技术领域technical field
本发明涉及生物技术领域，具体涉及一种利用通用引物多重PCR技术检测转基因番木瓜的方法。The invention relates to the field of biotechnology, in particular to a method for detecting transgenic papaya by using the multiplex PCR technology of general primers.
背景技术Background technique
番木瓜是我国重要的热带、亚热带优质水果，主要在云南、海南、广东、福建、广西、台湾等省区种植，不仅可以食用，还具有极其重要的工业应用价值。但由于病害严重，特别是番木瓜环斑病毒Papaya ring spot virus(PRSV)病,导致其品质下降，产量降低，极大地影响了番木瓜在市场的经济效益。20世纪90年代初，Fitch等把编码PRSV衣壳蛋白(Coat prote in,CP)基因转入番木瓜，获得了转CP基因的抗病品系，并于1997年获准进入商品化生产，在美国夏威夷广泛种植。但该品系对我国华南地区、台湾地区以及泰国等亚洲国家的PRSV株系无抗性效果。华南农业大学植物病毒研究室通过转番木瓜环斑病毒(papaya ring spot virus，PRSV)广东地区优势株系Ys复制酶(Rep)基因的方法，获得了高度抗PRSV的转基因番木瓜植株“华农一号”，并于2004年获准进行环境和食用安全评价，2006年获得农业部颁发的“转基因番木瓜华农一号在广东省应用”的安全证书[农基安证字(2006)第001号],这是我国第1例获准进行商品化的转基因果树作物，已进入市场进行商业化生产。Papaya is an important tropical and subtropical high-quality fruit in my country. It is mainly grown in Yunnan, Hainan, Guangdong, Fujian, Guangxi, Taiwan and other provinces and regions. It is not only edible, but also has extremely important industrial application value. However, due to serious diseases, especially the Papaya ring spot virus (PRSV) disease, the quality of papaya is reduced and the yield is reduced, which greatly affects the economic benefits of papaya in the market. In the early 1990s, Fitch et al. transferred the gene encoding PRSV capsid protein (Coat protein in, CP) into papaya, and obtained a disease-resistant strain of the transgenic CP gene, which was approved for commercial production in 1997. Widely planted. However, the strain has no resistance to PRSV strains in South my country, Taiwan, Thailand and other Asian countries. The Plant Virus Research Laboratory of South China Agricultural University has obtained the highly resistant PRSV transgenic papaya plant "Hua Nong No. In 2004, it was approved to conduct environmental and food safety assessments. In 2006, it obtained the safety certificate of "Transgenic Papaya Huanong No. 1 Application in Guangdong Province" issued by the Ministry of Agriculture [Nong Ji An Zheng Zi (2006) No. 001], This is the first transgenic fruit tree crop approved for commercialization in my country, and it has entered the market for commercial production.
基于转基因食品潜在安全的不确定性，世界各国政府都加强对转基因食品进行管理，以保障本国消费者的知情权。目前国内外对转基因作物进行检测，检测技术综合起来主要有两大类型：一是基于转入的外源基因核酸水平上的检测；二是基于转入外源基因表达产物蛋白质水平上的检测。国际上普遍采用以核酸为基础的PCR聚合酶链式反应(polymerase chain reaction，PCR)定性检测技术作为检测标准。Based on the uncertainty of the potential safety of genetically modified foods, governments around the world have strengthened the management of genetically modified foods to protect their consumers' right to know. At present, there are two main types of detection technologies for the detection of genetically modified crops at home and abroad: one is based on the detection of the nucleic acid level of the transferred exogenous gene; the other is based on the detection of the protein level of the expression product of the transferred exogenous gene. Nucleic acid-based PCR polymerase chain reaction (PCR) qualitative detection technology is generally used as the detection standard in the world.
随着转基因技术的迅猛发展，转基因作物的种类日益增多，品种达100多个，由转基因作物加工的食品和食品成分达4000多种,其外源基因成分也越来越复杂，需要同时对多个可能基因进行检测，检测任务逐渐增多，因此迫切需要研究开发出一种准确、快速、简便的多基因检测技术。但传统的多重PCR技术反应体系往往过于复杂，引物间存在排斥和抑制反应，以至于结果重复性不够好，一定程度上限制其高通量性。With the rapid development of genetically modified technology, the types of genetically modified crops are increasing day by day, with more than 100 varieties, and more than 4,000 kinds of food and food ingredients processed from genetically modified crops. Therefore, it is urgent to research and develop an accurate, fast and simple multi-gene detection technology. However, the reaction system of traditional multiplex PCR technology is often too complex, and there are exclusion and inhibition reactions between primers, so that the reproducibility of the results is not good enough, which limits its high-throughput to a certain extent.
发明内容Contents of the invention
本发明所要解决的技术问题是，为了克服传统多重PCR技术体系复杂、引物间排斥以及重现性差等缺陷，提供一种利用通用引物多重PCR技术检测转基因番木瓜的方法。该方法借助一条通用引物的以降低每条特异性引物的用量，可以克服传统多重PCR的引物间排斥、重现性差等缺点。The technical problem to be solved by the present invention is to provide a method for detecting transgenic papaya using the multiplex PCR technique with universal primers in order to overcome the defects of complex traditional multiplex PCR technique system, exclusion between primers and poor reproducibility. The method uses one universal primer to reduce the amount of each specific primer, and can overcome the disadvantages of traditional multiplex PCR such as exclusion between primers and poor reproducibility.
本发明所要解决的上述技术问题通过以下技术方案予以实现：The above-mentioned technical problems to be solved by the present invention are realized through the following technical solutions:
一种利用通用引物多重PCR技术检测转基因番木瓜的方法，包含如下步骤：A method for detecting transgenic papaya utilizing universal primer multiplex PCR technology, comprising the steps:
S1.设计1对通用引物和5对接有通用引物的特异性引物，所述引物的序列分别为：S1. Design 1 pair of universal primers and 5 specific primers docked with universal primers, the sequences of the primers are respectively:
Uni-Papain-F：CCTTCCTTCCTTCCACGCAATCTACAATCTTGCTAACCCTA；Uni-Papain-F: CCTTCCTTCCTTCCACGCAATCTACAAATCTTGCTAACCCTA;
Uni-Papain-R：CCTTCCTTCCTTCCACGCAAGTCATCTTGAGAATAACCCAC；Uni-Papain-R: CCTTCCTTCCTTCCACGCAAGTCATCTTGAGAATAACCCAC;
Uni-35s-F：CCTTCCTTCCTTCCACGCAAAGACGATCTACCCGAGCAA；Uni-35s-F: CCTTCCTTCCTTCCACGCAAAGACGATCTACCCGAGCAA;
Uni-35s-R：CCTTCCTTCCTTCCACGCAGAAGCAAGCCTTGAATCGTC；Uni-35s-R: CCTTCCTTCCTTCCACGCAGAAGCAAGCCTTGAATCGTC;
Uni-NptII-F：CCTTCCTTCCTTCCACGCACTGTGCTCGACGTTGTCACT；Uni-NptII-F: CCTTCCTTCCTTCCACGCACTGTGCTCGACGTTGTCACT;
Uni-NptII-R：CCTTCCTTCCTTCCACGCACTTCCATCCGAGTACGTGCT；Uni-NptII-R: CCTTCCTTCCTTCCACGCACTTCCATCCGAGTACGTGCT;
Uni-RP-F：CCTTCCTTCCTTCCACGCACTTTGGTGCGGAAAAGTTGT；Uni-RP-F: CCTTCCTTCCTTCCACGCACTTTGGTGCGGAAAAGTTGT;
Uni-RP-R：CCTTCCTTCCTTCCACGCACCATAGCCCACAGTCGAATT；Uni-RP-R: CCTTCCTTCCTTCCACGCACCATAGCCCACAGTCGAATT;
Uni-CP-F：CCTTCCTTCCTTCCACGCAAGAATGTTTGGAATGGACGG；Uni-CP-F: CCTTCCTTCCTTCCACGCAAGAATGTTTGGAATGGACGG;
Uni-CP-R：CCTTCCTTCCTTCCACGCAGCATACCCAGGAGAGAGTGC；Uni-CP-R: CCTTCCTTCCTTCCACGCAGCATACCCAGGAGAGAGTGC;
Universal-primer：CCTTCCTTCCTTCCACGCA；Universal-primer: CCTTCCTTCCTTCCACGCA;
S2.检测：以待检测番木瓜基因组DNA为模板，在PCR反应中加入S1.设计的所有引物，进行PCR、电泳，从而得知所检测的番木瓜是否为转基因番木瓜。S2. Detection: Using the genomic DNA of the papaya to be detected as a template, add all the primers designed in S1. to the PCR reaction, perform PCR and electrophoresis, so as to know whether the papaya to be detected is a transgenic papaya.
优选地，S2.中所述的PCR反应，其反应条件为：预变性94℃，3min；30个循环：变性94℃，30sec，退火62℃，30sec，延伸72℃，1min；终止延伸72℃，5min。Preferably, for the PCR reaction described in S2., the reaction conditions are: pre-denaturation at 94°C, 3min; 30 cycles: denaturation at 94°C, 30sec, annealing at 62°C, 30sec, extension at 72°C, 1min; stop extension at 72°C , 5min.
优选地，S2.中所述的PCR反应，其反应体系为30μL，含有2×MasterMix；引物Uni-Papain-F和Uni-Papain-R为0.15μL，引物Uni-35s-F和Uni-35s-R为0.2μL，引物Uni-NptII-F和Uni-NptII-R为0.4μL，引物Uni-RP-F和Uni-RP-R为0.3μL，引物Uni-CP-F和Uni-CP-R为0.3μL，通用引物Universal-primer为6μL，各引物的浓度为10μM；DNA模板为6μL，浓度为20ng/μL。Preferably, for the PCR reaction described in S2., the reaction system is 30 μL, containing 2×MasterMix; primers Uni-Papain-F and Uni-Papain-R are 0.15 μL, primers Uni-35s-F and Uni-35s- R is 0.2 μL, primers Uni-NptII-F and Uni-NptII-R are 0.4 μL, primers Uni-RP-F and Uni-RP-R are 0.3 μL, primers Uni-CP-F and Uni-CP-R are 0.3 μL, Universal-primer 6 μL, the concentration of each primer is 10 μM; DNA template is 6 μL, the concentration is 20 ng/μL.
优选地，S2.中使用的电泳为琼脂糖凝胶电泳。Preferably, the electrophoresis used in S2. is agarose gel electrophoresis.
更优选地，所述的琼脂糖凝胶电泳的方法为：取5μL PCR产物于质量体积比为2％的琼脂糖凝胶，1×TAE缓冲液中，稳压80V电泳30min。More preferably, the method of agarose gel electrophoresis is as follows: take 5 μL of the PCR product and place it on an agarose gel with a mass volume ratio of 2%, in 1×TAE buffer solution, and electrophoresis at a constant voltage of 80V for 30 minutes.
所述的方法，能同时对“Event 55-1”、“GM-YK”和“华农一号”三种转基因番木瓜进行检测。The method can simultaneously detect three kinds of transgenic papayas "Event 55-1", "GM-YK" and "Huanong No. 1".
优选地，所述的多重PCR技术为五重PCR技术。Preferably, the multiplex PCR technique is a five-fold PCR technique.
有益效果：(1)本发明首次成功开发了采用通用引物多重PCR技术检测转基因番木瓜；(2)本发明通用引物可提高多重PCR的扩增效率，大大地提高检测的灵敏度，同时还具备多重PCR减低检测成本的优点，在转基因番木瓜的检测的应用上来说，通用引物多重PCR技术具有很高的可行性；(3)本发明通用引物多重PCR的检测限为0.001ng，目标DNA或者更低，比普通引物的多重PCR效果提高了二个数量级以上。Beneficial effects: (1) The present invention has successfully developed for the first time the detection of transgenic papaya by using the multiplex PCR technique of the universal primer; The advantage of PCR reducing detection cost, in the application of the detection of transgenic papaya, universal primer multiplex PCR technology has very high feasibility; (3) the detection limit of universal primer multiplex PCR of the present invention is 0.001ng, target DNA or more Low, the effect of multiplex PCR is improved by more than two orders of magnitude compared with common primers.
附图说明Description of drawings
图1为通用引物及特异性引物(带接头)单重PCR特异性验证结果分析图。其中，图中各条带对应的情况为：1.引物CP；2.引物35S；3.引物NPTII；4.Papain引物；5.RP引物，6.带接头引物CP；7.带接头引物35S；8.带接头引物NPTII；9.带接头Papain引物；19.带接头RP；M.Marker。Figure 1 is an analysis diagram of the single-plex PCR specificity verification results of universal primers and specific primers (with adapters). Among them, the conditions corresponding to each band in the figure are: 1. Primer CP; 2. Primer 35S; 3. Primer NPTII; 4. Papain primer; 5. RP primer, 6. Adapter primer CP; ; 8. Primer NPTII with linker; 9. Primer Papain with linker; 19. RP with linker; M.Marker.
图2为通用引物多重PCR的验证图。其中，图中各条带对应的情况为：1.转基因番木瓜华农一号(带接头Papain引物)；2.转基因番木瓜“华农一号”(带接头35s引物)；3.转基因番木瓜“华农一号”(带接头35s引物和NtpII213引物)；4.转基因番木瓜“华农一号”(带接头Papain引物、35s引物和NtpII213引物)；5.转基因番木瓜GM-YK(含CP基因)(五种引物)；6.转基因番木瓜“华农一号”(含RP基因)(五种引物)；7转基因番木瓜GM-YK和“华农一号”混合(五种引物)；8空白；M Marker。Fig. 2 is a verification diagram of multiplex PCR with universal primers. Among them, the conditions corresponding to each band in the figure are: 1. Transgenic papaya Huanong No. 1 (with adapter Papain primer); 2. Transgenic papaya "Huanong No. 1" (with adapter 35s primer); 3. Transgenic papaya " "Huanong No.1" (with adapter 35s primer and NtpII213 primer); 4. Transgenic papaya "Huanong No.1" (with adapter Papain primer, 35s primer and NtpII213 primer); 5. Transgenic papaya GM-YK (with CP gene) (five primers); 6. Transgenic papaya "Huanong No. 1" (including RP gene) (five primers); 7 transgenic papaya GM-YK and "Huanong No. 1" mixed (five primers); 8 blank; M Marker.
图3为通用引物多重PCR灵敏度分析图。其中，图中各条带对应的情况为：Figure 3 is a graph showing the sensitivity analysis of the multiplex PCR of the universal primers. Among them, the situation corresponding to each strip in the figure is:
1.华农一号100ng，GM-YK 100ng；2.华农一号10ng，GM-YK 100ng；3.华农一号1ng，GM-YK 100ng；4.华农一号0.1ng，GM-YK 100ng；5.华农一号0.01ng，GM-YK 100ng；6.华农一号0.001ng，GM-YK 100ng；7.空白对照；M.Mark。1. Huanong No.1 100ng, GM-YK 100ng; 2. Huanong No.1 10ng, GM-YK 100ng; 3. Huanong No.1 1ng, GM-YK 100ng; 4. Huanong No.1 0.1ng, GM-YK 100ng; 5 .Huanong No.1 0.01ng, GM-YK 100ng; 6. Huanong No.1 0.001ng, GM-YK 100ng; 7. Blank control; M.Mark.
具体实施方式Detailed ways
本实施方式实施例中采用的通用引物单重或多重PCR技术检测转基因番木瓜的方法如下：The method for the detection of transgenic papaya by the universal primer single or multiplex PCR technology used in the embodiment of the present embodiment is as follows:
(1)待测试样品DNA的提取与纯化 (1) Extraction and purification of DNA from samples to be tested
采用三种转基因番木瓜(Event 55-1、GM-YK和华农一号)和普通番木瓜(台农2号)的新鲜叶片，取每种品种的新鲜番木瓜叶约100mg，加入液氮充分研磨，用DNA提取试剂盒提取各样品的DNA。紫外分光光度法来测定DNA提纯的浓度和纯度。Fresh leaves of three kinds of transgenic papaya (Event 55-1, GM-YK and Huanong No. 1) and ordinary papaya (Tainong No. 2) were used, and about 100 mg of fresh papaya leaves of each species were taken, and liquid nitrogen was added to fully Grind and extract DNA from each sample using a DNA extraction kit. UV spectrophotometry was used to determine the concentration and purity of purified DNA.
(2)引物设计(2) Primer design
1对通用引物和5对接有通用引物的特异性引物的序列如表1所示：The sequences of 1 pair of universal primers and 5 specific primers docked with universal primers are shown in Table 1:
表1检测转基因番木瓜内、外源基因的PCR引物Table 1 PCR primers for detection of endogenous and exogenous genes in transgenic papaya
注：引物浓度为10μMNOTE: Primer concentration is 10 μM
(3)PCR扩增条件(3) PCR amplification conditions
扩增采用反应体系为30μL，含2×MasterMix(包括0.1U Taq Polymerase/μL、500μM dNTP each、20mM Tris-HCl(pH8.3)、100mM KCl、3mM MgCl2、其他稳定剂和增强剂)，10μM引物，DNA模板浓度均为20ng/μL。The reaction system used for amplification is 30 μL, containing 2×MasterMix (including 0.1U Taq Polymerase/μL, 500 μM dNTP each, 20 mM Tris-HCl (pH8.3), 100 mM KCl, 3 mM MgCl2 , other stabilizers and enhancers), 10μM primers, DNA template concentration are 20ng/μL.
单重和多重PCR反应对扩增仪的设置为：预变性(94℃，3min)；30个循环：变性(94℃，30sec)，退火(62℃，30sec)，延伸(72℃，1min)；终止延伸(72℃，5min)。反应体系为30μL。The setting of the amplification instrument for single and multiplex PCR reactions is: pre-denaturation (94°C, 3min); 30 cycles: denaturation (94°C, 30sec), annealing (62°C, 30sec), extension (72°C, 1min) ; Termination of extension (72° C., 5 min). The reaction volume is 30 μL.
本发明通用引物多重PCR反应体系见表2：The multiplex PCR reaction system of the universal primer of the present invention is shown in Table 2:
表2通用引物多重PCR反应体系Table 2 Universal Primer Multiplex PCR Reaction System
(4)凝胶电泳(4) Gel electrophoresis
反应结束后取5μL PCR产物于2％的琼脂糖凝胶，1×TAE缓冲液中，稳压80V电泳30min，在紫外凝胶成像系统下检测。After the reaction, 5 μL of the PCR product was placed on 2% agarose gel in 1×TAE buffer, electrophoresed at a constant voltage of 80 V for 30 min, and detected under an ultraviolet gel imaging system.
实施例1通用引物单重PCR特异性验证结果分析Example 1 Universal primer single-plex PCR specificity verification result analysis
按照上述通用引物多重PCR方法进行实验，实验结果如图1所示：The experiment was carried out according to the above-mentioned general primer multiplex PCR method, and the experimental results are shown in Figure 1:
由图1可分析，因为复合引物是在原来普通引物的5′端加了一段通用引物序列，因此通用引物扩增得到的产物片段较普通引物扩增的产物片段大38bp，所以6、7、8、9、10泳道的条带较1、2、3、4、5泳道的条带位置略高。带上接头并没有对原来引物的特异性和扩增效果产生影响。而每一对引物都得到相应特异性的扩增条带，且复合引物的亮度更高，说明复合引物与普通引物对目的片段的特异性一致，相对而言，复合引物的扩增效果也更好。It can be analyzed from Fig. 1 that because the composite primer adds a general primer sequence to the 5' end of the original common primer, the product fragment amplified by the universal primer is 38bp larger than the product fragment amplified by the common primer, so 6, 7, The bands in lanes 8, 9, and 10 are slightly higher than those in lanes 1, 2, 3, 4, and 5. Bringing adapters did not affect the specificity and amplification effect of the original primers. However, each pair of primers can obtain corresponding specific amplification bands, and the brightness of the composite primers is higher, indicating that the specificity of the composite primers to the target fragment is consistent with that of the common primers. Relatively speaking, the amplification effect of the composite primers is also better. good.
实施例2通用引物多重PCR的验证Verification of embodiment 2 universal primer multiplex PCR
按照上述方法进行通用引物多重PCR反应，电泳结果如图2所示：According to the method described above, multiple PCR reactions with universal primers were carried out, and the electrophoresis results are shown in Figure 2:
图中结果表明，通用引物多重PCR的扩增结果(条带位置)、保持有较高的特异性。与普通引物的多重PCR相比，条带更为明亮清晰，表明扩增效果有所提高。The results in the figure show that the amplification results (band positions) of the multiplex PCR with the universal primers maintain high specificity. Compared with multiplex PCR with common primers, the bands are brighter and clearer, indicating that the amplification effect has been improved.
实施例3通用引物多重PCR灵敏度分析 Example 3 Universal Primer Multiplex PCR Sensitivity Analysis
按照上述方法进行通用引物多重PCR反应，进行灵敏度分析。灵敏度的验证结果如图3所示：The multiplex PCR reaction with universal primers was carried out according to the above method, and the sensitivity analysis was carried out. The verification results of the sensitivity are shown in Figure 3:
灵敏度验证结果显示，扩增效率随模板量减少而降低，同时条带的清晰度也随之下降，当模板量为0.001ng时，仍能到目的条带。所以，本发明通用引物 5重PCR的检测限为0.001ng目标DNA或者更低，比普通引物的多重PCR效果提高了二个数量级以上。Sensitivity verification results showed that the amplification efficiency decreased with the decrease of the template amount, and the clarity of the band also decreased. When the template amount was 0.001ng, the target band could still be obtained. Therefore, the detection limit of the 5-fold PCR of the universal primer of the present invention is 0.001ng target DNA or lower, which is more than two orders of magnitude higher than the multiplex PCR effect of common primers.
实施例4番木瓜汁中转基因成分的应用检测Application Detection of Transgenic Components in Example 4 Papaya Juice
以新鲜的3个转基因番木瓜华农1号和3个非转基因番木瓜，榨汁后采用优化改进的CTAB法提取的木瓜汁基因组DNA,并采用上述通用引物PCR方法检测番木瓜中的转基因成分，扩增结果见表3。Genomic DNA of papaya juice was extracted by the optimized and improved CTAB method after juicing three fresh transgenic papayas Huanong No. 1 and three non-transgenic papayas, and the above-mentioned general primer PCR method was used to detect the transgenic components in the papaya. The amplification results are shown in Table 3.
表3番木瓜汁中的转基因成分的扩增结果Amplification result of transgenic components in table 3 papaya juice
备注：+表示检出，-表示未检出。 Remarks: + means detected, - means not detected. the
从表3的扩增结果可以看出，6个鲜榨番木瓜汁中3个华农1号样品均检测到相对应内标基因(Papain)和外源基因(35S、NPTII、RP、CP)，另外3个非转基因番木瓜汁仅检测到内标基因Papian。表明此方法可以快速准确的应用于番木瓜中转基因成分的检测。From the amplification results in Table 3, it can be seen that the corresponding internal standard gene (Papain) and exogenous gene (35S, NPTII, RP, CP) were detected in 3 samples of Huanong No. 1 in 6 freshly squeezed papaya juices. The other three non-transgenic papaya juices only detected the internal standard gene Papian. It shows that this method can be quickly and accurately applied to the detection of genetically modified components in papaya.
本发明采用自行的通用引物及带有接头的特异性引物进行单重PCR及多重PCR实验，对此方法的可行性进行验证。与普通多重PCR体系相比，通用多重引物PCR(UP-M-PCR)体系中除了含有各对目的基因特异性引物之外，还含有一对通用引物Universal-primer要发挥作用，进行扩增，而在每条特异性引物的5’端需要连接上所选用的通用引物成为复合引物，而通用引物Universal-primer由于没有目标模板，无法进行扩增。当特异性引物用尽，扩增产物得到一定程度的积累，通用引物开始以得到的产物为模板，进行序列扩增(见图4)。The present invention uses its own universal primers and specific primers with adapters to carry out single-plex PCR and multiple PCR experiments, and verifies the feasibility of the method. Compared with the ordinary multiplex PCR system, the universal multiplex primer PCR (UP-M-PCR) system contains not only each pair of target gene-specific primers, but also a pair of universal primers to play a role in amplification, The 5' end of each specific primer needs to be connected with the selected universal primer to form a composite primer, and the universal primer cannot be amplified because there is no target template. When the specific primers are exhausted, the amplified product is accumulated to a certain extent, and the universal primer begins to use the obtained product as a template to perform sequence amplification (see Figure 4).
PCR反应体系的优化涉及一对通用引物及五对带有接头的特异性引物，相较而言增加了其复杂程度。通用引物的加入可以减少复合引物的用量，故30μL的反应体系中，以6μL通用引物、0.4μL每个引物为起始浓度，以0.1μL为浓度梯度进行优化，最终得出如表2的反应体系，而电泳结果及灵敏度验证结果显示引物特异性良好(图1)且在多重PCR体系下没有明显的竞争或者抑制现象(图2)，标明该体系的扩增效果比较成功。The optimization of the PCR reaction system involves a pair of universal primers and five pairs of specific primers with adapters, which increases its complexity in comparison. The addition of universal primers can reduce the amount of compound primers. Therefore, in the 30 μL reaction system, 6 μL of universal primers, 0.4 μL of each primer were used as the initial concentration, and 0.1 μL was used as the concentration gradient to optimize the final reaction as shown in Table 2. system, while the results of electrophoresis and sensitivity verification showed good primer specificity (Figure 1) and no obvious competition or inhibition in the multiplex PCR system (Figure 2), indicating that the amplification effect of the system was relatively successful.
对比不加入接头的特异性引物的PCR验证结果，发现带有接头的特异性引物无论在单重PCR或者多重PCR的检测中的电泳条带均更为明亮清晰，证明其扩增后的DNA含量比原特异性引物PCR得出的含量要更高；而由灵敏度的对比，则可以直接对比得出通用引物多重PCR灵敏度结果比普通引物的灵敏度提高了三个数量级以上。Compared with the PCR verification results of the specific primers without adapters, it was found that the electrophoretic bands of the specific primers with adapters were brighter and clearer in the detection of single-plex PCR or multiplex PCR, which proved the amplified DNA content The content obtained by PCR with the original specific primers is higher; and from the comparison of the sensitivity, it can be directly compared that the sensitivity of the multiplex PCR of the general primers is more than three orders of magnitude higher than that of the common primers.
| Application Number | Priority Date | Filing Date | Title |
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| CN201510075452.0ACN104673908A (en) | 2015-02-12 | 2015-02-12 | A method for detecting transgenic papaya using multiplex PCR technology with universal primers |
| Application Number | Priority Date | Filing Date | Title |
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| CN201510075452.0ACN104673908A (en) | 2015-02-12 | 2015-02-12 | A method for detecting transgenic papaya using multiplex PCR technology with universal primers |
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| CN201510075452.0APendingCN104673908A (en) | 2015-02-12 | 2015-02-12 | A method for detecting transgenic papaya using multiplex PCR technology with universal primers |
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