CN113063835B - cfDNA analysis method based on element labeling-inductively coupled plasma mass spectrometry detection strategy and application thereof - Google Patents

Abstract

The invention provides a metal probe and a method for detecting cfDNA based on ICP-MS. According to the invention, a compound containing rare earth metal is used for marking DNA to prepare a metal probe, base interaction and a sandwich model are used for marking free nucleic acid cfDNA of a specific sequence, and the marked target sequence quantifies rare earth metal ions on the metal probe through ICP-MS, so that indirect quantification of the target cfDNA is realized. The method can detect the specific fragment of the cfDNA extracted from the plasma or the effusion, greatly reduces the detection limit on the basis of the existing method, can accurately quantify the cfDNA to ng/L level, and has simpler and more efficient detection method and more accurate detection result.

Description

Translated fromChinese

一种基于元素标记-电感耦合等离子体质谱检测策略的cfDNA 分析方法及其应用A cfDNA analysis method based on element labeling-inductively coupled plasma mass spectrometry detection strategy and its application

技术领域technical field

本发明属于生物技术领域。更具体地，涉及一种金属探针及其基于ICP-MS 检测cfDNA的方法。The present invention belongs to the field of biotechnology. More specifically, it relates to a metal probe and a method for detecting cfDNA based on ICP-MS.

背景技术Background technique

细胞外游离核酸(cfDNA)是自身免疫性疾病的分子标志物，其对类风湿性 关节炎疾病的研究至关重要。cfDNA在生物体血浆内的含量非常低(200ug/L)， 但在类风湿性关节炎病人积液当中浓度会显著增加。目前传统的生物标志物检测 方法有：PCR、荧光染料法、荧光光谱法和电化学等方法。传统方法的检出限只 能达到mg/L，但实际应用过程中并达不能到检出限级别的准确定量。因此样品 常需要富集或者PCR处理才能检出，如中国专利申请CN201910180935.5公开一 种血浆cfDNA全局性甲基化检测方法，该方法在进行富集甲基化实验后需经过 PCR扩增，再进行后续的检测。但是，过多的前处理可能会造成定量结果的偏 差。综上，开发和研究更高要求的分析检测手段是cfDNA定量方法发展的趋势 之一。Extracellular free nucleic acid (cfDNA) is a molecular marker of autoimmune diseases, which is crucial for the study of rheumatoid arthritis disease. The content of cfDNA in the plasma of the organism is very low (200ug/L), but the concentration will be significantly increased in the effusion of rheumatoid arthritis patients. At present, traditional biomarker detection methods include PCR, fluorescent dye method, fluorescence spectroscopy and electrochemistry. The detection limit of the traditional method can only reach mg/L, but the accurate quantification cannot reach the detection limit level in the actual application process. Therefore, samples often need to be enriched or PCR processed to be detected. For example, Chinese patent application CN201910180935.5 discloses a method for global methylation detection of plasma cfDNA, which requires PCR amplification after enrichment methylation experiments. Carry out follow-up testing. However, excessive pretreatment may cause bias in quantitative results. In conclusion, the development and research of more demanding analytical detection methods is one of the trends in the development of cfDNA quantitative methods.

因此，亟待于提供一种前处理简单、检测方法简便高效和结果准确的cfDNA 的分析方法。Therefore, it is urgent to provide a cfDNA analysis method with simple pretreatment, simple and efficient detection method and accurate results.

发明内容SUMMARY OF THE INVENTION

本发明要解决的技术问题是克服现有技术的缺陷和不足，提供一种金属探针 及其基于ICP-MS检测cfDNA的方法。本发明所述金属探针可以对血浆或积液 中提取出来的cfDNA特定片段进行检测，结合有较大线性范围的ICP-MS检测， 实现浓度差距较大的样品同时检测，和对多种待测核酸同时检测，方法简单高效， 检测结果准确。The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art, and to provide a metal probe and a method for detecting cfDNA based on ICP-MS. The metal probe of the present invention can detect specific fragments of cfDNA extracted from plasma or effusion, combined with ICP-MS detection with a large linear range, to achieve simultaneous detection of samples with large concentration differences, and to detect a variety of samples to be Simultaneous detection of nucleic acid, the method is simple and efficient, and the detection results are accurate.

本发明的目的是提供一种金属探针的制备方法。The purpose of the present invention is to provide a preparation method of a metal probe.

本发明上述目的通过以下技术方案实现：The above-mentioned purpose of the present invention is achieved through the following technical solutions:

一种金属探针，所述金属探针中DNA-SH端上的-SH与螯合剂连接，螯合 剂螯合稀土金属离子；A metal probe, wherein -SH on the DNA-SH end in the metal probe is connected with a chelating agent, and the chelating agent chelates rare earth metal ions;

其中，所述金属探针中DNA序列为5'-CGGACAAACTTCTTGGCGTAAA-3'。Wherein, the DNA sequence in the metal probe is 5'-CGGACAAACTTCTTGGCGTAAA-3'.

本发明利用含有稀土金属的化合物标记DNA制备金属探针，利用碱基互配 和“三明治模型”对特定序列的游离核酸(cfDNA)进行标记，其原理简单，操作 便捷。标记后的目标序列通过ICP-MS对金属的定量，实现间接对cfDNA的定 量。本发明还可以对多种待测核酸片段进行检测，只要有相应的特征片段，就可 以任意组合。The invention utilizes compounds containing rare earth metals to label DNA to prepare metal probes, and utilizes base pairing and "sandwich model" to label free nucleic acids (cfDNA) of specific sequences. The principle is simple and the operation is convenient. The labeled target sequence is quantified by ICP-MS to achieve indirect quantification of cfDNA. The present invention can also detect a variety of nucleic acid fragments to be detected, which can be combined arbitrarily as long as there are corresponding characteristic fragments.

本发明所述金属探针的制备方法，包括以下步骤：The preparation method of the metal probe of the present invention comprises the following steps:

S1-1.将巯基修饰的DNA与三(2-氯乙基)磷酸酯反应，还原二硫键，得 到DNA-SH；S1-1. The thiol-modified DNA is reacted with tris(2-chloroethyl) phosphate, and the disulfide bond is reduced to obtain DNA-SH;

S1-2.加入螯合剂DOTA反应，得到螯合后的DNA-DOTA溶液；S1-2. Add the chelating agent DOTA to react to obtain the chelated DNA-DOTA solution;

S1-3.加入稀土金属离子溶液，与螯合后的DNA-DOTA溶液发生螯合反应， 得到金属探针；S1-3. adding rare earth metal ion solution, and chelating reaction with the chelated DNA-DOTA solution to obtain metal probe;

所述螯合剂DOTA的结构如式(I)所示：The structure of the chelating agent DOTA is shown in formula (I):

其中，式(I)所示螯合剂DOTA是一个双官能团的螯合物，稀土金属在多 元环内可形成稳定络合物，另一端的马来酰亚胺可与巯基修饰后的DNA发生反 应。Among them, the chelating agent DOTA represented by the formula (I) is a bifunctional chelate, the rare earth metal can form a stable complex in the multi-element ring, and the maleimide at the other end can react with the thiol-modified DNA .

优选地，步骤S1-3.中，所述稀土金属为铽、镧、铈或钆。Preferably, in step S1-3., the rare earth metal is terbium, lanthanum, cerium or gadolinium.

更优选地，步骤S1-3.中，所述稀土金属为铽。More preferably, in step S1-3., the rare earth metal is terbium.

优选地，步骤S1-2.中，所述螯合剂DOTA与DNA-SH的摩尔比为10～20： 1。Preferably, in step S1-2., the molar ratio of the chelating agent DOTA to DNA-SH is 10-20:1.

更优选地，步骤S1-2.中，所述螯合剂DOTA与DNA-SH的摩尔比为20：1。More preferably, in step S1-2., the molar ratio of the chelating agent DOTA to DNA-SH is 20:1.

优选地，步骤S1-3.中，所述稀土金属与巯基修饰的DNA的摩尔比为30～40： 1。Preferably, in step S1-3., the molar ratio of the rare earth metal to the thiol-modified DNA is 30-40:1.

更优选地，步骤S1-3.中，所述稀土金属与巯基修饰的DNA的摩尔比为40： 1。More preferably, in step S1-3., the molar ratio of the rare earth metal to the thiol-modified DNA is 40:1.

优选地，对制备得到的金属探针经进一步纯化，所述纯化方法是利用高效液 相色谱过柱进行洗脱，洗脱后得到的产物冻干。Preferably, the prepared metal probe is further purified, and the purification method is to use high performance liquid chromatography to elute through a column, and the product obtained after elution is lyophilized.

本发明同时还保护所述金属探针或所述制备方法在检测cfDNA含量中的应 用。The present invention also protects the application of the metal probe or the preparation method in detecting the content of cfDNA.

一种cfDNA含量的检测方法，包括以下步骤：A method for detecting cfDNA content, comprising the following steps:

S2-1.加入待测cfDNA、金属探针和捕获探针，进行杂交反应，得到“三明 治模型”；S2-1. Add the cfDNA to be tested, the metal probe and the capture probe, and carry out a hybridization reaction to obtain a "sandwich model";

S2-2.将“三明治模型”加入到有链亲霉素包被的96孔板中，固定cfDNA， 洗涤；S2-2. Add the "sandwich model" to a 96-well plate coated with streptavidin, fix cfDNA, and wash;

S2-3.加入硝酸，80～90℃下加热反应后，定容，ICP-MS检测，根据标准曲 线测定cfDNA含量；S2-3. Add nitric acid, heat the reaction at 80-90°C, make up the volume, detect by ICP-MS, and measure the cfDNA content according to the standard curve;

其中，所述捕获探针中DNA序列为5'-TTATGTGTCTGCCACTGGTGC-3'。Wherein, the DNA sequence in the capture probe is 5'-TTATGTGTTCTGCCACTGGTGC-3'.

电感耦合等离子体质谱(ICP-MS)作为一种痕量多元素分析手段，通过元 素标记策略对生物大分子进行检测可以充分利用ICP-MS的优势，将生物体中不 存在以及对ICP-MS检测没有干扰、灵敏度高的元素标记在生物大分子上，并通 过ICP-MS对标记的元素进行检测，从而实现对生物大分子的定性和定量分析。 该方法具有以下优点:(1)元素标记不需要探针具有光学、电化学等特性，标记 的元素可通过ICP-MS直接检测；(2)生物体稀土元素内含量低，基体效应小， 干扰因素少；(3)ICP-MS检出限低，灵敏度高；(4)针对不同片段DNA实 现多元素标记。Inductively coupled plasma mass spectrometry (ICP-MS), as a trace multi-element analysis method, can make full use of the advantages of ICP-MS for the detection of biological macromolecules by element labeling strategy. Elements with no interference and high sensitivity are labeled on biological macromolecules, and the labeled elements are detected by ICP-MS, thereby realizing the qualitative and quantitative analysis of biological macromolecules. This method has the following advantages: (1) element labeling does not require probes with optical and electrochemical properties, and the labeled elements can be directly detected by ICP-MS; (2) the content of rare earth elements in the organism is low, the matrix effect is small, and the interference There are few factors; (3) ICP-MS has low detection limit and high sensitivity; (4) realizes multi-element labeling for different DNA fragments.

本发明利用DNA的碱基互配，金属探针与目标DNA(cfDNA)、生物素标 记的捕获探针按照“三明治模型”进行杂交反应。利用链亲霉素对杂交反应后的 产物进行捕获，通过生物素与链亲霉素反应，实现cfDNA的固定，洗去多余的 金属探针，用硝酸消解的方式将稀土金属离子释放出来，通过ICP-MS对稀土金 属离子进行定量分析，再用稀土金属离子定量DNA。本发明将ICP-MS分析方 法和cfDNA检测通过金属探针联系在一起，可以准确定量特定序列核酸浓度。 此外，本发明不需要对样品进行过多处理，一般情况下不需要核酸扩增即可检测， 从采样到检测的用时较短；且本发明无需通过PCR扩增手段对样品中的特定序 列的游离核酸进行定量，减少了PCR扩增所带来的如非特异扩增、得不到扩增 产物等因素影响。The present invention utilizes the base pairing of DNA, and the metal probe performs hybridization reaction with target DNA (cfDNA) and biotin-labeled capture probe according to the "sandwich model". The product after the hybridization reaction is captured by streptavidin, the immobilization of cfDNA is realized by the reaction of biotin and streptavidin, the excess metal probes are washed away, and the rare earth metal ions are released by digestion with nitric acid. Rare earth metal ions were quantitatively analyzed by ICP-MS, and then DNA was quantified by rare earth metal ions. The invention combines the ICP-MS analysis method and the cfDNA detection through metal probes, and can accurately quantify the nucleic acid concentration of a specific sequence. In addition, the present invention does not require excessive processing of the sample, and generally does not require nucleic acid amplification for detection, and the time from sampling to detection is relatively short; and the present invention does not require PCR amplification to detect specific sequences in the sample. Free nucleic acid is quantified, which reduces the influence of factors such as non-specific amplification and the lack of amplification products caused by PCR amplification.

优选地，所述捕获探针的3’端具有生物素标记。Preferably, the 3' end of the capture probe is labeled with biotin.

优选地，步骤S2-3.中，所述标准曲线中，标准品溶液的浓度范围为0.1-5.0 pmol。Preferably, in step S2-3., in the standard curve, the concentration range of the standard solution is 0.1-5.0 pmol.

本发明同时还保护所述检测方法在cfDNA定量检测中的应用。The present invention also protects the application of the detection method in the quantitative detection of cfDNA.

本发明所述检测方法还可用于液体活检，对比传统的组织活检存在创伤性和 取材限制，液体活检在体液中检测生物标志物分子的技术方法作为临床检验新技 术正逐步推广应用，本发明作为一种便捷、非侵入性的cfDNA检测方法，可实 现多次取材，为开发可靠的、可重复的液体活检检测提供了新的方法。The detection method of the present invention can also be used for liquid biopsy. Compared with traditional tissue biopsy, there are invasiveness and limitations in material sampling. The technical method for detecting biomarker molecules in body fluids by liquid biopsy is gradually popularized and applied as a new clinical test technology. A convenient, non-invasive method for cfDNA detection that enables multiple sampling, providing a new way to develop reliable and reproducible liquid biopsy assays.

与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

本发明提供了一种金属探针及其基于ICP-MS检测cfDNA的方法。本发明 利用含有稀土金属的化合物标记DNA制备金属探针，通过ICP-MS对金属探针 上金属定量，间接定量cfDNA。本发明可以对血浆或积液中提取出来的cfDNA 特定片段进行检测，结合有较大线性范围的ICP-MS检测，实现浓度差距较大的 样品同时检测，和对多种待测核酸同时检测，且检出限在现有方法的基础上大大 降低，可以准确定量cfDNA到ng/L级别，方法简单高效，检测结果更加准确。The invention provides a metal probe and a method for detecting cfDNA based on ICP-MS. In the present invention, a metal probe is prepared by labeling DNA with a compound containing a rare earth metal, and the metal on the metal probe is quantified by ICP-MS to indirectly quantify cfDNA. The present invention can detect specific fragments of cfDNA extracted from plasma or effusion, combined with ICP-MS detection with a large linear range, to achieve simultaneous detection of samples with large concentration differences, and simultaneous detection of multiple nucleic acids to be detected, And the detection limit is greatly reduced on the basis of the existing method, which can accurately quantify cfDNA to ng/L level, the method is simple and efficient, and the detection result is more accurate.

附图说明Description of drawings

图1为DNA-SH的LC-MS图；Fig. 1 is the LC-MS picture of DNA-SH;

图2为DOTA-MMA-DNA的LC-MS图；Fig. 2 is the LC-MS diagram of DOTA-MMA-DNA;

图3为金属探针的LC-MS图；Fig. 3 is the LC-MS picture of metal probe;

图4为本发明金属探针纯化后的色谱图；Fig. 4 is the chromatogram after purification of the metal probe of the present invention;

图5为本发明金属探针纯化后的LC-MS图；Fig. 5 is the LC-MS figure after purification of the metal probe of the present invention;

图6为本发明“三明治模型”图；Fig. 6 is a "sandwich model" diagram of the present invention;

图7为目标cfDNA与Tb浓度的关系图；Figure 7 is a graph showing the relationship between target cfDNA and Tb concentration;

图8为“三明治模型”的有效性验证结果图。Figure 8 is a graph of the validation results of the "sandwich model".

具体实施方式Detailed ways

以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本 发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技 术领域常规试剂、方法和设备。The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the description, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

除非特别说明，以下实施例所用试剂和材料均为市购。Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

实验所用核酸购自生工生物工程(上海)股份有限公司。The nucleic acids used in the experiments were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

实施例1金属探针的合成Example 1 Synthesis of Metal Probes

1.溶液制备：1. Solution preparation:

(1)将5.3mg(10mmol)DOTA-MMA(cas：1006711-90-5)溶于2mL NH₄OAC 缓冲液(pH6.5)中，配置5M的DOTA-MMA储备液以待使用。(1) 5.3 mg (10 mmol) of DOTA-MMA (cas: 1006711-90-5) was dissolved in 2 mL of NH₄ OAC buffer (pH 6.5), and a 5 M DOTA-MMA stock solution was prepared for use.

(2)将5.3mg(20mmol)TbCl₃溶于2mL NH4OAC缓冲液(pH6.5)中，配 置10M的TbCl3的储备液以待使用。(2) 5.3 mg (20 mmol) TbCl₃ was dissolved in 2 mL of NH4OAC buffer (pH 6.5), and a 10 M stock solution ofTbCl 3 was prepared for use.

(3)将3mg(6mmol)三(2-氯乙基)磷酸酯(TCEP)溶于2mL NH4OAC 缓冲液(pH6.5)中，配置3M TCEP的储备液以待使用。(3) 3 mg (6 mmol) of tris(2-chloroethyl) phosphate (TCEP) was dissolved in 2 mL of NH4OAC buffer (pH 6.5), and a stock solution of 3M TCEP was prepared for use.

2.金属探针的制备：2. Preparation of metal probes:

为DNA-SH

for DNA-SH

(1)样品二硫键的还原：在2mL样品管中加入500μL巯基修饰的DNA (100uM)，再加入1mL TCEP储备液后在37℃下反应1小时，得，DNA-SH(化 合物A)，其检测结果如图1所示。(1) Reduction of sample disulfide bonds: add 500 μL of sulfhydryl-modified DNA (100 uM) to a 2 mL sample tube, and then add 1 mL of TCEP stock solution and react at 37° C. for 1 hour to obtain, DNA-SH (Compound A), The detection results are shown in Figure 1.

(2)马来酰亚胺与DNA巯基的反应：在上述样品管中加入200μL DOTA-MMA储备液，60℃下反应1小时。得到DOTA-MMA-DNA(化合物B)， 其检测结果如图2所示。(2) Reaction of maleimide with DNA sulfhydryl group: 200 μL of DOTA-MMA stock solution was added to the above sample tube, and the reaction was carried out at 60° C. for 1 hour. The DOTA-MMA-DNA (Compound B) was obtained, and the detection result was shown in FIG. 2 .

(3)环内鳌合金属Tb反应：在同一个样品管中加入200μL TbCl₃储备液(40 倍过量，2umol)，25℃下反应12小时或以上。得到金属探针，其检测结果如图3所示。(3) Reaction of chelated metal Tb in the ring: 200 μL of TbCl₃ stock solution (40-fold excess, 2 umol) was added to the same sample tube, and the reaction was carried out at 25° C. for 12 hours or more. A metal probe is obtained, and the detection result is shown in FIG. 3 .

(4)制备得到金属探针，因为产物里存在过量的Tb、DOTA-MMA或 DOTA-MMA-Tb。因此需要对产物进行纯化。纯化方法是利用高效液相色谱过柱 进行洗脱，洗脱后得到的产物冻干，配成100uM的储备液，检测结果如图4、 图5所示。(4) Metal probes were prepared because excess Tb, DOTA-MMA or DOTA-MMA-Tb existed in the product. Therefore purification of the product is required. The purification method is to use high performance liquid chromatography to pass through the column to elute, and the product obtained after elution is lyophilized and made into a 100uM stock solution. The detection results are shown in Figure 4 and Figure 5.

其中，高效液相色谱条件为：UV＝260nm，Thermo Hypersil GOLD，150× 4.6mm，5um，0.8ml/min的流动速率。流动相A为0.01％甲酸溶液，调PH值 到4.0，流动相B为甲醇；其中，0-2分钟，95％流动相A；2-7分钟，95-70％流 动相A；7-9分钟，70-1％流动相A；9-10分钟，1％-95％流动相A。Wherein, the high performance liquid chromatography conditions are: UV=260nm, Thermo Hypersil GOLD, 150×4.6mm, 5um, flow rate of 0.8ml/min. Mobile phase A is 0.01% formic acid solution, adjust pH to 4.0, mobile phase B is methanol; 0-2 minutes, 95% mobile phase A; 2-7 minutes, 95-70% mobile phase A; 7-9 min, 70-1% mobile phase A; 9-10 min, 1%-95% mobile phase A.

结果显示，图1为DNA原料的分子量，证明二硫键被还原；图2质谱结果 是DNA-DOTA的分子量，证明得到的产物为DNA-DOTA；图3为DNA-DOTA-Tb 的分子量，证明得到的产物为金属探针。图4为金属探针的纯化图；图5是对金 属探针的纯化后得到两个峰的产物进行质谱分析，得到第一个峰的产物分子量就 是金属探针的分子量，表明第一个峰的产物是金属探针。The results show that Figure 1 is the molecular weight of the DNA raw material, which proves that the disulfide bond is reduced; Figure 2 is the molecular weight of DNA-DOTA, which proves that the obtained product is DNA-DOTA; Figure 3 is the molecular weight of DNA-DOTA-Tb, which proves that The resulting product is a metal probe. Figure 4 is the purification diagram of the metal probe; Figure 5 is the mass spectrometry analysis of the product obtained after the purification of the metal probe to obtain two peaks, and the molecular weight of the product obtained from the first peak is the molecular weight of the metal probe, indicating that the first peak The product is a metal probe.

实施例2性能测试实验Example 2 Performance Test Experiment

1.三明治模型1. Sandwich model

利用DNA的碱基互配原理，将金属探针与目标DNA(cfDNA)、生物素标 记的捕获探针按照三明治模型进行杂交反应。然后，通过链亲霉素包被的孔板对 杂交产物进行捕获(生物素与链亲霉素反应)，实现目标cfDNA的固定，洗去多 余的金属探针，用硝酸消解的方式将金属铽(Tb)释放出来，再用ICP-MS定量 Tb，再用Tb定量DNA。具体过程如图6所示。Using the base pairing principle of DNA, the metal probe, target DNA (cfDNA), and biotin-labeled capture probe are hybridized according to the sandwich model. Then, the hybridization product is captured by the streptavidin-coated well plate (biotin reacts with streptavidin) to achieve the immobilization of the target cfDNA, the excess metal probes are washed away, and the metal terbium is digested with nitric acid. (Tb) was released, Tb was quantified by ICP-MS, and DNA was quantified by Tb. The specific process is shown in Figure 6.

2.金属探针、捕获探针、目标cfDNA的碱基序列设计与修饰2. Base sequence design and modification of metal probes, capture probes, and target cfDNA

表1各段DNA序列及其修饰Table 1 DNA sequences of each segment and their modifications

3.ICP-MS检测步骤3. ICP-MS detection steps

(1)首先在200μL PCR管中加入0、0.5、1、2、5pmol的目标cfDNA， 再依次加入15pmol的金属探针和10pmol的捕获探针，最后加入缓冲液A 10×PBS，0.1％吐温20(tween20))使得最终体积为60μL。每个样品分别做八 个平行样品。将所有PCR管放入PCR仪中使其中的样品进行碱基互配(升温程 序为0-3min，93℃；3-13min，50℃；13-43min，37℃；43-73min，25℃)。(1) First, add 0, 0.5, 1, 2, and 5 pmol of target cfDNA to a 200 μL PCR tube, then add 15 pmol of metal probe and 10 pmol of capture probe in turn, and finally add buffer A 10×PBS, 0.1% NaCl tween20) to make the final volume 60 μL. Eight parallel samples were made for each sample. Put all PCR tubes into the PCR machine to make the samples in them base match (the temperature program is 0-3min, 93℃; 3-13min, 50℃; 13-43min, 37℃; 43-73min, 25℃) .

(2)将反应后的产物转移至链亲霉素包被的孔板中，在孔板内孵育1小时， 使生物素与链亲霉素充分反应。结束反应后，用缓冲液B(10×PBS，0.1％tween 20，0.1％BSA)洗板五次，buffer A洗板5次，洗去未反应的金属探针。(2) Transfer the reacted product to a streptavidin-coated well plate, and incubate in the well plate for 1 hour to fully react biotin and streptavidin. After the reaction, the plate was washed five times with buffer B (10×PBS, 0.1% tween 20, 0.1% BSA) and five times with buffer A to remove unreacted metal probes.

(3)孔板中加入300μL 50％(v/v)HNO₃，90℃下加热1小时，将金属释 放出来。最后将产物转移至2mL的样品管中，产物用5％(v/v)的HNO₃定容至 1mL。(3) Add 300 μL of 50% (v/v) HNO₃ to the well plate, and heat at 90° C. for 1 hour to release the metal. Finally, the product was transferred to a 2 mL sample tube, and the product was made up to 1 mL with 5% (v/v) HNO₃ .

(4)采用Bi为内标(化学性质与Tb相近且样品溶液中不含Bi)，用ICP-MS 检测样品中所含Tb的量，得到标准曲线。结果如图7所示。(4) Using Bi as the internal standard (the chemical properties are similar to Tb and the sample solution does not contain Bi), the amount of Tb contained in the sample is detected by ICP-MS, and the standard curve is obtained. The results are shown in Figure 7.

其中，ICP-MS检测条件为：Among them, the ICP-MS detection conditions are:

4.方法的有效性性验证4. Validation of the method

在1pmol的目标cfDNA和非目标cfDNA中分别加入过量金属探针和捕获探 针，按照ICP-MS检测步骤重复试验，得到结果如图8所示。Excess metal probes and capture probes were added to 1 pmol of target cfDNA and non-target cfDNA, respectively, and the experiment was repeated according to the ICP-MS detection steps. The results are shown in Figure 8.

其中，目标DNA序列：Among them, the target DNA sequence:

GCACCAGTGGCAGACACATAATTACGCCAAGAAGTTTGTCCG；GCACCAGTGGCAGACACATAATTACGCCAAGAAGTTTGTCCG;

非目标DNA的序列：Sequence of non-target DNA:

GCACCAGTGGCAATCACATAATTACGCCAAGAAGTTTGTCCG (SEQ ID NO.5)。GCACCAGTGGCAATCACATAATTACGCCAAGAAGTTTGTCCG (SEQ ID NO. 5).

由图8结果可知，该方法具有特异性差异，具有特异性识别cfDNA的作用， 在实际样品中可以排除干扰。It can be seen from the results in Figure 8 that this method has specific differences and can specifically recognize cfDNA, which can eliminate interference in actual samples.

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 embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

序列表sequence listing

<120> 一种基于元素标记-电感耦合等离子体质谱检测策略的cfDNA分析方法及其应用<120> A cfDNA analysis method based on element labeling-inductively coupled plasma mass spectrometry detection strategy and its application

<160> 5<160> 5

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 22<211> 22

<212> DNA<212> DNA

<213> Artificial sequence<213> Artificial sequence

<400> 1<400> 1

cggacaaact tcttggcgta aa 22cggacaaact tcttggcgta aa 22

<210> 2<210> 2

<211> 21<211> 21

<212> DNA<212> DNA

<213> Artificial sequence<213> Artificial sequence

<400> 2<400> 2

ttatgtgtct gccactggtg c 21ttatgtgtct gccactggtg c 21

<210> 3<210> 3

<211> 42<211> 42

<212> DNA<212> DNA

<213> Artificial sequence<213> Artificial sequence

<400> 3<400> 3

gcaccagtgg cagacacata attacgccaa gaagtttgtc cg 42gcaccagtgg cagacacata attacgccaa gaagtttgtc cg 42

<210> 4<210> 4

<211> 400<211> 400

<212> DNA<212> DNA

<213> Artificial sequence<213> Artificial sequence

<400> 4<400> 4

agagcaagac tccatctcaa aaaaaaaaaa aaagtttata aatttgtgtt gggcctcatt 60agagcaagac tccatctcaa aaaaaaaaaa aaagtttata aatttgtgtt gggcctcatt 60

caaagctgca ctgggccaca ggtcggacaa acttggcgta aaatattttt gggttgtcac 120caaagctgca ctgggccaca ggtcggacaa acttggcgta aaatattttt gggttgtcac 120

aattaactaa cggggaggtt gctgctggcc tccagtcggt ggaggccaag gatgctgcta 180aattaactaa cggggaggtt gctgctggcc tccagtcggt ggaggccaag gatgctgcta 180

agcatcctgc aatgcacagg aaacaaccaa ttaaccccac agtgaagaat tatccagccc 240agcatcctgc aatgcacagg aaacaaccaa ttaaccccac agtgaagaat tatccagccc 240

ccaatcccag tgaccaagac tgaaaaaccc gggctgggag agacagtgct gccatcgatt 300ccaatcccag tgaccaagac tgaaaaaccc gggctgggag agacagtgct gccatcgatt 300

gcttatgtct gccactggtg cagacagggt ggtggtgatg cctgcaatcc taatcccacc 360gcttatgtct gccactggtg cagacagggt ggtggtgatg cctgcaatcc taatcccacc 360

cggcagactg tcctgaatgt ataacccatc tcacctccca 400cggcagactg tcctgaatgt ataacccatc tcacctccca 400

<210> 5<210> 5

<211> 42<211> 42

<212> DNA<212> DNA

<213> Artificial sequence<213> Artificial sequence

<400> 5<400> 5

gcaccagtgg caatcacata attacgccaa gaagtttgtc cg 42gcaccagtgg caatcacata attacgccaa gaagtttgtc cg 42

Claims (9)

1. A metal probe is characterized in that-SH at the end of DNA-SH in the metal probe is connected with a chelating agent which chelates rare earth metal ions;

wherein the DNA sequence in the metal probe is 5'-CGGACAAACTTCTTGGCGTAAA-3';

the preparation method of the metal probe comprises the following steps:

s1-1, reacting the DNA modified by sulfydryl with tris (2-chloroethyl) phosphate, and reducing a disulfide bond to obtain DNA-SH;

s1-2, adding a chelating agent DOTA for reaction to obtain a chelated DNA-DOTA solution;

s1-3, adding a rare earth metal ion solution, and carrying out chelation reaction with the chelated DNA-DOTA solution to obtain a metal probe;

the structure of the chelating agent DOTA is shown as the formula (I):

formula (I).

2. The metal probe according to claim 1, wherein in step S1-3, the rare earth metal is terbium, lanthanum, cerium, or gadolinium.

3. The metal probe according to claim 1, wherein in step S1-2, the molar ratio of the chelating agent DOTA to the DNA-SH is (10-20): 1.

4. the metal probe according to claim 1, wherein in step S1-3, the molar ratio of the rare earth metal to the chelated DNA-DOTA is (30-40): 1.

5. use of the metal probe of any one of claims 1 to 4 in detecting cfDNA content.

6. A method for detecting the content of cfDNA is characterized by comprising the following steps:

s2-1, adding cfDNA to be tested, the metal probe of any one of claims 1-4 and a capture probe, and carrying out hybridization capture reaction to obtain a 'sandwich model';

s2-2, adding the sandwich model into a 96-well plate coated with streptomycin for reaction, fixing cfDNA and washing;

s2-3, adding nitric acid, heating at 80-90 ℃, performing constant volume, performing ICP-MS detection, and determining the cfDNA content according to a standard curve;

wherein the DNA sequence in the capture probe is 5'-TTATGTGTCTGCCACTGGTGC-3'.

7. The detection method according to claim 6, wherein in step S2-3, the concentration of the standard solution in the standard curve is in the range of 0.1 to 5.0 pmol.

8. The detection method according to claim 6, wherein the capture probe has a biotin label at its 3' end.

9. Use of the detection method according to any one of claims 6 to 8 in cfDNA quantitative detection.

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