CN107505298B - Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe - Google Patents

Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe Download PDF

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CN107505298B
CN107505298B CN201710705105.0A CN201710705105A CN107505298B CN 107505298 B CN107505298 B CN 107505298B CN 201710705105 A CN201710705105 A CN 201710705105A CN 107505298 B CN107505298 B CN 107505298B
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cyromazine
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邢海波
胡晓钧
周世英
田富箱
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Shanghai Institute of Technology
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    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract

The invention discloses a method for detecting cyromazine in milk based on a G-quadruplet aptamer fluorescent probe. When the detection system does not contain cyromazine, fluorescein FAM modified on the aptamer releases fluorescence at 520 nm; when cyromazine is added into a system, cyromazine is combined with thymine on functional nucleic acid to promote the functional nucleic acid to form a G-quadruplex structure, so that the fluorescence release at 520nm is inhibited, and the quantitative detection of cyromazine in milk can be realized by measuring the fluorescence signal based on that the released fluorescence signal is in inverse proportion to the concentration of cyromazine. The method can be used for detecting the cyromazine in the milk, and has the advantages of high detection sensitivity, low detection limit of 0.68ppb, good selectivity, simple and convenient operation, simple pretreatment and no need of large-scale instruments.

Description

一种基于G-四联体核酸适配体荧光探针检测牛奶中环丙氨嗪 的方法A G-quadruplex nucleic acid aptamer-based fluorescent probe for the detection of cyromazine in milk Methods

技术领域technical field

本发明属于环丙氨嗪检测技术领域,特别涉及一种基于G-四联体核酸适配体荧光探针检测牛奶中环丙氨嗪的方法。The invention belongs to the technical field of cyromazine detection, in particular to a method for detecting cyromazine in milk based on a G-quadruplex nucleic acid aptamer fluorescent probe.

背景技术Background technique

环丙氨嗪(Cyromazine,Cyr)的化学名为2-环丙胺基-4,6-二氨基三嗪,与阿特拉津、西玛津、莠灭津、扑灭津等同属三嗪类或均三氮苯化合物,是一种高效抑制昆虫生长剂、杀寄生虫类杀虫剂,在畜禽养殖过程中作为饲料添加剂使用。研究表明环丙氨嗪进入动物体内绝大部分以原药或代谢物形式通过奶或者粪尿排泄,随后再经由畜禽粪便暴露在土壤和水环境中。土壤或水环境中的环丙氨嗪通过环境转归再次进入食物链,对不同营养级生物和人体的健康造成潜在隐患。环丙氨嗪在动物和植物体内经脱烷基化作用代谢为三聚氰胺(Melamine,Mel),而三聚氰胺的主要代谢产物为三聚氰酸(Cyanuric acid,CA)、三聚氰酸一酰胺(Ammelide,Amd)和三聚氰酸二酰胺(Ammeline,Amn)。长期涉入三聚氰胺会导致膀胱结石,膀胱癌的发生率明显提高。The chemical name of Cyromazine (Cyr) is 2-cyclopropylamino-4,6-diaminotriazine, which belongs to triazine or atrazine, simazine, atrazine and permethazine. Triazepine compound is a kind of high-efficiency insect growth inhibitor and parasitic insecticide, which is used as a feed additive in the process of livestock and poultry breeding. Studies have shown that most of cyromazine enters the body of animals in the form of original drug or metabolites, which are excreted through milk or feces and urine, and then exposed to soil and water environment through livestock manure. Cypromazine in soil or water environment re-enters the food chain through environmental fate, causing potential risks to the health of organisms at different trophic levels and human health. Cyromazine is metabolized to melamine (Mel) by dealkylation in animals and plants, and the main metabolites of melamine are cyanuric acid (CA), cyanuric acid amide (Ammelide). , Amd) and ammeline (Ammeline, Amn). Long-term exposure to melamine can lead to bladder stones and significantly increase the incidence of bladder cancer.

对牛奶中环丙氨嗪及其代谢产物的快速检测,可以有效避免有毒有害物质进入人体。环丙氨嗪及其代谢物三聚氰胺是一组分子量小、且极性强的化合物,要想将环丙氨嗪及其相关代谢物从实际样品中提取出来非常困难。美国EPA严格规定了环丙氨嗪必须作为饲料添加剂且只能放置在厩舍喂饲槽中使用,但仍有通喷洒用于饲养场所、堆肥、垃圾等处的灭蝇。环丙氨嗪经动物口服后绝大部分以原药或代谢产物三聚氰胺形式经动物尿粪排泄,在动物体内残留很少。美国EPA和PRC制定了环丙氨嗪在一系列动植物食品中的最高残留标准,其中牛奶为0.05mg/kg。The rapid detection of cyromazine and its metabolites in milk can effectively prevent toxic and harmful substances from entering the human body. Cyromazine and its metabolite melamine are a group of small molecular weight and highly polar compounds. It is very difficult to extract cyromazine and its related metabolites from actual samples. The US EPA strictly stipulates that cyromazine must be used as a feed additive and can only be used in feeding troughs in stables, but there are still flies that can be sprayed for feeding places, composting, garbage, etc. After oral administration of cyromazine, most of it is excreted in the form of original drug or metabolite melamine through animal urine and feces, and there is very little residue in animals. The US EPA and PRC have established the maximum residue standard for cyromazine in a series of animal and plant foods, of which milk is 0.05mg/kg.

根据环丙氨嗪及其代谢产物三聚氰胺在食品中的限量标准要求,目前常用于环丙氨嗪及其代谢产物残留检测的方法有容量分析法;色谱分析方法,包括高效液相色谱法;免疫化学分析法;光学分析法等。在GB 29704-2013中采用超高效液相色谱-串联质谱法来测定动物性食品中环丙氨嗪的残留。随着环丙氨嗪类兽药残留检测列入生乳收购及乳制品产品出厂必检项目,而由于上述检测方法和条件的限制,面对庞大的检测数量会导致成本巨大且工作量重,难以满足现场快速检测的需求,这就在客观上要求改进现有的检测方法,开发出高通量、快速、高灵敏的环丙氨嗪检测方法,用于日常监控。According to the limit standard requirements of cyromazine and its metabolite melamine in food, currently commonly used methods for the detection of cyromazine and its metabolite residues include volumetric analysis; chromatographic analysis methods, including high performance liquid chromatography; immunoassay Chemical analysis; optical analysis, etc. In GB 29704-2013, ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine the residues of cyromazine in animal food. With the detection of veterinary drug residues of cyromazine included in the mandatory inspection items for raw milk purchase and dairy products, due to the limitations of the above-mentioned detection methods and conditions, the huge number of tests will lead to huge costs and heavy workload, which is difficult to meet. The demand for rapid on-site detection requires the improvement of existing detection methods and the development of high-throughput, rapid, and highly sensitive detection methods for cyromazine for daily monitoring.

核酸适配体是通过指数富集配体系统体外进化(SELEX)技术,从大量寡聚核苷酸库中筛选出对靶物质具有高特异性和高结核性的核酸片段。但与蛋白质类抗体和生物酶相比,核酸适配体具有更高的亲和力、稳定性和特异性,且易于标记设计出传感器,已经用于核酸、蛋白、无机金属离子及病毒颗粒和细胞的检测。尽管胸腺嘧啶可以与环丙氨嗪的代谢产物三聚氰胺通过氢键结合已有报道,但可与环丙氨嗪特异性结合形成G-四联体的适配体还未有筛选或合成出来,并且基于此使用荧光分光光度计用于检测也未见报道。Nucleic acid aptamers are selected from a large number of oligonucleotide libraries with high specificity and high tuberculosis of nucleic acid fragments through exponential enrichment ligand system in vitro evolution (SELEX) technology. However, compared with protein antibodies and biological enzymes, nucleic acid aptamers have higher affinity, stability and specificity, and are easy to label and design sensors. They have been used for nucleic acid, protein, inorganic metal ions, virus particles and cells. detection. Although it has been reported that thymine can bind to melamine, the metabolite of cyromazine, through hydrogen bonding, aptamers that can specifically bind to cyromazine to form G-quadruplexes have not been screened or synthesized. Based on this, the use of fluorescence spectrophotometer for detection has not been reported.

发明内容SUMMARY OF THE INVENTION

本发明的目的是利用不同浓度的环丙氨嗪可以不同程度的淬灭检测液中荧光素的荧光强度的现象,提供一种灵敏度高、选择性好、成本低的牛奶中环丙氨嗪的检测方法。The object of the present invention is to utilize the cyromazine of different concentrations to quench the phenomenon of the fluorescence intensity of fluorescein in the detection solution to varying degrees, and to provide a kind of detection of cyromazine in the milk with high sensitivity, good selectivity and low cost method.

为了达到上述目的,本发明提供了一种利用荧光素FAM修饰的可形成G-四联体的核酸适配体检测液检测牛奶中环丙氨嗪的方法。荧光素FAM修饰在核酸适配体的5’端,形成稳定检测液,在激发光波长为480nm时,会在520nm处释放出稳定的荧光。In order to achieve the above object, the present invention provides a method for detecting cyromazine in milk by using a nucleic acid aptamer detection solution modified by fluorescein FAM that can form a G-quadruplex. Fluorescein FAM is modified on the 5' end of the nucleic acid aptamer to form a stable detection solution. When the excitation light wavelength is 480nm, it will release stable fluorescence at 520nm.

本发明的原理是:根据环丙氨嗪的结构特征,结合环丙氨嗪可以和胸腺嘧啶通过氢键相结合的性质,设计合成以胸腺嘧啶为主,富含鸟嘌呤的环丙氨嗪高特异性适配体并在5’端修饰荧光素FAM。环丙氨嗪的存在会使适配体空间结构改变,形成稳定的G-四联体结构。由于适配体空间结构的改变,导致上面连接的荧光基团与环丙氨嗪的距离变小,从而淬灭荧光。通过环丙氨嗪的浓度和荧光淬灭率的关系,即淬灭率F0/F的值与一定范围内环丙氨嗪的浓度呈线性关系,所以通过分析荧光淬灭率F0/F的变化,可以实现牛奶中环丙氨嗪的定量检测。The principle of the invention is: according to the structural characteristics of cyromazine, combined with the property that cyromazine can be combined with thymine through hydrogen bonds, the design and synthesis of thymine is mainly, and guanine-rich cyromazine is high Specific aptamer and modified fluorescein FAM at the 5' end. The presence of cyromazine can change the spatial structure of the aptamer and form a stable G-quadruplex structure. Due to the change of the spatial structure of the aptamer, the distance between the fluorophore attached above and cyromazine becomes smaller, thereby quenching the fluorescence. Through the relationship between the concentration of cyromazine and the fluorescence quenching rate, that is, the value of the quenching rate F 0 /F has a linear relationship with the concentration of cyromazine within a certain range, so by analyzing the fluorescence quenching rate F 0 /F The change of cyromazine can realize the quantitative detection of cyromazine in milk.

本发明的技术方案具体介绍如下。The technical solutions of the present invention are specifically introduced as follows.

本发明提供一种基于G-四联体核酸适配体荧光探针检测牛奶中环丙氨嗪的方法,具体步骤如下:The invention provides a method for detecting cyromazine in milk based on a G-quadruplex nucleic acid aptamer fluorescent probe. The specific steps are as follows:

(1)将若干相同浓度的检测液和若干经前处理的添加了不同浓度环丙氨嗪标准液的牛奶样品在24~26℃的温度下,混合2min后,得到环丙氨嗪含量维持在0-10ppb之间的标准溶液;其中:所述检测液由Tris-乙酸缓冲液将FAM修饰的核酸适配体稀释得到,所述FAM修饰的核酸适配体为5’-FAM-GGTTGGTTGGTTGGTTTT-3;(1) at the temperature of 24~26 ℃, after mixing 2min, obtain that the cyromazine content is maintained at A standard solution between 0-10ppb; wherein: the detection solution is obtained by diluting a FAM-modified nucleic acid aptamer with Tris-acetic acid buffer, and the FAM-modified nucleic acid aptamer is 5'-FAM-GGTTGGTTGGTTGGTTTT-3 ;

(2)取上述制备的标准溶液,置于比色皿中,设置激发波长为480nm后,用荧光分光光度计测定其在520nm处的荧光强度;检测体系中存在环丙氨嗪测得的荧光强度记作F,不存在环丙氨嗪测得的荧光强度记作F0,计算淬灭率(F0-F)/F0(2) get the standard solution prepared above, place in the cuvette, after setting the excitation wavelength to be 480nm, measure its fluorescence intensity at 520nm with a fluorescence spectrophotometer; there is the fluorescence that cyromazine measures in the detection system The intensity is denoted as F, the fluorescence intensity measured in the absence of cyromazine is denoted as F 0 , and the quenching rate (F 0 -F)/F 0 is calculated;

(3)将不同浓度的环丙氨嗪与对应的淬灭率(F0-F)/F0作图,绘制标准曲线;(3) cyromazine of different concentrations and corresponding quenching rate (F 0 -F)/F 0 are plotted, and standard curve is drawn;

(4)将待检测牛奶样品前处理后,与步骤(1)相同浓度的检测液混合,充分混匀后反应1~3min,用荧光分光光度计在480nm的激发波长下测试混合液在520nm处的荧光强度,再计算其淬灭率(F0-F)/F0(4) After pre-treatment of the milk sample to be detected, mix it with the detection solution of the same concentration in step (1), fully mix and react for 1-3 minutes, and use a fluorescence spectrophotometer to test the mixture at an excitation wavelength of 480 nm at 520 nm. The fluorescence intensity of , and then calculate its quenching rate (F 0 -F)/F 0 ;

(5)根据待检测牛奶样品的淬灭率(F0-F)/F0,查标准曲线,求得待检测牛奶样品中环丙氨嗪含量。(5) According to the quenching rate (F 0 -F)/F 0 of the milk sample to be detected, check the standard curve to obtain the content of cyromazine in the milk sample to be detected.

本发明中,检测液的浓度为20~30nmol/L。In the present invention, the concentration of the detection solution is 20-30 nmol/L.

本发明中,牛奶样品的前处理的步骤如下:牛奶样品的前处理的步骤如下:先将4~6mL的1wt%醋酸和1.0mL的牛奶样品混合,静置5分钟;然后8000~12000r/min离心8~12分钟,取上清后,用1.5~2.5mol/L NaOH调节pH到8.0。In the present invention, the steps of the pretreatment of the milk sample are as follows: the steps of the pretreatment of the milk sample are as follows: first, mix 4-6 mL of 1wt% acetic acid and 1.0 mL of the milk sample, and let stand for 5 minutes; then 8000-12000 r/min Centrifuge for 8-12 minutes, take the supernatant, and adjust the pH to 8.0 with 1.5-2.5 mol/L NaOH.

和现有技术相比,本发明的有益效果在于:本方法提供的检测手段灵敏度高选择性好,最低检测限0.68ppb,操作简便,前处理简单,不需要大型仪器,可用于牛奶中环丙氨嗪的检测。Compared with the prior art, the present invention has the beneficial effects that the detection means provided by the method has high sensitivity and good selectivity, the lowest detection limit is 0.68ppb, the operation is simple, the pretreatment is simple, no large-scale instrument is required, and the detection method can be used for cyclopropylamine in milk. detection of oxazine.

附图说明Description of drawings

图1是基于G-四联体核酸适配体荧光探针快速检测环丙氨嗪的原理示意图。Figure 1 is a schematic diagram of the principle of rapid detection of cyromazine based on G-quadruplex nucleic acid aptamer fluorescent probe.

图2是检测体系的圆二色谱图。Figure 2 is a circular dichroism chromatogram of the detection system.

图3是温度对检测体系的影响图示。Figure 3 is a graphical representation of the effect of temperature on the detection system.

图4是反应时间和K+对检测体系的影响图示。Figure 4 is a graphical representation of the effect of reaction time and K + on the detection system.

图5是体系选择性实验结果。Fig. 5 is the experimental result of system selectivity.

图6是环丙氨嗪浓度与荧光淬灭率(F0-F)/F0的关系。Figure 6 is the relationship between cyromazine concentration and fluorescence quenching ratio (F 0 -F)/F 0 .

具体实施方式Detailed ways

下面结合附图和实施例对本发明的技术方案进行详细介绍。The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

图1是基于G-四联体核酸适配体荧光探针快速检测环丙氨嗪的原理示意图。当检测体系中没有环丙氨嗪时,修饰在适配体上的荧光素FAM会在520nm处释放出荧光;当体系中加入环丙氨嗪后,环丙氨嗪和功能核酸上的胸腺嘧啶结合,促进功能核酸形成G-四联体结构,抑制了520nm处的荧光释放,基于释放出的荧光信号与环丙氨嗪浓度呈反比,通过测定荧光信号可实现牛奶中环丙氨嗪的定量检测。Figure 1 is a schematic diagram of the principle of rapid detection of cyromazine based on G-quadruplex nucleic acid aptamer fluorescent probe. When there is no cyromazine in the detection system, the fluorescein FAM modified on the aptamer will release fluorescence at 520 nm; when cyromazine is added to the system, thymine on cyromazine and functional nucleic acid Combined, it promotes the formation of G-quadruplex structure of functional nucleic acid, and inhibits the fluorescence release at 520nm. Based on the fact that the released fluorescence signal is inversely proportional to the concentration of cyromazine, the quantitative detection of cyromazine in milk can be realized by measuring the fluorescence signal. .

实施例1Example 1

(1)体系圆二色谱检测:准备两个离心管,分别加入核酸适配体,核酸适配体混合环丙氨嗪,再通过圆二色谱在220nm~320nm波长上进行扫描,证明G-四联体结构的形成。图2是检测体系的圆二色谱图。(1) System circular dichroism detection: prepare two centrifuge tubes, add nucleic acid aptamers respectively, mix the nucleic acid aptamers with cyromazine, and then scan by circular dichroism at the wavelength of 220nm ~ 320nm to prove that G-tetra Formation of conjoined structures. Figure 2 is a circular dichroism chromatogram of the detection system.

(2)温度对检测体系的影响:配制环丙氨嗪最终浓度分别为0ppb,6ppb,15ppb,40ppb,100ppb,200ppb的环丙氨嗪检测液混合体系,设置激发波长为480nm后用荧光分光光度计分别在25℃,35℃,45℃和55℃的条件下测定其在520nm处的荧光强度。检测体系中存在环丙氨嗪测得的荧光强度记作F,不存在环丙氨嗪测得的荧光强度记作F0,计算F0/F。图3是温度对检测体系的影响图示,由图所示温度在25℃最佳。(2) The influence of temperature on the detection system: prepare a mixed system of cyromazine detection solution with the final concentrations of cyromazine as 0ppb, 6ppb, 15ppb, 40ppb, 100ppb and 200ppb respectively, set the excitation wavelength to 480nm and use fluorescence spectrophotometry The fluorescence intensity at 520nm was measured by meter at 25℃, 35℃, 45℃ and 55℃, respectively. The fluorescence intensity measured in the presence of cyromazine in the detection system is recorded as F, and the fluorescence intensity measured in the absence of cyromazine is recorded as F 0 , and F 0 /F is calculated. Figure 3 is a graph showing the effect of temperature on the detection system, and the temperature shown in the figure is optimal at 25°C.

(3)K+对检测体系的影响:分别配制0、10、50、100ppb K+溶液,与25nM的核酸适配体混合,与检测液反应时间在0~360秒的过程中,设置激发波长为480nm用荧光分光光度计测定其在520nm处的荧光强度。检测体系中存在K+测得的荧光强度记作F,不存在K+测得的荧光强度记作F0,计算淬灭率(F0-F)/F0。图4所示,K+对检测体系的影响图,K+浓度的增大并不影响体系的荧光淬灭率。(3) The influence of K + on the detection system: respectively prepare 0, 10, 50, 100ppb K + solutions, mix with 25nM nucleic acid aptamer, and set the excitation wavelength during the reaction time with the detection solution for 0 to 360 seconds. The fluorescence intensity at 520 nm was measured with a spectrofluorometer for 480 nm. The fluorescence intensity measured in the presence of K + in the detection system is recorded as F, and the fluorescence intensity measured in the absence of K + is recorded as F 0 , and the quenching rate (F 0 -F)/F 0 is calculated. Figure 4 shows the effect of K + on the detection system. The increase of K + concentration does not affect the fluorescence quenching rate of the system.

(4)反应时间对检测体系的影响:分别配制0、10ppb、50ppb、100ppb的环丙氨嗪溶液,与检测液反应时间在0~360秒的过程中,设置激发波长为480nm用荧光分光光度计测定其在520nm处的荧光强度。检测体系中存在环丙氨嗪测得的荧光强度记作F,不存在环丙氨嗪测得的荧光强度记作F0,计算淬灭率(F0-F)/F0。图4所示为反应时间和对检测体系的影响图,由图可知体系最佳反应时间在2分钟以后。(4) Influence of reaction time on detection system: prepare 0, 10ppb, 50ppb, 100ppb cyromazine solutions respectively, and set the excitation wavelength to 480nm and use fluorescence spectrophotometry during the reaction time of 0 to 360 seconds with the detection solution. The fluorescence intensity at 520 nm was measured by a meter. The fluorescence intensity measured in the presence of cyromazine in the detection system is denoted as F, and the fluorescence intensity measured in the absence of cyromazine is denoted as F 0 , and the quenching rate (F 0 -F)/F 0 is calculated. Figure 4 shows the reaction time and the influence on the detection system. It can be seen from the figure that the optimal reaction time of the system is after 2 minutes.

(5)检测体系的选择性测试:在牛奶样品中分别添加15ppb环丙氨嗪,以及50ppb的牛奶中常见残留污染物土霉素、甲砜霉素、绿霉素、双甲脒、氯羟吡啶、阿维菌素、左旋咪唑、土霉素,设置激发波长为480nm用荧光分光光度计测定其分别在520nm处的荧光强度。检测体系中存在兽药测得的荧光强度记作F,空白测得的荧光强度记作F0,计算淬灭率(F0-F)/F0。图5是检测体系选择性测定结果图,由图可知本检测体系有较好的选择性。(5) Selectivity test of detection system: 15ppb cyromazine was added to milk samples, and 50ppb of common residual pollutants in milk were oxytetracycline, thiamphenicol, chloramphenicol, amitraz, and chlorhydrin. Pyridine, abamectin, levamisole, oxytetracycline, set the excitation wavelength to 480nm, and measure their fluorescence intensity at 520nm with a spectrofluorometer. The fluorescence intensity measured by the presence of veterinary drugs in the detection system was recorded as F, and the fluorescence intensity measured by blank was recorded as F 0 , and the quenching rate (F 0 -F)/F 0 was calculated. Fig. 5 is a graph showing the results of the determination of the selectivity of the detection system. It can be seen from the figure that the detection system has good selectivity.

实施例2Example 2

(1)制备检测液:在序列为GGTTGGTTGGTTGGTTTT的ssDNA的5’端连接荧光素FAM,得到最终的环丙氨嗪核酸适配体5’-FAM-GGTTGGTTGGTTGGTTTT-3’。用Tris-乙酸缓冲液(10mM,pH 8.0)将适配体含量稀释到浓度为25nM,得到检测液。(1) Preparation of detection solution: connect fluorescein FAM to the 5' end of the ssDNA whose sequence is GGTTGGTTGGTTGGTTTT to obtain the final cyromazine aptamer 5'-FAM-GGTTGGTTGGTTGGTTTT-3'. The aptamer content was diluted to a concentration of 25 nM with Tris-acetic acid buffer (10 mM, pH 8.0) to obtain a detection solution.

(2)牛奶样品的前处理:在10mL的离心管中,添加5mL 1%醋酸和1.0mL的牛奶样品,静置5分钟。然后10000r离心10分钟,取上清后,用2.0mol/L NaOH调节pH到8.0。(2) Pretreatment of milk samples: In a 10 mL centrifuge tube, add 5 mL of 1% acetic acid and 1.0 mL of milk samples, and let stand for 5 minutes. Then centrifuge at 10,000 r for 10 minutes, and after taking the supernatant, adjust the pH to 8.0 with 2.0 mol/L NaOH.

(3)制备已知环丙氨嗪浓度的检测体系:取10支包含检测液的离心管,分别加入前处理后的添加了不同浓度环丙氨嗪标准液的牛奶样品,使得整个检测体系中环丙氨嗪含量维持在0-10ppb,在25℃反应2min后测定。(3) prepare the detection system of the known cyromazine concentration: get 10 centrifuge tubes containing the detection solution, add the milk samples of the cyromazine standard solutions of different concentrations after the pretreatment respectively, so that the ring in the whole detection system is The content of imiazine was maintained at 0-10ppb, and was measured after reacting at 25°C for 2min.

(4)取制备的标准溶液,置于比色皿中,设置激发波长为480nm后用荧光分光光度计测定其在520nm处的荧光强度。检测体系中存在环丙氨嗪测得的荧光强度记作F,不存在环丙氨嗪测得的荧光强度记作F0,计算淬灭率(F0-F)/F0(4) Take the prepared standard solution, place it in a cuvette, set the excitation wavelength to 480 nm, and measure its fluorescence intensity at 520 nm with a fluorescence spectrophotometer. The fluorescence intensity measured in the presence of cyromazine in the detection system is denoted as F, and the fluorescence intensity measured in the absence of cyromazine is denoted as F 0 , and the quenching rate (F 0 -F)/F 0 is calculated.

(5)以不同浓度的环丙氨嗪与对应的淬灭率(F0-F)/F0作图,绘制标准曲线。图6是环丙氨嗪浓度与荧光淬灭率(F0-F)/F0的标准曲线图,其中环丙氨嗪浓度分别为0,1,2,3,4,5,6,7,8,9,10,15,20,40,60,80,100and 200ppb,Tcy2浓度为25nM。(5) Draw a standard curve by plotting different concentrations of cyromazine and the corresponding quenching rate (F 0 -F)/F 0 . Figure 6 is a standard curve diagram of cyromazine concentration and fluorescence quenching rate (F 0 -F)/F 0 , wherein the cyromazine concentrations are 0, 1, 2, 3, 4, 5, 6, 7, respectively , 8, 9, 10, 15, 20, 40, 60, 80, 100 and 200ppb, the Tcy2 concentration was 25nM.

(6)制备样品待测体系:前处理后,与检测液混合,充分混匀后反应2min,按步骤(4)测定其淬灭率(F0-F)/F0(6) Preparation of the sample system to be tested: after pretreatment, mix with the detection solution, fully mix and react for 2 minutes, and measure its quenching rate (F 0 -F)/F 0 according to step (4).

(7)根据样品所得淬灭率(F0-F)/F0,查标准曲线,可以求得样品中环丙氨嗪含量。(7) According to the quenching rate (F 0 -F)/F 0 obtained by the sample, and checking the standard curve, the content of cyromazine in the sample can be obtained.

(8)验证:用本发明方法测定四份含环丙氨嗪浓度分别为0.1、0.2、0.5、1.0ppm的牛奶,得到的回收率分别为120%、95%、106%、106%,证明了本方法的可靠性。(8) Verification: Four milks containing cyromazine with concentrations of 0.1, 0.2, 0.5, and 1.0 ppm were measured by the method of the present invention, and the recoveries obtained were 120%, 95%, 106%, and 106%, respectively, proving that reliability of this method.

(9)本发明方法测定牛奶中环丙氨嗪的最低检测限为0.68ppb。(9) The minimum detection limit of the method of the present invention for measuring cyromazine in milk is 0.68ppb.

Claims (3)

1.一种基于G-四联体核酸适配体荧光探针检测牛奶中环丙氨嗪的方法,其特征在于,具体步骤如下:1. a method for detecting cyromazine in milk based on G-quadruplex nucleic acid aptamer fluorescent probe, is characterized in that, concrete steps are as follows: (1)将若干相同浓度的检测液和若干经前处理的添加了不同浓度环丙氨嗪标准液的牛奶样品在24~26℃的温度下,混合2min后,得到环丙氨嗪含量维持在0-10ppb之间的标准溶液;其中:所述检测液由Tris-乙酸缓冲液将FAM修饰的核酸适配体稀释得到,所述FAM修饰的核酸适配体为5’-FAM-GGTTGGTTGGTTGGTTTT-3;(1) at the temperature of 24~26 ℃, after mixing 2min, obtain that the cyromazine content is maintained at A standard solution between 0-10ppb; wherein: the detection solution is obtained by diluting a FAM-modified nucleic acid aptamer with Tris-acetic acid buffer, and the FAM-modified nucleic acid aptamer is 5'-FAM-GGTTGGTTGGTTGGTTTT-3 ; (2)取上述制备的标准溶液,置于比色皿中,设置激发波长为480nm后,用荧光分光光度计测定其在520nm处的荧光强度;检测体系中存在环丙氨嗪测得的荧光强度记作F,不存在环丙氨嗪测得的荧光强度记作F0,计算淬灭率(F0-F)/F0(2) get the standard solution prepared above, place in the cuvette, after setting the excitation wavelength to be 480nm, measure its fluorescence intensity at 520nm with a fluorescence spectrophotometer; there is the fluorescence that cyromazine measures in the detection system The intensity is denoted as F, the fluorescence intensity measured in the absence of cyromazine is denoted as F 0 , and the quenching rate (F 0 -F)/F 0 is calculated; (3)将不同浓度的环丙氨嗪与对应的淬灭率(F0-F)/F0作图,绘制标准曲线;(3) cyromazine of different concentrations and corresponding quenching rate (F 0 -F)/F 0 are plotted, and standard curve is drawn; (4)将待检测牛奶样品前处理后,与步骤(1)相同浓度的检测液混合,充分混匀后反应1~3min,用荧光分光光度计在480nm的激发波长下测试混合液在520nm处的荧光强度,再计算其淬灭率(F0-F)/F0(4) After pre-treatment of the milk sample to be detected, mix it with the detection solution of the same concentration in step (1), fully mix and react for 1-3 minutes, and use a fluorescence spectrophotometer to test the mixture at an excitation wavelength of 480 nm at 520 nm. The fluorescence intensity of , and then calculate its quenching rate (F 0 -F)/F 0 ; (5)根据待检测牛奶样品的淬灭率(F0-F)/F0,查标准曲线,求得待检测牛奶样品中环丙氨嗪含量。(5) According to the quenching rate (F 0 -F)/F 0 of the milk sample to be detected, check the standard curve to obtain the content of cyromazine in the milk sample to be detected. 2.根据权利要求1所述的方法,其特征在于,检测液的浓度为20~30nmol/L。2 . The method according to claim 1 , wherein the concentration of the detection solution is 20-30 nmol/L. 3 . 3.根据权利要求1所述的方法,其特征在于,牛奶样品的前处理的步骤如下:先将4~6mL的1wt%醋酸和1.0mL的牛奶样品混合,静置5分钟;然后8000~12000r/min离心8~12分钟,取上清后,用1.5~2.5mol/L NaOH调节pH到8.0。3. method according to claim 1, is characterized in that, the step of pretreatment of milk sample is as follows: first mix the 1wt% acetic acid of 4~6mL and the milk sample of 1.0mL, let stand for 5 minutes; Then 8000~12000r /min centrifugation for 8-12 minutes, after taking the supernatant, adjust the pH to 8.0 with 1.5-2.5mol/L NaOH.
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