包括含转基因元件与转基因品系的番木瓜。Specifically, transgenic papaya includes papaya containing transgenic elements and transgenic lines.
在一些实施方式中，所述引物对组合包括：In some embodiments, the primer pair combination comprises:
特异性扩增Rainbow-left的引物对，其核苷酸序列如SEQ ID NO.17至SEQ IDNO.18所示；A primer pair for specific amplification of Rainbow-left, the nucleotide sequences of which are shown in SEQ ID NO.17 to SEQ ID NO.18;
特异性扩增Rainbow-right的引物对，其核苷酸序列如SEQ ID NO.19至SEQ IDNO.20所示；A primer pair for specific amplification of Rainbow-right, the nucleotide sequences of which are shown in SEQ ID NO.19 to SEQ ID NO.20;
和/或，特异性扩增华农1号的引物对，其核苷酸序列如SEQ ID NO.21至SEQ IDNO.22所示。And/or, a primer pair for specific amplification of Huanong No. 1, whose nucleotide sequence is shown in SEQ ID NO.21 to SEQ ID NO.22.
优选的，开发了12对引物，所述引物互相间不影响，可以通过多重PCR进行高效的扩增。所述多重PCR引物组合可以用于开发番木瓜转基因元件及品系检测试剂盒；同时，每对引物由正向引物和反向引物组成。Preferably, 12 pairs of primers are developed, which do not affect each other and can be amplified efficiently by multiplex PCR. The multiplex PCR primer combination can be used to develop a papaya transgenic element and strain detection kit; at the same time, each pair of primers consists of a forward primer and a reverse primer.
本申请实施例中，所述引物对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；避免了现有Real-time技术一次只能检测一个靶标分子，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，同时避免了该技术出现的假阳性或假阴性结果，一次实验可以实现9个以上的PCR反应，提高了检测效率、通量与灵敏度，并兼顾了检测成本。在一些实施方式中，所述引物对组合包括：In the embodiment of the present application, the amplified product of the primer pair combination can be subjected to a high-throughput sequencing and analysis to obtain multiple test results, including transgenic components, determining whether the sample to be tested contains target molecules, and determining the number of copies of the reference gene and the target molecule in the sample to be tested, so as to determine the content of the exogenous gene; it avoids the existing Real-time technology that can only detect one target molecule at a time, and requires multiple amplifications and tests to cover multiple target transgenic components in the sample, while avoiding the false positive or false negative results of the technology, and one experiment can achieve more than 9 PCR reactions, thereby improving the detection efficiency, throughput and sensitivity, and taking into account the detection cost. In some embodiments, the primer pair combination includes:
上述引物与其扩增的上述番木瓜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 above papaya transgenic elements and lines amplified by them, the numbers of the corresponding primer pairs and the nucleotide sequences of the primer pairs 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 and primer sequences screened by the present invention.
在引物设计时，为了增强所述引物的适用性和灵敏性，所设引物长度介于在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。When designing primers, in order to enhance the applicability and sensitivity of the primers, the length of the primers is set between 18-30bp, the primers do not interfere with each other, and all 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. It has been confirmed by use that they have high sensitivity and strong applicability.
在一些实施方式中，所述引物对组合还包括用于扩增番木瓜内参基因CaP_papain的引物对。In some embodiments, the primer pair combination also includes a primer pair for amplifying the papaya internal reference gene CaP_papain.
为了实现对样品中检测番木瓜转基因元件及品系的目的，在选用了上述番木瓜转基因元件或品系时，加入对番木瓜内参基因的检测引物，以实现转基因成分含量的定量检测。In order to detect papaya transgenic elements and strains in samples, when the above papaya transgenic elements or strains are selected, detection primers for papaya internal reference genes are added to achieve quantitative detection of the transgenic component content.
在一些实施方式中，扩增番木瓜内参基因CaP_papain的引物对，其核苷酸序列如SEQ ID NO.23至SEQ ID NO.24所示。In some embodiments, the primer pair for amplifying the papaya internal reference gene CaP_papain has a nucleotide sequence as shown in SEQ ID NO.23 to SEQ ID NO.24.
选用2对内参基因引物对的原因在于避免内参基因的不稳定和低含量样品中1对内参基因引物无法把目标内参基因有效检出。The reason for selecting two pairs of internal reference gene primers is to avoid the instability of the internal reference genes and the inability of one pair of internal reference gene primers to effectively detect the target internal reference gene in low-content samples.
在一些实施方式中，所述引物对数范围为：1-12对之间根据具体检测样品的情况适当调整。后期可根据新收集的转基因元件或品系进行定期增加，引物组合尝试过3000对，扩增效果依然很好。为了实现对转基因番木瓜的检测，收集了番木瓜中8个常用的转基因元件和3种转基因品系，覆盖了市面上大多数转基因番木瓜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，包括但不限于：2、3、4、5、6、7、8、9、10和11对；相比常规的6对以下的特异性多重PCR，具有检测通量和灵敏度高的优势。In some embodiments, the number of primer pairs ranges from 1 to 12 pairs, which can be appropriately adjusted according to the specific conditions of the test sample. In the later stage, it can be regularly increased according to the newly collected transgenic elements or strains. The primer combination has been tried for more than 3,000 pairs, and the amplification effect is still very good. In order to detect transgenic papaya, 8 commonly used transgenic elements and 3 transgenic strains in papaya were collected, covering the commonly used transgenic elements of most transgenic papaya strains on the market. The number of pairs of the multiplex PCR primers ranges from 1 to 12 pairs, including but not limited to: 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 pairs; compared with the conventional specific multiplex PCR with less than 6 pairs, it has the advantages of high detection throughput and sensitivity.
在一些实施方式中，所述引物对组合包括特异性扩增选自下组的番木瓜转基因元件的引物对：p35S、t35S、pNOS、tNOS、NPtII、PRSV_CP、replicase和GUS；所述引物对组合还包括特异性扩增选自下组的番木瓜转基因品系特异性序列的引物对：Rainbow-left、Rainbow-right和华农1号。In some embodiments, the primer pair combination includes a primer pair that specifically amplifies a papaya transgenic element selected from the following group: p35S, t35S, pNOS, tNOS, NPtII, PRSV_CP, replicase and GUS; the primer pair combination also includes a primer pair that specifically amplifies a papaya transgenic line-specific sequence selected from the following group: Rainbow-left, Rainbow-right and Huanong No. 1.
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番木瓜转基因元件的其他引物对引物的对数组合，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。On the basis of the existing primer pair combinations, other primer pairs that specifically amplify the same papaya transgenic elements can also be included. The multiple PCR primer pair arrays can also be regularly added according to the newly collected transgenic elements in the later stage, which has been verified to be up to 3,000 pairs.
第二方面，本申请提供了一种检测转基因番木瓜的试剂盒，所述试剂盒包括第一方面所述的检测转基因番木瓜的所述引物对组合。In a second aspect, the present application provides a kit for detecting transgenic papaya, the kit comprising the primer pair combination for detecting transgenic papaya described in the first aspect.
在一些实施方式中，所述试剂盒包括第一容器，所述第一容器内含有所述引物对组合。In some embodiments, the kit includes a first container containing the primer pair combination.
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 comprises a multiplex PCR premix.
具体的，多重PCR预混液的组分包括所述扩增转基因番木瓜元件、品系和内参基因的各引物组时，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，具体实施例中，每条引物浓度是2-2.5nM。优选的，每条引物浓度为2.1、2.2和2.3nM。Specifically, when the components of the multiplex PCR premix include the primer sets for amplifying transgenic papaya elements, strains and internal reference genes, each primer is premixed at a ratio of 1:1, and the primers are mixed according to different experimental purposes. In a specific embodiment, the concentration of each primer is 2-2.5 nM. Preferably, the concentration of each primer is 2.1, 2.2 and 2.3 nM.
第三方面，本申请提供了第一方面所述的引物对组合、第二方面的所述的检测试剂盒在检测转基因番木瓜及其相关产品中的应用。In a third aspect, the present application provides the use of the primer pair combination described in the first aspect and the detection kit described in the second aspect in detecting transgenic papaya and related products thereof.
第四方面，本申请提供了一种检测转基因番木瓜的方法，所述方法包括以下步骤：In a fourth aspect, the present application provides a method for detecting transgenic papaya, the method comprising the following steps:
得到待测番木瓜的DNA和第一方面所述的引物对组合；Obtaining DNA of papaya to be tested and the primer pair combination described in the first aspect;
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 a reaction system, performing an amplification reaction, and obtaining an amplified 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，得到检测转基因番木瓜的结果。The gene sequence in the high-throughput library is analyzed to obtain the result of detecting the transgenic papaya.
具体地，高通量测序可以是二代测序，也可以是三代测序，得到的高通量文库可以从多个维度分析番木瓜中转基因成分，包括但不限于实施案例中的番木瓜转基因元件或品系。Specifically, high-throughput sequencing can be second-generation sequencing or third-generation sequencing, and the resulting high-throughput library can analyze the transgenic components in papaya from multiple dimensions, including but not limited to the transgenic elements or strains of papaya in the implementation case.
在一些实施例中，使用上述方法，可一次性对多样本的所有靶标转基因成分进行检测，具有高通量、高灵敏、准确和快速等优势，可应用于转基因番木瓜品系及其制品的转基因成分的定性与定量检测。In some embodiments, the above method can be used to detect all target transgenic components of multiple samples at one time, which has the advantages of high throughput, high sensitivity, accuracy and rapidity, and can be applied to the qualitative and quantitative detection of transgenic components of transgenic papaya lines and their products.
本申请实施例中，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TouchDowncycle，(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 embodiment of the present application, the environment/procedure of the amplification reaction includes: pre-denaturation at 94°C for 5 minutes; the first step of amplification reaction, denaturation at 94°C for 15 seconds, annealing at 62°C to 56°C for 30 seconds, and 12 TouchDown cycles (the temperature of annealing and extension is reduced by 0.5°C in each cycle); the second step of amplification reaction, denaturation at 94°C for 15 seconds, annealing at 57°C for 30 seconds, and 22 cycles.
本申请实施例中，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；剩余的用水补充；所述高通量文库的浓度大于2ng/ul为合格。In the embodiment of the present application, the reaction system includes: 40 μl of the total system, 2.5 μl of primer premix, 2×buffer: 20 μl, and multiplex PCR amplification enzyme: 0.5 μl; the rest is supplemented with water; the rest is supplemented with water; the concentration of the high-throughput library is qualified if it is greater than 2 ng/ul.
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TouchDown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/procedure of the amplification reaction in the method described 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 TouchDown cycles (the temperature of annealing and extension is reduced by 0.8°C in each cycle); the second step of 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；所述高通量文库的浓度大于2ng/ul为合格。More preferably, the reaction system of the method comprises: a total system of 30 μl, primer pairs: 2 μl, 2×buffer: 15 ul, multiplex amplification enzyme: 0.5 μl; the remainder is supplemented with water; the concentration of the high-throughput library is qualified if it is greater than 2 ng/ul.
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％的番木瓜转基因产品。The kit provided by the invention can sensitively detect transgenic papaya products with a transgenic 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＝98.6％。In the reproducibility test of the present invention, the reproducibility rate of the detection results between different libraries of each sample and between different library construction batches is r=100%, and the accuracy rate is a=98.6%.
本发明的所述试剂盒在复杂模板中检测多种番木瓜转基因品系或样品的多种转基因元件，具有高度特异性。The kit of the present invention detects multiple transgenic elements of multiple papaya transgenic lines or samples in a complex template with high specificity.
上述引物与其扩增的上述番木瓜转基因元件及品系的核苷酸序列即靶标分子与相应引物对的编号及引物的核苷酸序列具体对应关系如表1所示。The specific correspondence between the primers and the nucleotide sequences of the papaya transgenic elements and strains amplified by them, that is, the numbers of the target molecules and the corresponding primer pairs and the nucleotide sequences of the primers, is shown in Table 1.
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 with reference to embodiments, 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 Screening of target transgenic elements and 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番木瓜内参基因，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 papaya reference genes used in the examples are mainly collected from commonly used transgenic databases, national standards, industry standards or existing literature, and are collected as completely as possible to ensure the specificity and accuracy of the detection. The names of the transgenic elements, strains and reference genes screened are shown in Table 1.
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 embodiment of the present application, multiple PCR primers are designed using Primer3Plus, and the length of the primers is between 18-30bp, and the primers do not interfere with each other. This mainly evaluates the dimers between primers, or the hairpin structure inside the primers, and the non-specific amplification of non-target sequences. All primers after evaluation can be combined into a primer pool for multiple PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction. Specific primer sequences include: SEQ ID NO.1 to SEQ ID NO.24.
实施例2检测番木瓜样品是否含有转基因成分Example 2 Detection of whether papaya samples contain genetically modified ingredients
1.实验材料：转基因材料华农1号。实验材料转入了p35S、NPtII、pNOS、tNOS、replicase，其转基因含量为10％，用其作为研究材料。1. Experimental materials: Transgenic material Huanong No. 1. p35S, NPtII, pNOS, tNOS, and replicase were introduced into the experimental material, and its transgenic content was 10%, and it was used as research material.
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 genomes were extracted using CTAB or the high-efficiency plant genome DNA extraction kit (DP350) of Tiangen Biochemical Technology (Beijing) Co., Ltd. In this example, the DNA of the test samples was extracted using the Tiangen DNA extraction kit, and three biological replicates were performed for each sample.
3.PCR扩增、文库构建与测序3. PCR amplification, library construction and sequencing
利用22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。The genomic DNA of the sample is amplified using 22 pairs of multiplex PCR amplification primers; the amplified products of each sample are connected to the sequencing adapter and the specific sample DNA barcode and then mixed to form a high-throughput sequencing library; the high-throughput sequencing library is detected using a high-throughput sequencing platform and the high-throughput sequencing data is quality controlled. This step requires the adjustment of 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 to the third-generation sequencing to achieve complementary advantages between the second-generation sequencing and the third-generation sequencing.
4.结果的判定4. Determination 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元件或品系的信号指数P判定污染是否可接受，其中：1) Determine whether the contamination is acceptable based on the signal index S of the transgenic strain in the test sample and the signal index P of the transgenic element or strain in the blank control, 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，转基因元件或品系的测序片段的数量和总测序片段数量。The blank control noise index P = nc/Nc, where nc and Nc represent the number of sequenced fragments of the transgenic element or strain and the total number of sequenced fragments 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，转基因元件或品系的测序片段的数量和总测序片段数量。The signal index S of the test sample is nt/Nt, wherein nt and Nt represent the number of sequenced fragments of the transgenic element or line and the total number of sequenced fragments in the test sample, respectively.
信噪比＝S/PSignal-to-Noise Ratio = S/P
2)转基因结果的判断2) Determination of transgenic 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或品系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 and homology alignment of the sample to be tested, each sequencing fragment is assigned to each target position of each target species, and the targets include transgenic elements, strains and internal reference genes. According to the number of sequencing sequences at each target position, the absolute quantification of transgenic elements or strains is achieved. When the sequencing sequences of the internal reference gene and the transgenic element or strain exceed the specified threshold, it is qualitatively determined that the sample contains transgenic components; when the sample contains transgenic components, the content of the exogenous gene in the sample is quantitatively determined based on the ratio of the sequencing sequences of the transgenic element and strain to the internal reference gene. The calculation formula of the transgenic content in this embodiment is shown in (A):
CtestDNA——测试样品的转基因含量CtestDNA - Testing samples for GMO content
tTi——测试样品中的每种转基因元件和品系的测序序列数目tTi - the number of sequences sequenced for each transgenic element and line in the test sample
tRi——测试样品中检出的每种内参基因片段的测序序列目tRi——Sequencing sequence of each reference gene fragment detected in the test sample
m——测试样品中检出的内参基因片段的总数m——The total number of internal reference gene fragments detected in the test sample
n——标准品中检出的转基因元件和品系片段的总数n——The total number of transgenic elements and strain fragments detected in the standard
根据本实例，共检测了2个样品，1个转基因品系华农1号和一个阴性样品，每个样品做了三个生物重复，结果如表2和图1所示：阴性样品中常用的启动子和终止子在阴性番木瓜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。当样品中转基因元件或品系的信噪比大于10，判定样本中检出转基因元件或品系的核酸。According to this example, a total of 2 samples were tested, 1 transgenic line Huanong No. 1 and a negative sample, and three biological replicates were made for each sample. The results are shown in Table 2 and Figure 1: Several sequences of the promoters and terminators commonly used in negative samples will also be detected in negative papayas, and sequences with less than 5 sequencing reads are required to be filtered out. The present invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be determined that the contamination in the detection system is acceptable. When the signal-to-noise ratio of the transgenic element or line in the sample is greater than 10, it is determined that the nucleic acid of the transgenic element or line 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 Genetically modified test results of the samples tested in Example 2.
由表可知，华农1号中的所对应的各转基因元件和品系在三个重复实验中均被有效检出，而且含量接近于其实际含量；从该表说明番木瓜转基因试剂盒可以用来检测转基因产品。As can be seen from the table, the corresponding transgenic elements and strains in Huanong No. 1 were effectively detected in three repeated experiments, and the content was close to its actual content; the table shows that the papaya transgenic kit can be used to detect transgenic products.
实施例3准确性、特异性与灵敏度评估Example 3 Accuracy, specificity and sensitivity evaluation
转基因番木瓜品种华农1号和YK1601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，具体地把转基因番木瓜华农1号和YK1601用阴性番木瓜分别稀释成10％，1％，0.1％，0.05％，0.025％和0.01％的样本，分别对应于转基因品系华农1号的稀释样本编号(A1，A2，A3，A4，A5，A6)和转基因品系YK1601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，能够检出的转基因元件或品系的最低含量，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。Transgenic papaya varieties Huanong No. 1 and YK1601 transgenic standards were used to prepare transgenic samples with different mass percentages to evaluate the accuracy, specificity and sensitivity of the developed technology. Specifically, the transgenic content of each sample was diluted by mass percentage. Specifically, transgenic papaya Huanong No. 1 and YK1601 were diluted with negative papaya 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 Huanong No. 1 and the dilution sample numbers (B1, B2, B3, B4, B5, B6) of the transgenic line YK1601. The accuracy of qualitative detection refers to the proportion of true positives to true negatives, and the quantitative accuracy refers to the degree to which the average value of multiple measurements is consistent with the true value, expressed as an error. Specificity is also called the true negative rate, which is the percentage of true negatives detected by multiple tests to all negatives. Sensitivity refers to the minimum content of transgenic elements or strains that can be detected at 95% confidence level, that is, the detection limit. The test was performed according to the method of Example 2, with three biological replicates for each sample. The results are shown in Table 3.
表3本发明方法的准确性与灵敏度评估Table 3 Accuracy and sensitivity evaluation of the method of the present invention
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: + represents detected, - represents undetected, A1 and B1 represent 10% GMO content, A2 and B2 represent 1% GMO content, A3 and B3 represent 0.1% GMO content, A4 and B4 represent 0.05% GMO content, 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或品系，而在阴性的样本中最多检出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。As can be seen from the table, the kit can stably detect various transgenic elements or strains in samples with a transgenic content of 0.05%, and can detect at most one transgenic component in negative samples, indicating that the kit has strong specificity and can clearly distinguish between samples with a transgenic content of 0.05% and negative samples, and has technical stability and detection sensitivity of a transgenic content of 0.05%.
实施例4Example 4
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143份转基因成分未知的番木瓜叶片样本进行检测，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143份测试样品中，只有2份样品的结果不一致，检测结果的一致性高达98.6％，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 its role in the detection of genetically modified organisms in batch samples, the laboratory selected 143 papaya leaf samples of unknown genetically modified organisms from a company for detection, and used the detection method of Example 2 to compare the detection results with the company's storage type, and statistically analyzed the consistency of the results. The analysis results showed that among the 143 test samples, only 2 samples had inconsistent results, and the consistency of the detection results was as high as 98.6%, thus well proving the accuracy of the method.
本发明实施例中的一个或多个技术方案，至少还具有如下技术效果或优点：One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
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 detection efficiency of transgenic products;
2)检验对象全，包含了番木瓜当前常见的8种转基因元件和3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 objects are complete, including 8 common transgenic elements and 3 transgenic strains of papaya, and new detection target sequences can be easily added to avoid the failure of single target amplification, thus improving the specificity, accuracy and sensitivity of the test;
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，适用于转基因番木瓜及其制品的大规模检测。3) The kit integrates the second-generation sequencing platform to sequence the amplified products, which improves the throughput and repeatability of the detection system, and the test results can be directly digitized, which is suitable for large-scale detection of genetically modified papaya and its products.
因此，本申请克服了现有检测技术费时费力成本高的缺陷，提供的番木瓜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。Therefore, the present application overcomes the defects of existing detection technologies, such as being time-consuming, labor-intensive and costly. The papaya transgenic variety detection kit provided is simple to operate, rapid and sensitive, has a large detection throughput, good repeatability of detection results, and low cost for multi-sample and multi-target sequence detection. It has important applications in the detection of transgenic products at seed stations, agricultural science institutes and customs import and export ports.
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, relational 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 any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the existence of other identical elements in the process, method, article or equipment including the elements.
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 foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210221988.9ACN114622028B (en) | 2022-03-07 | 2022-03-07 | Primer pair combination, kit and detection method for detecting transgenic papaya |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210221988.9ACN114622028B (en) | 2022-03-07 | 2022-03-07 | Primer pair combination, kit and detection method for detecting transgenic papaya |
| Publication Number | Publication Date |
|---|---|
| CN114622028A CN114622028A (en) | 2022-06-14 |
| CN114622028Btrue CN114622028B (en) | 2023-12-22 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210221988.9AActiveCN114622028B (en) | 2022-03-07 | 2022-03-07 | Primer pair combination, kit and detection method for detecting transgenic papaya |
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