技术领域technical field
本发明属于生物检测技术领域，涉及一种基于共有引物介导的非对称PCR的试纸条检测植物病原菌的方法及试剂盒。The invention belongs to the technical field of biological detection, and relates to a method and a kit for detecting plant pathogenic bacteria with a test strip based on shared primer-mediated asymmetric PCR.
背景技术Background technique
我国是农业大国，保障和促进农业的可持续发展一直是国家非常重视的问题。植物病原菌严重危害各种农作物的生长，是现代农业生产的最主要威胁之一。植物病害造成的经济损失是多方面的。首先，对生产者来说，由于病害引起的产量减少和产品质量降低，导致收入减少；由于防治植物病害（比如喷洒农药）所产生的防治费用而增加了成本；有时不得不种植抗病但产量不高的品种或者改种其他经济效益低的作物。其次，对消费者来说，病害引起的减产会导致农产品价格上涨，使他们生活开支增加，生活品质下降。据联合国粮农组织（FAO）估计，植物因受病害损失平均为总产量的10 % - 15 %。全球因此造成的经济损失平均每年高达2000亿美元。因此，无论是病害预测还是病害防治，都亟需发展一种快速、灵敏、特异、安全的检测植物病原菌的方法。my country is a big agricultural country, and the guarantee and promotion of sustainable development of agriculture has always been a matter that the country attaches great importance to. Plant pathogens seriously harm the growth of various crops and are one of the most important threats to modern agricultural production. The economic losses caused by plant diseases are manifold. First, for producers, there is a loss of income due to reduced yields and lower product quality due to disease; increased costs due to control expenses incurred to control plant diseases (such as spraying pesticides); sometimes having to plant disease-resistant but yielding Varieties that are not high or replanted with other crops with low economic benefits. Secondly, for consumers, the reduction in production caused by diseases will lead to an increase in the price of agricultural products, which will increase their living expenses and reduce their quality of life. According to the estimates of the Food and Agriculture Organization of the United Nations (FAO), the average loss of plants due to diseases is 10% - 15% of the total output. The global economic loss caused by this is as high as 200 billion U.S. dollars per year on average. Therefore, whether it is disease prediction or disease control, it is urgent to develop a rapid, sensitive, specific and safe method for detecting plant pathogens.
对发病植物进行快速准确的病害诊断，对无病状植物材料和植物生长环境中病原物的及时检测和监测是控制植物病害流行和成灾的重要基础。在植物病害检测中，传统的检测方法一般是在植物出现症状以后，通过观察其症状表现，进行病原物的分离等一系列繁琐的过程，最终才得到鉴定结果，而且有一些病原物的分离鉴定比较困难，尤其是大多数专性寄生物难以人工离体培养，这就使得病原物检测和病害诊断过程存在检测周期长、工作量大、结果不准确等缺点。近年来随着分子生物学技术的发展，一些新的检测技术得到了迅速的发展和应用，比如核酸试纸条检测技术，其源于成熟的免疫层析技术。免疫层析技术的原理是将特异的抗体先固定于硝酸纤维素膜的某一区带，当该干燥的硝酸纤维素一端浸入样品后，由于毛细管作用，样品将沿着该膜向前移动，当移动至固定有抗体的区域时，样品中相应的抗原即与该抗体发生特异性结合，若用免疫胶体金或免疫酶染色可使该区域显示一定的颜色，从而实现特异性的免疫诊断。核酸试纸条技术的原理则是基于核酸杂交而不是抗原抗体特异性结合，其与免疫试纸条相比，具有成本低、应用范围广、灵敏度高、准确性好的特点，因而已被应用于转基因生物的检测、细菌感染的分子诊断和基因分型等领域，同时在控制植物病害的传播、成灾中也发挥了重要作用。Rapid and accurate disease diagnosis of diseased plants, timely detection and monitoring of pathogens in disease-free plant materials and plant growth environments are important foundations for controlling the prevalence and disasters of plant diseases. In the detection of plant diseases, the traditional detection method is generally after the symptoms of the plants appear, by observing the symptoms, carrying out a series of tedious processes such as the isolation of the pathogen, and finally obtaining the identification results, and some pathogens are isolated and identified It is relatively difficult, especially most obligate parasites are difficult to culture in vitro, which makes the process of pathogen detection and disease diagnosis have disadvantages such as long detection cycle, heavy workload, and inaccurate results. In recent years, with the development of molecular biology technology, some new detection technologies have been rapidly developed and applied, such as nucleic acid test strip detection technology, which is derived from mature immunochromatography technology. The principle of immunochromatography is to immobilize the specific antibody on a certain zone of the nitrocellulose membrane. When one end of the dry nitrocellulose is immersed in the sample, the sample will move forward along the membrane due to capillary action. When moving to the area where the antibody is immobilized, the corresponding antigen in the sample will specifically bind to the antibody. If stained with immunocolloidal gold or immunoenzyme, the area can display a certain color, thereby realizing specific immunodiagnosis. The principle of nucleic acid test strip technology is based on nucleic acid hybridization rather than antigen-antibody specific binding. Compared with immune test strips, it has the characteristics of low cost, wide application range, high sensitivity and good accuracy, so it has been applied It is used in the detection of genetically modified organisms, molecular diagnosis and genotyping of bacterial infections, and also plays an important role in controlling the spread and disaster of plant diseases.
发明内容Contents of the invention
本发明的首要目的在于克服现有技术的缺点与不足，提供一种基于共有引物介导的非对称PCR与核酸试纸条检测结合的快速、灵敏、准确、操作简便的检测植物病原菌的方法。The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a method for detecting plant pathogenic bacteria based on the combination of common primer-mediated asymmetric PCR and nucleic acid test strip detection that is fast, sensitive, accurate and easy to operate.
本发明的另一目的在于提供一种使用上述方法检测致病疫霉的试剂盒。Another object of the present invention is to provide a kit for detecting Phytophthora infestans using the above method.
本发明的目的通过下述技术方案实现：The object of the present invention is achieved through the following technical solutions:
基于共有引物介导的非对称PCR结合核酸试纸条检测植物病原菌的方法，具体包括以下步骤：The method for detecting plant pathogenic bacteria based on asymmetric PCR mediated by common primers combined with nucleic acid test strips specifically includes the following steps:
（1）模板的准备：提取植物病原菌的DNA。(1) Template preparation: extract the DNA of plant pathogenic bacteria.
（2）设计扩增引物：包括一条共有引物以及一对能够特异地扩增植物病原菌保守序列的上游引物和下游引物。共有引物为与植物病原菌基因组不能完全互补配对的一段序列。上游引物的5’端添加了一段与共有引物序列相同的序列。(2) Design amplification primers: including a consensus primer and a pair of upstream primers and downstream primers that can specifically amplify the conserved sequences of plant pathogenic bacteria. The consensus primer is a sequence that cannot be completely complementary to the genome of the plant pathogenic bacteria. A sequence identical to the consensus primer sequence was added to the 5' end of the upstream primer.
（3）非对称PCR反应：将步骤（1）制备的模板、步骤（2）设计合成的引物进行非对称PCR扩增反应，得到单链DNA产物。(3) Asymmetric PCR reaction: The template prepared in step (1) and the primers designed and synthesized in step (2) are subjected to asymmetric PCR amplification reaction to obtain single-stranded DNA products.
（4）探针的设计：根据单链DNA产物的序列设计三条捕捉探针，探针1和探针2分别和单链DNA产物的两端互补，探针3与探针1完全互补；三条探针的一端都修饰有功能基团。(4) Probe design: Design three capture probes according to the sequence of the single-stranded DNA product, probe 1 and probe 2 are complementary to both ends of the single-stranded DNA product, and probe 3 is completely complementary to probe 1; One end of the probe is modified with a functional group.
（5）纳米金探针的制备：将步骤（4）中所设计合成的探针1与纳米金连接制备纳米金探针，并用包埋缓冲液重悬纳米金探针得到用于包埋的纳米金探针。(5) Preparation of nano-gold probes: connect probe 1 designed and synthesized in step (4) to nano-gold probes to prepare nano-gold probes, and resuspend nano-gold probes with embedding buffer to obtain embedding Nanogold Probes.
（6）胶体金核酸试纸条的制备(6) Preparation of colloidal gold nucleic acid test strips
胶体金核酸试纸条由底板、样品板、金垫、硝酸纤维素膜（NC膜）和吸水板构成，将样品板、金垫、硝酸纤维素膜（NC膜）和吸水板依次搭接固定于底板上（如图1B所示结构）组装得到胶体金核酸试纸条。The colloidal gold nucleic acid test strip is composed of a base plate, a sample plate, a gold pad, a nitrocellulose membrane (NC membrane) and a water-absorbing plate, and the sample plate, gold pad, nitrocellulose membrane (NC membrane) and the water-absorbing plate are lapped and fixed in sequence Assemble the colloidal gold nucleic acid test strip on the bottom plate (structure shown in Figure 1B).
所述的金垫包埋有步骤（5）制备的纳米金探针；所述的硝酸纤维素膜上有含链霉亲和素和探针3的控制线（C线）和含链霉亲和素和探针2的检测线（T线），检测线靠近金垫，控制线靠近吸水板。The gold pad is embedded with the nano-gold probe prepared in step (5); the nitrocellulose membrane has a control line (C line) containing streptavidin and probe 3 and a control line (C line) containing streptavidin The detection line (T line) of Hesu and probe 2, the detection line is close to the gold pad, and the control line is close to the water-absorbing plate.
（7）检测：将由步骤（3）得到的DNA产物与检测缓冲液（4×柠檬酸钠缓冲液+5% 甲酰胺+1% Triton X-100）混合，混合液滴加到胶体金核酸试纸条的样品板上，再滴加检测缓冲液，读取结果，检测线和控制线都变成红色表明有靶序列存在，即有待测植物病原菌，仅有控制线变成红色表明没有待测植物病原菌。(7) Detection: Mix the DNA product obtained in step (3) with the detection buffer (4×sodium citrate buffer + 5% formamide + 1% Triton X-100), and add the mixture dropwise to the colloidal gold nucleic acid test On the sample plate of the paper strip, add the detection buffer solution dropwise, and read the result. Both the detection line and the control line turn red, indicating that there is a target sequence, that is, there are plant pathogens to be tested, and only the control line turns red, indicating that there is no target sequence. Detection of plant pathogens.
步骤（1）中所述植物病原菌包括致病疫霉（Phytophthora infestans）、立枯丝核菌（Rhizoctonia solani）、茄科雷尔氏菌（Ralstonia solanacearum）、稻瘟菌（Magnaporthe grisea）、玉米锈病菌（Puccinia sorghiSchw.）、小麦壳针孢叶枯病菌（Zymoseptoria tritici）、柑桔溃疡病菌（Xanthomonas axonopodispv.citri,Xac）和马铃薯早疫病菌（Alternaria solani）等。The plant pathogens described in step (1) includePhytophthora infestans ,Rhizoctonia solani ,Ralstonia solanacearum ,Magnaporthe grisea , corn rustPuccinia sorghhi Schw .,Zymoseptoria tritici ,Xanthomonas axonopodis pv.citri , Xac andAlternaria solani , etc.
步骤（2）中所述植物病原菌保守序列优选为植物病原菌的16S 核糖体RNA基因（16S rRNA基因）保守区、基因组重复序列或毒力基因片段，如致病疫霉O8重复序列、立枯丝核菌的内毒素ENDO1基因和稻瘟菌的16S rRNA基因等。The conserved sequence of the plant pathogen described in step (2) is preferably the 16S ribosomal RNA gene (16S rRNA gene) conserved region, genome repeat sequence or virulence gene fragment of the plant pathogen, such as the repeat sequence of Phytophthora infestans O8, the The endotoxin ENDO1 gene of Sclerotinia and the 16S rRNA gene of Magnaporthe oryzae, etc.
步骤（3）中所述非对称PCR反应的体系包含：模板DNA、聚合酶、聚合酶缓冲液、共有引物、上游引物、下游引物、dNTPs和双蒸水；所述的共有引物的终浓度为300～500 nM；所述的上游引物的终浓度为30～60nM；所述的下游引物的终浓度为30～60nM。The system of the asymmetric PCR reaction described in step (3) includes: template DNA, polymerase, polymerase buffer, consensus primers, upstream primers, downstream primers, dNTPs and double distilled water; the final concentration of the consensus primers is 300-500 nM; the final concentration of the upstream primer is 30-60 nM; the final concentration of the downstream primer is 30-60 nM.
步骤（3）所述非对称PCR反应的条件优选为每25 μL反应体系的组成如下：模板DNA1 ng/μL、共有引物300 nM，上游引物30nM、下游引物30 nM、聚合酶0.5U、聚合酶缓冲液、dNTPs 0.1 mM（每种脱氧核糖核苷三磷酸的浓度为0.1 mM）；The conditions of the asymmetric PCR reaction in step (3) are preferably as follows: template DNA 1 ng/μL, common primer 300 nM, upstream primer 30 nM, downstream primer 30 nM, polymerase 0.5U, polymerase Buffer, dNTPs 0.1 mM (concentration of each deoxyribonucleoside triphosphate is 0.1 mM);
步骤（3）所述扩增过程优选为：95 ℃ 反应3min；接着95℃反应30s，52℃反应30s，72℃反应30s，共反应45个循环；72℃反应10min。The amplification process in step (3) is preferably: react at 95°C for 3 minutes; then react at 95°C for 30s, 52°C for 30s, and 72°C for 30s, for a total of 45 cycles; react at 72°C for 10 minutes.
步骤（3）中所述单链DNA产物的长度优选为100～300 nt。The length of the single-stranded DNA product in step (3) is preferably 100-300 nt.
步骤（4）中所述探针1和探针2的长度优选为20～30 nt。The length of probe 1 and probe 2 in step (4) is preferably 20-30 nt.
步骤（4）中所述探针1的功能基团优选为巯基，探针2和探针3的功能基团优选为生物素。The functional group of probe 1 in step (4) is preferably thiol, and the functional groups of probe 2 and probe 3 are preferably biotin.
步骤（5）中所述纳米金的粒径优选为13-20 nm。The particle size of the gold nanoparticles described in step (5) is preferably 13-20 nm.
步骤（5）中所述包埋缓冲液为：Na3PO4 20 mM，BSA（牛血清白蛋白）质量百分比5%，Tween 20体积百分比0.25%，蔗糖质量百分比10%。The embedding buffer described in step (5) is: Na3 PO4 20 mM, BSA (bovine serum albumin) 5% by mass, Tween 20 0.25% by volume, and sucrose 10% by mass.
步骤（6）中所述的底板的材料优选为PVC塑料。The material of the bottom plate described in step (6) is preferably PVC plastic.
步骤（6）中所述样品板的材料优选为玻璃纤维，其处理方法为：用样品板处理缓冲液浸润，置于干燥器中室温保存；所述样品板处理缓冲液为：pH 8.0，体积百分比0.25%的Triton X-100，0.05 M Tris-HCl，0.15 M NaCl。The material of the sample plate in step (6) is preferably glass fiber, and the treatment method is: soak with the sample plate treatment buffer, store in a desiccator at room temperature; the sample plate treatment buffer is: pH 8.0, volume Percentage 0.25% Triton X-100, 0.05 M Tris-HCl, 0.15 M NaCl.
步骤（6）中所述金垫的材料优选为玻璃纤维，其处理方法为：用20 μL步骤（5）中所述的用于包埋的纳米金探针喷在其上，室温下干燥，干燥器中4 ℃保存。The material of the gold pad described in step (6) is preferably glass fiber, and the treatment method is: spray 20 μL of the nano-gold probe for embedding described in step (5) on it, and dry it at room temperature. Store in a desiccator at 4°C.
步骤（6）中所述硝酸纤维素膜的处理方法优选为：用喷膜仪将5 μL链霉亲和素溶液和探针2混合液喷到检测线（T线）的位置，将5 μL链霉亲和素溶液和探针3混合液喷到控制线（C线）的位置，置于室温下干燥1 h，并于4 ℃干燥保存；所述的链霉亲和素溶液浓度为1mg/mL，所述的探针2和探针3的浓度为100 μM；所述的检测线与控制线间隔6mm；所述的检测线宽1 mm与金垫相隔6 mm，所述的控制线宽1 mm与金垫相隔12 mm。The treatment method of the nitrocellulose membrane described in step (6) is preferably as follows: spray 5 μL of the streptavidin solution and probe 2 mixture to the position of the detection line (T line) with a membrane sprayer, and spray 5 μL Spray the mixture of streptavidin solution and probe 3 onto the position of the control line (C line), let it dry at room temperature for 1 hour, and store it in a dry place at 4°C; the concentration of the streptavidin solution is 1mg /mL, the concentration of the probe 2 and probe 3 is 100 μM; the detection line and the control line are separated by 6 mm; the detection line width of 1 mm is separated from the gold pad by 6 mm, and the control line 1 mm wide and 12 mm apart from the gold pad.
步骤（6）中所述吸水板的材料优选为吸水纤维。The material of the water-absorbing board in step (6) is preferably water-absorbing fiber.
步骤（6）中所述组装优选为：底板在最下层，NC膜粘贴在底板上的中间部位，金垫位于NC膜的上部的一侧并与之重叠2 mm，样品板位于金垫的上部与之重叠2 mm，吸水板位于NC膜的上部相对与金垫和样品板的另一侧并与NC膜重叠2 mm，最后用切条机切成4 mm宽的条。The assembly described in step (6) is preferably as follows: the base plate is on the bottom layer, the NC film is pasted on the middle part of the base plate, the gold pad is located on one side of the upper part of the NC film and overlaps it by 2 mm, and the sample plate is located on the upper part of the gold pad With an overlap of 2 mm, the water-absorbing plate is located on the upper part of the NC membrane opposite to the gold pad and the other side of the sample plate and overlaps with the NC membrane by 2 mm, and finally cut into 4 mm-wide strips with a strip cutter.
步骤（7）中所述检测的过程优选为：将25 μL由步骤（3）得到的DNA产物与125 μL检测缓冲液组成的溶液滴加到胶体金核酸试纸条的样品板上，10 min后，再滴加50 μL 检测缓冲液，15 min之内读取结果。The detection process described in step (7) is preferably: add 25 μL of the DNA product obtained in step (3) and 125 μL of detection buffer to the sample plate of the colloidal gold nucleic acid test strip for 10 min Then, add 50 μL detection buffer dropwise, and read the result within 15 min.
所述的植物病原菌为致病疫霉时，其保守序列为O8重复序列；When the plant pathogen is Phytophthora infestans, its conserved sequence is O8 repeat sequence;
体系中扩增保守序列的引物为：The primers for amplifying conserved sequences in the system are:
共有引物：5’-CCAATCCAATCCAATCCCCCC-3’；Consensus primer: 5'-CCAATCCAATCCAATCCCCCC-3';
上游引物：5’- CCAATCCAATCCAATCCCCCCAAGATGATGTTGGATGATTG -3’；Upstream primer: 5'- CCAATCCAATCCAATCCCCCCAAGATGATGTTGGATGATTG -3';
下游引物：5’- TGCCTGATTTCTACCTTCT -3’；Downstream primer: 5'- TGCCTGATTTCTACCTTCT -3';
单链DNA产物的序列为：5’-CCAATCCAATCCAATCCCCCCAAGATGATGTTGGATGATTGGAGAGCTCGGATCCACGGCGAGTGTACAATTACAGTGAAGTGGATCCAAGCGACCTGGATCCGGATGAGTGTACCCATAATAGGGTGACCATGATGATTACGTCATGGATCCGGTGGATCTGAAGATAATACAGCTGGGATGATGGGTCAAGGGAAGAAGGGACATGCAGCCGAGCCTGGTAAGGGATTTAGCAGATGAAAGACATAGAAGGTAGAAATCAGGCA -3’；单下划线部分为与探针1互补配对的，双下划线表示的是探针2结合的区域；探针3与探针1完全互补；单链DNA产物的序列为：5'-CCAATCCAATCCAATCCC CCCAAGATGATGTTGGATGATTGGAGAGCTCGGATCCACGGCGAGTGTACAATTACAGTGAAGTGGATCCAAGCGACCTGGATCCGGATGAGTGTACCCATAATAGGGTGACCATGATGATTACGTCATGGATCCGGTGGATCTGAAGATAATACAGCTGGGATGATGGGTCAAGGGAAGAAGGGACATGCAGCCGAGCCTGGTAAGGGATTTAGCAGATGAAAGACATAGAAGGTAGAAATCAGGCA -3'；单下划线部分为与探针1互补配对的，双下划线表示的是探针2结合的区域；探针3与探针1完全complementary;
探针1为：5’-GGGATTGGATTGGATTGGTTTTTTTTTTTTTTT-SH-3’ （SH表示巯基）；Probe 1 is: 5'-GGGATTGGATTGGATTGGTTTTTTTTTTTTTTTT-SH-3' (SH stands for thiol);
探针2为：5’-Bio- TTTTTTTTTTTTTGCCTGATTTCTACCTTCT -3’（Bio表示生物素）；Probe 2 is: 5'-Bio- TTTTTTTTTTTTTGCCTGATTTCTACCTTCT -3' (Bio means biotin);
探针3为：5’-CCAATCCAATCCAATCCCTTTTTTTTTTTT-Bio-3’（Bio表示生物素）。Probe 3 is: 5'-CCAATCCAATCCAATCCCTTTTTTTTTTTT-Bio-3' (Bio means biotin).
使用上述方法检测致病疫霉的试剂盒，包含A、B两个分试剂盒；A kit for detecting Phytophthora infestans using the above method, including two sub-kits, A and B;
A分试剂盒包含引物、聚合酶、聚合酶缓冲液、dNTPs、双蒸水，其中：Part A kit contains primers, polymerase, polymerase buffer, dNTPs, double distilled water, of which:
所述的引物包括共有引物、上游引物和下游引物：Described primer comprises consensus primer, upstream primer and downstream primer:
共有引物：5’-CCAATCCAATCCAATCCCCCC-3’；Consensus primer: 5'-CCAATCCAATCCAATCCCCCC-3';
上游引物：5’- CCAATCCAATCCAATCCCCCCAAGATGATGTTGGATGATTG -3’；Upstream primer: 5'- CCAATCCAATCCAATCCCCCCAAGATGATGTTGGATGATTG -3';
下游引物：5’- TGCCTGATTTCTACCTTCT -3’；Downstream primer: 5'- TGCCTGATTTCTACCTTCT -3';
B分试剂盒包含探针、检测缓冲液、胶体金核酸试纸条；Part B kit includes probes, detection buffer, colloidal gold nucleic acid test strips;
所述的探针包括探针1、2和3：The probes include probes 1, 2 and 3:
探针1为：5’-GGGATTGGATTGGATTGGTTTTTTTTTTTTTTT-SH-3’ （SH表示巯基）；Probe 1 is: 5'-GGGATTGGATTGGATTGGTTTTTTTTTTTTTTTT-SH-3' (SH stands for thiol);
探针2为：5’-Bio- TTTTTTTTTTTTTGCCTGATTTCTACCTTCT -3’（Bio表示生物素）；Probe 2 is: 5'-Bio- TTTTTTTTTTTTTGCCTGATTTCTACCTTCT -3' (Bio means biotin);
探针3为：5’-CCAATCCAATCCAATCCCTTTTTTTTTTTT-Bio-3’（Bio表示生物素）；Probe 3 is: 5'-CCAATCCAATCCAATCCCTTTTTTTTTTTT-Bio-3' (Bio means biotin);
所述的检测缓冲液为含5% 甲酰胺和1% Triton X-100的4×SSC缓冲液。The detection buffer is 4×SSC buffer containing 5% formamide and 1% Triton X-100.
所述的胶体金核酸试剂条包含附着于底板（材料为PVC塑料）上且依次搭接的样品板（材料为玻璃纤维）、金垫（材料为玻璃纤维）、硝酸纤维素膜和吸水板（材料为吸水纤维）；金垫包埋有连接探针1的纳米金探针；硝酸纤维素膜上有含链霉亲和素和探针3的控制线（C线）和含链霉亲和素和探针2的检测线（T线），检测线靠近金垫，控制线靠近吸水板。The colloidal gold nucleic acid reagent strip comprises a sample plate (material is glass fiber), a gold pad (material is glass fiber), a nitrocellulose membrane and a water-absorbing plate ( The material is water-absorbing fiber); the gold pad is embedded with a nano-gold probe connected to probe 1; the control line (C line) containing streptavidin and probe 3 and the control line (C line) containing streptavidin on the nitrocellulose membrane The detection line (T line) of prime and probe 2, the detection line is close to the gold pad, and the control line is close to the water-absorbing plate.
本发明的基本原理（如图1所示）：当检测体系中存在目的基因片段时，上游引物和下游引物会与目的片段结合，进行对称PCR扩增（指数扩增），产生双链产物。当上下游引物耗尽后，共有引物与双链产物的其中一条链结合，进行非对称PCR扩增（线性扩增），产生大量的单链DNA产物（图1A）。所得的DNA产物滴加到样品板上时，通过层析作用，先与金垫处已经包埋的纳米金探针杂交，当其到达已包埋有与DNA产物互补的探针2的检测线（T线）处时，DNA、纳米金探针和探针2形成“三明治结构”，就会形成一条红色的线，过量的纳米金探针继续层析，到达已包埋有探针3的控制线（C线）处时形成第二条红色的线(图1C)。The basic principle of the present invention (as shown in Figure 1): when there is a target gene fragment in the detection system, the upstream primer and downstream primer will combine with the target fragment to perform symmetric PCR amplification (exponential amplification) to generate a double-stranded product. When the upstream and downstream primers are exhausted, the consensus primer binds to one strand of the double-stranded product for asymmetric PCR amplification (linear amplification), resulting in a large amount of single-stranded DNA product (Figure 1A). When the resulting DNA product is dropped onto the sample plate, it will first hybridize with the gold nanometer probe embedded in the gold pad through chromatography, and when it reaches the detection line embedded with the probe 2 complementary to the DNA product (T line), DNA, nano-gold probe and probe 2 form a "sandwich structure", and a red line will be formed. A second red line was formed at the control line (line C) (Figure 1C).
而不含目的基因片段时，检测线（T线）处因不能形成“三明治”结构的杂交产物，而没有红色的线出现，只有过量的纳米金探针继续层析，到达已包埋有探针3的控制线（C线）处时形成一条红色的线(图1C)。When the target gene fragment is not included, the detection line (T line) cannot form a hybrid product with a "sandwich" structure, and no red line appears. A red line is formed at the control line (C line) of needle 3 (Figure 1C).
若检测线（T线）和控制线（C线）处都无红色的线出现，则表明试纸条已经失效，检测失败，样品需要重新检测。If there is no red line at the test line (T line) and control line (C line), it means that the test strip has expired, the test has failed, and the sample needs to be re-tested.
本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
（1）共有引物介导的非对称PCR特异性高，不易出现假阳性结果；(1) Asymmetric PCR mediated by common primers has high specificity and is less prone to false positive results;
（2）共有引物介导的非对称PCR产物为单链，因此无需任何预处理就可以用于试纸条检测，缩短了检测时间；(2) The asymmetric PCR product mediated by the consensus primer is single-stranded, so it can be used for test strip detection without any pretreatment, shortening the detection time;
（3）将共有引物介导的非对称PCR与试纸条检测结合，可以实现定性或者定量检测，结果稳定；(3) Combining common primer-mediated asymmetric PCR with test strip detection can achieve qualitative or quantitative detection with stable results;
（4）检测速度快，特异性好，灵敏度高；(4) Fast detection speed, good specificity and high sensitivity;
（5）设备简单，成本低，无需昂贵的仪器；(5) The equipment is simple, the cost is low, and no expensive instruments are needed;
（6）探针设计简单，操作步骤简短，易于推广；(6) The design of the probe is simple, the operation steps are short, and it is easy to promote;
（7）本发明没有溴化已锭、同位素等有害物质的使用，没有安全隐患，使用安全。(7) The present invention does not use harmful substances such as ammonium bromide, isotopes, etc., has no potential safety hazards, and is safe to use.
附图说明Description of drawings
图1是本发明原理图，A：共有引物介导的非对称PCR扩增原理，B：试纸条各部分包埋情况，C：试纸条检测原理。Fig. 1 is a principle diagram of the present invention, A: the principle of asymmetric PCR amplification mediated by common primers, B: the embedding situation of each part of the test strip, C: the detection principle of the test strip.
图2是基于共有引物介导的非对称PCR扩增产物的琼脂糖凝胶电泳结果。Figure 2 is the result of agarose gel electrophoresis based on the asymmetric PCR amplification products mediated by consensus primers.
图3是13 nm胶体金的吸收光谱图。Figure 3 is the absorption spectrum of 13 nm colloidal gold.
图4是基于共有引物介导的非对称PCR的试纸条检测致病疫霉结果图，阳性结果（1号试纸条）和阴性结果（2号试纸条）。Figure 4 is a graph showing the results of the test strips for detecting Phytophthora infestans based on common primer-mediated asymmetric PCR, positive results (test strip No. 1) and negative results (test strip No. 2).
图5是基于共有引物介导的非对称PCR的试纸条检测致病疫霉灵敏度结果图，A：试纸条检测结果，B：琼脂糖凝胶电泳结果。Fig. 5 is a graph showing the sensitivity results of test strips for detection of Phytophthora infestans based on common primer-mediated asymmetric PCR, A: test strip detection results, B: agarose gel electrophoresis results.
图6是基于共有引物介导的非对称PCR的试纸条检测致病疫霉特异性结果图，A：试纸条检测结果，B：琼脂糖凝胶电泳结果。Fig. 6 is a graph showing the specificity results of the test strip detection of Phytophthora infestans based on the common primer-mediated asymmetric PCR, A: test strip detection results, B: agarose gel electrophoresis results.
具体实施方式Detailed ways
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
将本发明的方法应用于致病疫霉的检测。如图1所示为基于共有引物介导的非对称PCR的试纸条检测植物病原菌的原理图。实施例中所用试剂均购自北京全式金生物技术有限公司、宝生物工程（大连）有限公司和生工生物工程（上海）股份有限公司，试纸条材料及设备均购自上海金标生物有限公司。The method of the present invention is applied to the detection of Phytophthora infestans. Figure 1 is a schematic diagram of the detection of plant pathogenic bacteria by a test strip based on common primer-mediated asymmetric PCR. The reagents used in the examples were purchased from Beijing Quanshijin Biotechnology Co., Ltd., Bao Bioengineering (Dalian) Co., Ltd. and Sangon Bioengineering (Shanghai) Co., Ltd., and the test strip materials and equipment were purchased from Shanghai Jinbiao Biotechnology Co., Ltd. Ltd.
实施例1 检测方法的建立Embodiment 1 The establishment of detection method
（1） 致病疫霉基因组DNA的提取： 采用HP Fungal DNA kit抽提致病疫霉基因组DNA。(1) Extraction of Phytophthora infestans genomic DNA: HP Fungal DNA kit was used to extract Phytophthora infestans genomic DNA.
（2） 引物及探针设计：根据致病疫霉的O8重复序列设计一对引物，另设计一条共有引物，再在上游引物的5’端添加一段与共有引物序列相同的序列，最后的得到的单链DNA产物大小为266 nt。根据扩增所得的产物来设计试纸条检测所需的探针。所用到的引物和探针如表1所示：(2) Primer and probe design: Design a pair of primers based on the O8 repeat sequence of Phytophthora infestans, and design another consensus primer, then add a sequence identical to the sequence of the consensus primer to the 5' end of the upstream primer, and finally get The single-stranded DNA product has a size of 266 nt. Probes required for test strip detection are designed according to the amplified products. The primers and probes used are shown in Table 1:
表1 引物及探针列表Table 1 List of primers and probes
（3） 非对称PCR反应：(3) Asymmetric PCR reaction:
25μL的扩增体系如表2：The 25μL amplification system is shown in Table 2:
表2 PCR 体系中各组分浓度Table 2 The concentration of each component in the PCR system
反应条件：95℃反应3min；接着95℃反应30s，54℃反应30s，72℃反应30s，共反应40个循环；最后72℃反应10min。Reaction conditions: react at 95°C for 3min; then react at 95°C for 30s, 54°C for 30s, and 72°C for 30s, a total of 40 cycles; finally react at 72°C for 10min.
琼脂糖凝胶电泳检测的结果如图2所示（M为2K marker，C为空白对照，1为致病疫霉目的片段扩增产物），得到的产物可直接用于试纸条检测。The results of agarose gel electrophoresis are shown in Figure 2 (M is a 2K marker, C is a blank control, and 1 is a fragment amplification product of Phytophthora infestans). The obtained products can be directly used for test strip detection.
（4） 纳米金的制备(4) Preparation of gold nanoparticles
采用柠檬酸盐还原法制备纳米金，将100 mL 1 mM的HAuCl4加热沸腾，快速搅拌下迅速加入10 mL 38.8 mM的柠檬酸钠溶液，2 min内溶液颜色由金黄色→灰色→酒红色→透亮的红色，继续搅拌20 min，冷却至室温得到纳米金胶体溶液，用紫外吸收光谱仪测得其吸收峰在520 nm左右处（图3所示），即得到13 nm的纳米金胶体溶液。Gold nanoparticles were prepared by the citrate reduction method. 100 mL of 1 mM HAuCl4 was heated to boiling, and 10 mL of 38.8 mM sodium citrate solution was quickly added under rapid stirring. The color of the solution changed from golden yellow→gray→wine red→ Translucent red, continue to stir for 20 min, cool to room temperature to obtain a nano-gold colloid solution, and use an ultraviolet absorption spectrometer to measure its absorption peak at around 520 nm (as shown in Figure 3), that is, to obtain a 13 nm nano-gold colloid solution.
（5） 探针1与纳米金的连接(5) Connection between probe 1 and gold nanoparticles
向PCR管中加入20 μL 200 μM 的5’端修饰有巯基的探针1，接着向PCR管中加入0.66 μL 500 μM 的醋酸缓冲液（pH 5.2）和1 μL 10 μM TCEP（磷酸三氯乙酯）以活化巯基，室温、避光孵育1小时；取洁净的离心管，加入1 mL 10 nM的纳米金胶体溶液，然后在不断的摇动下，加入TCEP处理过的探针1溶液，盖上管盖，室温避光放置至少16小时，用振荡器600 rpm加速反应；缓慢震荡下向管中逐滴加入20 μL 500 mM的Tris醋酸缓冲液（pH 8.2），Tris醋酸缓冲液的终浓度为5 mM；再向管中逐滴加入200 μL 1 M NaCl，避光孵育一天；12000rpm，4 ℃离心30分钟，取出离心管，纳米粒子沉积在管底，轻轻吸掉上清，用1 mL包埋缓冲液（20 mM Na3PO4，质量百分比5% BSA，体积百分比0.25% Tween X-100，质量百分比10%sucrose）重悬，用于试纸条金垫处的包埋。Add 20 μL of 200 μM thiol-modified probe 1 at the 5’ end to the PCR tube, followed by adding 0.66 μL of 500 μM acetate buffer (pH 5.2) and 1 μL of 10 μM TCEP (trichloroethyl phosphate ester) to activate sulfhydryl groups, and incubate at room temperature for 1 hour in the dark; take a clean centrifuge tube, add 1 mL of 10 nM nano-gold colloid solution, and then add TCEP-treated probe 1 solution under constant shaking, and cover Cover the tube and place it in the dark at room temperature for at least 16 hours. Accelerate the reaction with a shaker at 600 rpm; add 20 μL of 500 mM Tris acetate buffer (pH 8.2) dropwise to the tube under slow shaking, and the final concentration of Tris acetate buffer is 5 mM; add 200 μL 1 M NaCl dropwise to the tube, and incubate in the dark for one day; centrifuge at 12,000 rpm at 4 °C for 30 minutes, take out the centrifuge tube, the nanoparticles are deposited at the bottom of the tube, gently suck off the supernatant, and use 1 mL Embedding buffer (20 mM Na3 PO4 , 5% BSA by mass, 0.25% Tween X-100 by volume, 10% sucrose by mass) was resuspended for embedding at the gold pad of the test strip.
（6） 胶体金核酸试纸条的组装与准备(6) Assembly and preparation of colloidal gold nucleic acid test strips
①样品板：用样品板处理缓冲液（pH 8.0、体积百分比0.25%的Triton X-100，0.05 MTris-HCl，0.15 M NaCl）浸润，置于干燥器中室温保存；① Sample plate: infiltrate with sample plate treatment buffer (pH 8.0, 0.25% by volume Triton X-100, 0.05 MTris-HCl, 0.15 M NaCl), store in a desiccator at room temperature;
②金垫：将20 μL标记好的纳米金-探针1溶液喷在其上，室温下干燥，干燥器中4℃保存；② Gold pad: Spray 20 μL of the labeled nano-gold-probe 1 solution on it, dry it at room temperature, and store it in a desiccator at 4°C;
③NC膜：用喷膜仪将6 μL 1 mg/mL的链霉亲和素和100 μM 5’端修饰有生物素的探针2的混合液喷到检测线（T线）的位置，将6 μL 1 mg/mL的链霉亲和素溶液和100 μM 3’端修饰有生物素的探针3的混合液喷到控制线（C线）的位置，C线和T线处划线宽度为1 mm，两条线相隔6 mm，包埋好探针的NC膜置于室温下干燥1 h，并于4 ℃干燥保存；③ NC membrane: Spray 6 μL of 1 mg/mL streptavidin and 100 μM 5' end-modified probe 2 to the position of the detection line (T line) with a film sprayer, and 6 A mixture of μL of 1 mg/mL streptavidin solution and 100 μM probe 3 modified with biotin at the 3’ end was sprayed onto the position of the control line (line C), and the width of the lines at the lines C and T was 1 mm, and the distance between the two lines is 6 mm. The probe-embedded NC membrane was dried at room temperature for 1 h, and stored in a dry place at 4 °C;
组装：按图1B所示结构组装，底板在最下层，NC膜粘贴在底板上的中间部位，金垫位于NC膜的上部的一侧并与之重叠，样品板位于金垫的上部与之重叠，吸水板位于硝酸纤维素膜的上部相对于金垫和样品板的另一侧并与硝酸纤维素膜重叠；每部分之间重叠2 mm，最后切成4 mm宽的条。 Assembly: Assemble according to the structure shown in Figure 1B, the base plate is at the bottom layer, the NC film is pasted on the middle part of the base plate, the gold pad is located on the upper side of the NC film and overlaps it, and the sample plate is located on the upper part of the gold pad and overlaps it , the water-absorbing plate is located on the upper part of the nitrocellulose membrane on the other side of the gold pad and the sample plate and overlaps the nitrocellulose membrane; each part overlaps 2 mm, and finally cut into 4 mm wide strips.
（7） 样品准备与检测(7) Sample preparation and testing
将25 μL由步骤（3）得到的DNA产物与125 μL 检测缓冲液组成的溶液滴加到胶体金核酸试纸条的样品板上，10 min后，再滴加50 μL 检测缓冲液，15 min之内读取结果。检测结果如图4所示：1号试纸条（致病疫霉阳性）在C线和T线处都形成红线，2号试纸条（空白对照）只在C线处形成红线，表明该试纸条能正确检测样品中是否含有目的基因片段，进而确定所检测植物病原菌。Add 25 μL of the DNA product obtained in step (3) and 125 μL of detection buffer dropwise onto the sample plate of the colloidal gold nucleic acid test strip. After 10 min, add 50 μL of detection buffer dropwise for 15 min. Read the result within. The test results are shown in Figure 4: No. 1 test strip (positive for Phytophthora infestans) formed red lines at both C and T lines, and No. 2 test strip (blank control) only formed red lines at C line, indicating that the The test strip can correctly detect whether the sample contains the target gene fragment, and then determine the detected plant pathogenic bacteria.
实施例2 致病疫霉检测灵敏度实验Example 2 Detection Sensitivity Experiment of Phytophthora infestans
分别取 10 pg/μL、1 pg/μL、0.1 pg/μL、10 fg/μL致病疫霉基因组DNA，按照实施例1所建立的方法所得的检测结果如图5所示（1：10 pg/μL；2：1 pg/μL；3：0.1 pg/μL；4：10 fg/μL；5：空白对照），由图5A可知，当致病疫霉基因组DNA浓度为0.1 pg/μL时，试纸条上的C线和T线处都能形成明显的红线，与图5B的琼脂糖凝胶电泳结果一致，因此可以判断本发明检测目的基因具有非常好的灵敏度。Take 10 pg/μL, 1 pg/μL, 0.1 pg/μL, and 10 fg/μL Phytophthora infestans genomic DNA respectively, and the detection results obtained according to the method established in Example 1 are shown in Figure 5 (1:10 pg /μL; 2: 1 pg/μL; 3: 0.1 pg/μL; 4: 10 fg/μL; 5: blank control), as shown in Figure 5A, when the genomic DNA concentration of Phytophthora infestans was 0.1 pg/μL, Both the C line and the T line on the test strip can form obvious red lines, which are consistent with the agarose gel electrophoresis results in Figure 5B, so it can be judged that the present invention has very good sensitivity for detecting the target gene.
实施例3 致病疫霉检测特异性实验Example 3 Phytophthora infestans detection specificity experiment
按照实施例1所建立的方法分别检测大豆疫霉、辣椒疫霉、恶疫霉、烟草疫霉和致病疫霉的基因组DNA，检测结果如图6所示（1：大豆疫霉；2：辣椒疫霉；3：恶疫霉；4：烟草疫霉；5：空白对照；6：致病疫霉），由图6A可知，1—5号试纸条只有在C线处形成红线，6号试纸条在C线和T线都形成红线，与图6B的琼脂糖凝胶电泳结果一致，因此可以判断本发明检测目的基因具有非常好的特异性。According to the method established in Example 1, the genomic DNA of Phytophthora sojae, Phytophthora capsici, Phytophthora victoria, Phytophthora tabacum and Phytophthora infestans were detected respectively, and the detection results were as shown in Figure 6 (1: Phytophthora sojae; 2: Phytophthora capsici; 3: Phytophthora victoria; 4: Phytophthora nicotiana; 5: blank control; 6: Phytophthora infestans), as can be seen from Figure 6A, test strips 1-5 only form a red line at line C, 6 No. test strips form red lines at the C line and the T line, which is consistent with the agarose gel electrophoresis results in Figure 6B, so it can be judged that the detection target gene of the present invention has very good specificity.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 福建农林大学<110> Fujian Agriculture and Forestry University
<120> 一种非对称PCR的试纸条检测植物病原菌的方法及试剂盒<120> A method and kit for detecting plant pathogenic bacteria with asymmetric PCR test strips
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tttttttttt tttgcctgat ttctaccttc t 31tttttttttttttgcctgat ttctaccttc t 31
<210> 6<210> 6
<211> 30<211> 30
<212> DNA<212>DNA
<213> 人工序列<213> Artificial sequence
<400> 6<400> 6
ccaatccaat ccaatccctt tttttttttt 30ccaatccaat ccaatccctttttttttttt 30
<210> 7<210> 7
<211> 266<211> 266
<212> DNA<212>DNA
<213> 人工序列<213> Artificial sequence
<400> 7<400> 7
ccaatccaat ccaatccccc caagatgatg ttggatgatt ggagagctcg gatccacggc 60ccaatccaat ccaatccccc caagatgatg ttggatgatt ggagagctcg gatccacggc 60
gagtgtacaa ttacagtgaa gtggatccaa gcgacctgga tccggatgag tgtacccata 120gagtgtacaa ttacagtgaa gtggatccaa gcgacctgga tccggatgag tgtacccata 120
atagggtgac catgatgatt acgtcatgga tccggtggat ctgaagataa tacagctggg 180atagggtgac catgatgatt acgtcatgga tccggtggat ctgaagataa tacagctggg 180
atgatgggtc aagggaagaa gggacatgca gccgagcctg gtaagggatt tagcagatga 240atgatgggtc aagggaagaa gggacatgca gccgagcctg gtaagggatt tagcagatga 240
aagacataga aggtagaaat caggca 266aagacataga aggtagaaat caggca 266
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810035479.0ACN107937490A (en) | 2018-01-15 | 2018-01-15 | The method and kit of a kind of ELISA test strip phytopathogen of asymmetric pcr |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810035479.0ACN107937490A (en) | 2018-01-15 | 2018-01-15 | The method and kit of a kind of ELISA test strip phytopathogen of asymmetric pcr |
| Publication Number | Publication Date |
|---|---|
| CN107937490Atrue CN107937490A (en) | 2018-04-20 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810035479.0APendingCN107937490A (en) | 2018-01-15 | 2018-01-15 | The method and kit of a kind of ELISA test strip phytopathogen of asymmetric pcr |
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| CN (1) | CN107937490A (en) |
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20180420 | |
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