CN110823846A - Application of novel fluorescent dye AccuBlue in detection of aptamer - Google Patents
Application of novel fluorescent dye AccuBlue in detection of aptamer Download PDFInfo
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Abstract
本发明涉及一种新型荧光染料AccuBlue在核酸适配体检测法中的应用。将荧光染料AccuBlue在自身游离状态，或与单链DNA共存时只发出微弱的荧光，与双链DNA共存时会与之结合使得荧光信号显著增强的特性，与核酸适配体的高亲和力和高特异性的特性结合，建立基于AccuBlue的核酸适配体检测方法。同时，由于AccuBlue不具膜穿透性，不会与细胞核内的DNA发生识别，不会发生干扰反应，本发明所述方法可应用于生物检测，食品安全、污染物分析，致病菌检测以及药物研究等多个领域，对无机有机分子、蛋白质、致病菌、生物毒素等进行高效、快速、特异性强的检测。本发明所述方法克服了现有技术造价高，干扰大，应用范围有局限性的不足，具有简单便捷，应用范围广，灵敏度高，造价低，经济适用的优点。The invention relates to the application of a novel fluorescent dye AccuBlue in a nucleic acid aptamer detection method. The fluorescent dye AccuBlue emits only weak fluorescence in its free state, or when it coexists with single-stranded DNA, and when it coexists with double-stranded DNA, it will bind to it to significantly enhance the fluorescence signal. Combined with specific characteristics, an AccuBlue-based nucleic acid aptamer detection method was established. At the same time, since AccuBlue does not have membrane penetrability, will not recognize DNA in the nucleus, and will not interfere with the reaction, the method of the present invention can be applied to biological detection, food safety, pollutant analysis, pathogenic bacteria detection and medicine. Research and other fields, efficient, rapid and specific detection of inorganic organic molecules, proteins, pathogenic bacteria, biological toxins, etc. The method of the invention overcomes the disadvantages of high cost, large interference and limited application range in the prior art, and has the advantages of simplicity and convenience, wide application range, high sensitivity, low cost and economical application.
Description
技术领域technical field
本发明涉及一种新型荧光染料AccuBlue在核酸适配体检测法中的应用，属于分析检测领域。The invention relates to the application of a novel fluorescent dye AccuBlue in a nucleic acid aptamer detection method, and belongs to the field of analysis and detection.
背景技术Background technique
核酸适配体的检测方法是近年来食品、药品检测方法中研究的热点及重点。核酸适配体(Aptamer)是能够与药物，无机或有机分子、蛋白等目标物发生高亲合性和特异性结合的一类单链核苷酸(DNA or RNA)。通常由10~100个碱基组成。由于单链核苷酸空间结构的多样性，通过链内碱基互补配对和氢键作用，使自身形成G-四链体、凸环等复杂的三级结构。核酸适配体可与靶标发生高特异性，高亲和力的结合，且核酸适配体可被多种标记物进行标记，连接生物传感器后，核酸适配体检测法可在基础研究、分析检测以及药物的研发与临床诊断中应用。核酸适配体的检测方法中，主要利用对核酸适配体进行纳米物质、荧光物质、相关酶类材料、生物素及特定用途的蛋白质的修饰，而后通过酶联免疫反应、放射免疫测定、荧光方法等进行检测。The detection method of nucleic acid aptamer is the hot spot and focus of research in food and drug detection methods in recent years. Aptamers are a class of single-stranded nucleotides (DNA or RNA) that can bind to targets such as drugs, inorganic or organic molecules, and proteins with high affinity and specificity. Usually consists of 10 to 100 bases. Due to the diversity of the spatial structure of single-stranded nucleotides, it forms complex tertiary structures such as G-quadruplexes and convex rings through complementary base pairing and hydrogen bonding in the chain. Nucleic acid aptamers can bind to targets with high specificity and high affinity, and nucleic acid aptamers can be labeled with a variety of labels. After connecting with biosensors, nucleic acid aptamer detection methods can be used in basic research, analysis and detection. Application in drug development and clinical diagnosis. In the detection method of nucleic acid aptamers, nucleic acid aptamers are mainly modified by nano-materials, fluorescent substances, related enzyme materials, biotin and proteins for specific purposes, and then through enzyme-linked immunoreaction, radioimmunoassay, fluorescence method, etc. for testing.
近年来，基于核酸适配体的荧光传感器已有较多的报道，但对于具有广泛应用性的荧光染料的研究较少。目前发现并常用的PicoGreen、SYBR Green等染料具有在游离或是与单链 DNA 共存的情况下，只发出微弱的荧光，而与双链 DNA 共存时，荧光强度显著增强的特性，基于此种染料构建的适配体的免标记检测技术，无需特殊的荧光染料，也无需特殊的空间构象，因而应用范围十分广泛。然而上述两种染料都具有膜穿透性，当目标物为细菌、细胞时，则会与细胞核内的 DNA 发生结合，从而对检测结果产生极大的干扰，因而不适合用于细菌或细胞的检测。In recent years, there have been many reports of fluorescent sensors based on nucleic acid aptamers, but there are few studies on fluorescent dyes with wide applicability. The currently discovered and commonly used dyes such as PicoGreen and SYBR Green only emit weak fluorescence when they are free or coexist with single-stranded DNA, while the fluorescence intensity is significantly enhanced when they coexist with double-stranded DNA. Based on this dye The label-free detection technology of the constructed aptamers does not require special fluorescent dyes or special spatial conformations, so it has a wide range of applications. However, both of the above-mentioned dyes have membrane penetrability. When the target is bacteria or cells, they will combine with DNA in the nucleus, which will greatly interfere with the detection results, so they are not suitable for bacteria or cells. detection.
因此本发明中提出一种新型的荧光染料 AccuBlue，它不但具有在游离状态，与单链 DNA 共存时只发出微弱的荧光，与双链 DNA 共存时会与之发生结合使得荧光信号显著增强的特性，而且还不具有膜穿透性，从而不会与细胞核内的 DNA 发生识别，因此可广泛的应用于构建不同物质的核酸适配体荧光检测中。该方法的建立，可为生物检测，食品安全、污染物分析，致病菌检测以及药物研究等领域提供一种快速、便捷、经济、高效的切实可行的检测方式，打开检测领域新一扇大门。Therefore, a new type of fluorescent dye, AccuBlue, is proposed in the present invention, which not only emits weak fluorescence when coexisting with single-stranded DNA in the free state, but will combine with double-stranded DNA to significantly enhance the fluorescence signal when coexisting with double-stranded DNA. , and it does not have membrane penetration, so it will not recognize DNA in the nucleus, so it can be widely used in the fluorescence detection of nucleic acid aptamers of different substances. The establishment of this method can provide a fast, convenient, economical and efficient detection method for biological detection, food safety, pollutant analysis, pathogen detection and drug research and other fields, opening a new door in the field of detection .
为了更为清晰的介绍本专利涉及的检测方法，本发明以动物源性食品中的恩诺沙星的检测为示范例进行试验，实验结果表明，动物源性食品（奶粉、牛肉、虾肉）中的恩诺沙星残留检测线性范围为20～1000μg/L，检出限为9.96μg/L。检测方法可用于恩诺沙星的检测。该方法因利用新型荧光染料AccuBlue，具备经济、快速、灵敏、准确等优点，可广泛用于检测各类食品危害物，为保障食品安全提供了新思路，新方法。In order to introduce the detection method involved in this patent more clearly, the present invention takes the detection of enrofloxacin in animal-derived food as an example to conduct the test. The experimental results show that animal-derived food (milk powder, beef, shrimp meat) The linear range of enrofloxacin residue detection was 20-1000 μg/L, and the detection limit was 9.96 μg/L. The detection method can be used for the detection of enrofloxacin. The method utilizes a new fluorescent dye AccuBlue, which has the advantages of economy, rapidity, sensitivity and accuracy, and can be widely used in the detection of various food hazards, providing a new idea and method for ensuring food safety.
发明内容SUMMARY OF THE INVENTION
本发明所要解决的技术问题是提出一种新型荧光染料，可应用于核酸适配体检测法中。该染料可与核酸适配体发生高亲和高特异性的结合，用于检测多种物质。The technical problem to be solved by the present invention is to propose a new type of fluorescent dye, which can be applied to the detection method of nucleic acid aptamers. The dye can bind to nucleic acid aptamers with high affinity and high specificity, and is used to detect various substances.
为实现上述目的，本发明是通过以下的技术方案实现的。其特点是：In order to achieve the above objects, the present invention is achieved through the following technical solutions. Its characteristics are:
适配体与目标物在室温下孵育一段时间后形成适配体-目标物的复合物，接着将与适配体互补的DNA和AccuBlue染料加入到该复合物中，这样未能与目标物结合的游离的适配体则会与互补DNA 杂交形成双链DNA(dsDNA)，AccuBlue 染料能够插入到dsDNA中从而引起荧光信号的显著增强。理论上，目标物的浓度越高，体系中未能与目标物结合的游离的适配体则越少，这样能够与互补DNA杂交形成的dsDNA 也就越少，使得插入到dsDNA中的AccuBlue 染料也越少，从而荧光信号就越小，据此可进行定量分析。The aptamer and the target are incubated at room temperature for a period of time to form an aptamer-target complex, and then DNA and AccuBlue dye complementary to the aptamer are added to the complex, which fails to bind to the target. The free aptamer will hybridize with complementary DNA to form double-stranded DNA (dsDNA), and the AccuBlue dye can be inserted into the dsDNA to cause a significant increase in fluorescence signal. Theoretically, the higher the concentration of the target, the less free aptamers that cannot bind to the target in the system, and the less dsDNA that can be hybridized with complementary DNA, so that the AccuBlue dye inserted into the dsDNA will be less. Also the less, and thus the smaller the fluorescent signal, from which quantitative analysis can be performed.
其中所述适配体包括但不局限于人工合成或PCR扩增后单链化获得。The aptamer includes, but is not limited to, artificial synthesis or single-stranded obtained after PCR amplification.
所述目标物包括但不局限于蛋白质、有机物、生物污染物、抗生素，腺苷，毒素、致病菌等。The targets include but are not limited to proteins, organic matter, biological pollutants, antibiotics, adenosine, toxins, pathogenic bacteria and the like.
本发明的优点：Advantages of the present invention:
本发明所述的检测方法中的核酸适配体可因目标物的改变而改变，核酸适配体的来源可根据使用者的需求进行定制或筛选，具有灵活的可变范围。The nucleic acid aptamer in the detection method of the present invention can be changed due to the change of the target, and the source of the nucleic acid aptamer can be customized or screened according to the needs of the user, with a flexible variable range.
AccuBlue作为一种无膜穿透性的荧光染料，可与核酸适配体进行高亲和力结合，不仅适用于蛋白质、有机分子、无机分子等的检测，还可应用于细胞或致病菌等的检测，检测方法适用性更加广泛。As a non-membrane penetrating fluorescent dye, AccuBlue can bind to nucleic acid aptamers with high affinity. It is not only suitable for the detection of proteins, organic molecules, inorganic molecules, etc., but also for the detection of cells or pathogenic bacteria. , the detection method is more widely applicable.
基于AccuBlue的核酸适配体检测方法简单便捷，应用范围广，灵敏度高，造价低，经济适用。The nucleic acid aptamer detection method based on AccuBlue is simple and convenient, has a wide application range, high sensitivity, low cost, and is economical and applicable.
附图说明Description of drawings
结合附图对本发明是具体实施方式进行进一步详细说明The specific embodiments of the present invention will be further described in detail in conjunction with the accompanying drawings
图1. 基于核酸适配体与荧光染料AccuBlue检测恩诺沙星的原理图Figure 1. Schematic diagram of the detection of enrofloxacin based on nucleic acid aptamer and fluorescent dye AccuBlue
图2. 本发明方法检测不同浓度的恩诺沙星与荧光变化的线性曲线图Figure 2. The method of the present invention detects the linear curve diagram of different concentrations of enrofloxacin and fluorescence changes
具体实施方式：Detailed ways:
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明进一步详细说明。In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
以下实例将本发明的操作方法进行具体化，但不能作为本发明的限定。The following examples embody the operation method of the present invention, but cannot be regarded as a limitation of the present invention.
实施例1：本发明的检测方法在检测恩诺沙星中的应用Embodiment 1: the application of the detection method of the present invention in the detection of enrofloxacin
将恩诺沙星配成1g/L的ENR母液备用。然后分别稀释成质量浓度为 0，20，50，100，500，1000和2000μg/L的溶液。取10μL不同浓度的 ENR溶液与10μL 10nmol/L ENR核酸适配体-2溶液在孔内混匀，25℃孵育10min，再加入10μL10nmol/L互补链DNA-2溶液，25℃继续反应10min，然后加入200μL AccuBlue溶液，检测荧光强度。当ENR核酸适配体的量一定时，ENR浓度越大，体系中能与互补链结合的核酸适配体就越少，形成的dsDNA越少，AccuBlue识别dsDNA产生的荧光强度F值就越小。溶液中没有ENR药物存在时，核酸适配体完全与其对应的互补链结合，荧光强度F0最大，F0为定值，F/F0的值与F值的大小成正比，F越大，F/F0就越大。因此，在最优的实验条件下，该方法的标准曲线为Y= -0.1868x+1.186（如图2），R2=0.9457。检出限为9.96μg/L，线性范围为20～1000μg/L。Make enrofloxacin into 1g/L ENR mother solution for later use. Then it was diluted into solutions with mass concentrations of 0, 20, 50, 100, 500, 1000 and 2000 μg/L, respectively. Take 10 μL of ENR solutions of different concentrations and 10 μL of 10 nmol/L ENR aptamer-2 solution and mix them in the well, incubate at 25°C for 10 min, then add 10 μL of 10 nmol/L complementary strand DNA-2 solution, continue to react at 25°C for 10 min, and then 200 μL of AccuBlue solution was added, and the fluorescence intensity was detected. When the amount of ENR aptamer is constant, the higher the ENR concentration is, the less aptamer can bind to the complementary strand in the system, the less dsDNA is formed, and the smaller the F value of the fluorescence intensity generated by AccuBlue's recognition of dsDNA . When there is no ENR drug in the solution, the nucleic acid aptamer is completely bound to its corresponding complementary chain, and the fluorescence intensity F 0 is the largest, and F 0 is a fixed value. The value of F/F 0 is proportional to the size of the F value. F/F 0 is larger. Therefore, under the optimal experimental conditions, the standard curve of this method is Y= -0.1868x+1.186 (as shown in Figure 2), and R 2 =0.9457. The detection limit was 9.96μg/L, and the linear range was 20-1000μg/L.
所述基于新型荧光染料AccuBlue的核酸适配体检测动物源性食品中恩诺沙星的方法中，所述的恩诺沙星适配体序列为：In the method for detecting enrofloxacin in animal-derived food based on the nucleic acid aptamer of the novel fluorescent dye AccuBlue, the sequence of the enrofloxacin aptamer is:
5’- CCCATCAGGGGGCTAGGCTAACACGGTTCGGCTCTCTGAGCCCGGGTTATT5’- CCCATCAGGGGGCTAGGCTAACACGGTTCGGCTCTCTGAGCCCGGGTTATT
TCAGGGGGA -3’；由生工生物工程( 上海) 有限公司合成。TCAGGGGGA-3'; synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
实施例2：本发明的检测方法在检测奶粉、虾肉和牛肉中恩诺沙星的应用Embodiment 2: the application of the detection method of the present invention in the detection of enrofloxacin in milk powder, shrimp meat and beef
奶粉：称取脱脂奶粉5.0g，加入10mL的1×PBS，放置于25mL的离心管内，14000rpm离心20min，取出上清作为空白样品。Milk powder: Weigh 5.0 g of skim milk powder, add 10 mL of 1×PBS, place it in a 25 mL centrifuge tube, centrifuge at 14,000 rpm for 20 min, and take out the supernatant as a blank sample.
虾肉和牛肉：选取可食用部分剁碎混匀，准确称取5.0 g肉样，加入无水硫酸钠5.0g研磨，研磨均匀后倒入10 mL离心管中，加入提取溶剂( 4mL乙腈、0.04 mL冰乙酸)，高速涡旋2min，于3000 rpm离心12min，取出上清液；将残渣重复上述操作再提取1次，合并上清液，用氮气吹干，再用Tris缓冲液溶解残渣，备用。Shrimp meat and beef: Chopped and mixed the edible parts, accurately weighed 5.0 g of meat sample, added 5.0 g of anhydrous sodium sulfate to grind, grinded evenly, poured into a 10 mL centrifuge tube, and added extraction solvent (4 mL of acetonitrile, 0.04 mL of glacial acetic acid), vortexed at high speed for 2 min, centrifuged at 3000 rpm for 12 min, and took out the supernatant; repeat the above operation to extract the residue once again, combine the supernatant, dry with nitrogen, and dissolve the residue with Tris buffer for use. .
向空白样品中添加恩诺沙星终浓度为200μg/kg、500μg/kg、1000μg/kg和每个浓度作3个平行，经优化好的方法处理后，测定荧光强度值，每次测定重复3次。根据公式计算添加回收率。检测结果如表1所示。Add enrofloxacin to the blank sample at final concentrations of 200 μg/kg, 500 μg/kg, 1000 μg/kg and make 3 parallels for each concentration. After the optimized method is processed, the fluorescence intensity value is determined, and each measurement is repeated 3 times. Second-rate. Calculate the recovery rate of addition according to the formula. The test results are shown in Table 1.
表1 荧光法测定样品的加标回收结果(n=3；µg/kg)Table 1 The recovery results of samples measured by fluorescence method (n=3; µg/kg)
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