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WO2020093308A1 - 一种复合靶标-肿瘤血清核酸配基检测方法及试剂盒 - Google Patents

一种复合靶标-肿瘤血清核酸配基检测方法及试剂盒 Download PDF

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WO2020093308A1
WO2020093308A1 PCT/CN2018/114555 CN2018114555W WO2020093308A1 WO 2020093308 A1 WO2020093308 A1 WO 2020093308A1 CN 2018114555 W CN2018114555 W CN 2018114555W WO 2020093308 A1 WO2020093308 A1 WO 2020093308A1
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nucleic acid
acid ligand
tumor
ligand
serum
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廖世奇
袁红霞
曾家豫
栗怡
廖正宇
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Priority to DE112018007595.8T priority Critical patent/DE112018007595T5/de
Priority to CN201880003648.0A priority patent/CN109804081A/zh
Priority to JP2020571670A priority patent/JP2022511203A/ja
Priority to GB2018973.4A priority patent/GB2591009A/en
Priority to PCT/CN2018/114555 priority patent/WO2020093308A1/zh
Priority to US16/658,096 priority patent/US20200149117A1/en
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12N2320/00—Applications; Uses
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention relates to the field of biomedical detection, in particular to a composite target-tumor serum nucleic acid ligand detection method and kit.
  • nucleic acid ligands are used as a new ligand molecule with strong specificity, wide application range, and easy modification. It is used in disease detection and treatment, drug screening and application, food and environmental safety monitoring, Biological testing and other aspects.
  • Nucleic acid beacon ligand is a new detection molecule derived on the basis of nucleic acid ligand, and a double-stranded nucleotide sequence with a fluorescently labeled probe is connected to a known specificity at the 5 'end of the ligand It is prepared by the nucleic acid ligand of the target molecule, which has the recognition ability of the nucleic acid ligand and the function of signal storage, transmission and amplification. It is a new type of easy to construct, strong specificity, high sensitivity and wide range of use. Detect molecules.
  • the increase of the beacon sequence of the nucleic acid beacon ligand can change the nucleic acid molecular sequence of the ligand, which affects the molecular spatial structure and the binding force of the ligand ligand. Therefore, if the structure of the nucleic acid ligand is not changed, real-time quantitative PCR can be used. Detection will be more convenient and practical.
  • Magnetic beads as a new type of multi-functional materials, because of their good biocompatibility and surface functional groups make it an ideal carrier for nucleic acid ligand detection, and its applications in food, medicine, environment and biological separation More and more widely.
  • Serum is the most easily obtained specimen in clinical work, and can provide a large amount of body function information. Almost all cells in the body communicate directly or indirectly with the blood, so any disease may leave traces in serum proteins, causing changes in certain characteristics.
  • the traditional detection protein is mostly enzyme-linked immunoassay (ELISA), the capture antibody is combined with the antigen in the serum, and then the detection antibody with the coupled enzyme is added to form a capture antibody-antigen-detection antibody "sandwich” complex, and finally the couple
  • ELISA enzyme-linked immunoassay
  • the ligase activity shows the detection result, but the detection range is limited by the Kd value of the reaction between the capture antibody and the antigen, and the sensitivity is not good.
  • nucleic acid beacon ligand detection technology There are currently two specific methods for nucleic acid beacon ligand detection technology.
  • One is nucleic acid beacon ligand-mediated immuno-PCR detection, the process is similar to ELISA, the difference is that the detection antigen is not the enzyme-labeled secondary antibody corresponding to the capture antibody, but the specific nucleic acid beacon ligand The complex formed with the capture antibody is detected by real-time quantitative PCR; the other is a nucleic acid beacon ligand-mediated PCR detection method, which directly uses the nucleic acid beacon ligand corresponding to the target molecule as the detection molecule , Combined with target molecules, and then eluted and separated, using real-time quantitative-PCR for detection.
  • the target molecule is specifically identified by the nucleic acid beacon ligand molecule, then the signal is transmitted, and the signal amplification function is completed by real-time quantitative PCR to detect the target molecule.
  • the nucleic acid beacon ligand method has the advantages of rapid detection, high sensitivity and strong specificity.
  • Real-time quantitative nucleic acid ligand-PCR detection technology is an improved type of nucleic acid beacon ligand detection technology.
  • the nucleic acid beacon ligand is a double-stranded beacon sequence added at both ends of the ligand sequence, and its purpose is to bind the target through the ligand
  • the molecule transmits the signal to the beacon sequence through the ligand, and then detects the beacon by real-time quantitative-PCR, thereby indirectly detecting the target molecule.
  • the target molecule of the ligand action is not necessarily completed by a single chain, but by several chains.
  • the double-strand of the beacon also affects the spatial structure of the ligand, so the beacon sequence will not only Influencing the ligand structure will also affect the synergy between the ligands, thereby affecting the binding of the ligand to the target molecule and the detection results. Therefore, it is necessary to improve this deficiency and improve the detection technology.
  • the present invention provides a composite target-tumor serum nucleic acid ligand detection method and kit.
  • the present invention is achieved through the following technical solution: a composite target-tumor serum nucleic acid ligand detection kit, including a capture magnetic bead reagent, a blocking solution, a detection reagent, a washing solution, and a real-time quantitative-PCR reaction solution, in which:
  • the capture magnetic bead reagent has dissolved capture magnetic beads with a particle size of 5 to 5000 nm;
  • the blocking solution is a protein blocking solution
  • the real-time quantitative-PCR reaction solution includes primers and fluorescent probes for nucleic acid ligands.
  • the capture magnetic bead reagent may be specifically selected as 5 mL of 0.01 M Binding buffer with a pH value of 7.4, and 50% capture magnetic bead microparticles with a particle size of 5 to 5000 nm;
  • the detection reagent is gastric cancer (G-seq), Tumor serum specific nucleic acid ligand groups such as liver cancer (H-seq), lung cancer (L-seq) and non-tumor (N-seq) serum specific nucleic acid ligands, each ligand in the detection reagent is 10 9 molecular copy number, 0.1 ⁇ Binding buffer mixed solution;
  • the above washing solution preferably includes a first washing solution and a second washing solution, the first washing solution may be specifically selected as 10 mL, pH 7.4 0.01M Binding buffer (containing 0.17% Tween), the second washing solution may specifically be 10 mL 3 ⁇ SSC, with citric acid and sodium chloride dissolved therein; the above-mentioned
  • the tumor- and non-tumor serum-specific nucleic acid ligand groups are all obtained by bidirectional thermal cycle reduction SELEX technology. Specifically, it can be obtained by using two-way (or more) reduction SELEX technology screening with gastric cancer, liver cancer and lung cancer serum (separate mixtures of more than 10 cases) and non-tumor serum (mixtures of more than 10 cases) as mutual reduction targets. Gastric cancer, liver cancer and lung cancer serum specific nucleic acid ligand group, and non-tumor serum specific nucleic acid ligand group.
  • the content of each serum-specific nucleic acid ligand in the detection reagent is preferably 10 6-10 9 molecular copy number.
  • the nucleic acid ligands in the tumor and non-tumor serum specific nucleic acid ligand groups correspond to the fluorescent probes one by one, that is, the fluorescent probes are designed for each nucleic acid ligand sequence in a one-to-one correspondence Fluorescent probe.
  • the fluorescent probe preferably includes at least one of an MGB probe, a TaqMan probe, and a molecular beacon. The sequence is 5-25 bp in length, and its 3 ′ end and 5 ′ end are labeled with a fluorescent group and a quenching group, respectively.
  • Group fluorescent group: FAM, HEX, TET; quenching group: TAMRA, BHQ and other fluorescent quenching materials), can assist in real-time quantitative-PCR detection.
  • the capture magnetic bead has a functional group or a capture molecule that can be coupled with the target molecule on the surface, and the functional group includes at least one of an epoxy group, a carboxyl group, an amino group, and an NHS, and the target molecule is chemically coupled.
  • the capture molecule is one or more of antigen, antibody, affinity protein and nucleic acid aptamer, etc., which can specifically bind to the target molecule, bind to the surface of the magnetic bead through a functional group, and pass through immunological binding, protein ligand ligand Methods such as ligand binding to nucleic acid ligands bind to target molecules; the target molecules include at least one or more of nucleic acids, proteins, lipids, amino acids, and other biological molecules.
  • the primer is a ligand primer
  • its probe is a 5-25 base sequence on the ligand sequence, with a quenching group and a fluorescent group at the 3 'and 5' ends of the sequence.
  • the blocking liquid includes skimmed milk powder and casein, or bovine blood albumin.
  • the present invention provides a method for detecting a composite target-tumor serum nucleic acid ligand using the above kit.
  • the method specifically includes the following steps:
  • Capture and detect target molecules mix the capture magnetic bead reagent with the sample to be detected, and incubate at 37 °C for 1h to form a magnetic bead-target molecule complex (if non-specific binding needs to be blocked with a blocking solution at 37 °C for 1 hour), first The washing solution was washed 3 times, 3 minutes / time, magnetically separated, and magnetic beads were taken.
  • Binding beacon ligand heating the detection reagent at 95 ° C for 5min, then placing in ice water and rapidly cooling for 5min, adding magnetic beads to combine at 37 ° C for 1 hour, magnetic separation, and discarding the supernatant.
  • Washing add 0.5ml of the second washing solution, wash once, 3 minutes / time, magnetic separation; then add 0.5ml of the first washing solution, wash 3 times, 3 minutes / time, magnetic separation, take magnetic beads.
  • Ligand detection suck up 2 ⁇ L of the supernatant in step 5) into 18 ⁇ L real-time quantitative-PCR reaction solution, perform PCR, collect and process data (or use gene sequencing to complete the qualitative quantification of multi-target ligands).
  • the above step 6) is preferably performed in the following manner: if necessary, multiple real-time quantitative-PCR detection, multiple library screening, multiple primer detection, gene sequencing and other methods are used to detect the ligand or ligand group specifically bound on the capture magnetic beads. Through multiple real-time quantitative-PCR (gene sequencing, etc.) detection of nucleic acid ligand groups, qualitative and quantitative detection of specific marker groups can be achieved.
  • the nucleic acid ligand real-time quantitative-PCR detection method does not need to add any sequence on the ligand, does not change the ligand spatial structure, but the real-time quantitative-PCR detection probe sequence is a 5-25 base sequence on the ligand sequence. This does not require modification of the ligand structure, the binding force between the ligand and the ligand, and improves the detection sensitivity.
  • the detection kit of the present invention is for the detection of a composite target, and the specific nucleic acid ligand group is combined with the serum specific target to convert the composite target serum marker protein signal into a nucleic acid signal, which can be performed by real-time quantitative-PCR Dynamic quantitative detection.
  • the detection method can convert signals of various target molecules into nucleic acid signals through nucleic acid ligands, and has the characteristics of rapid, high sensitivity, strong specificity, and simultaneous detection of multiple ligands.
  • the present invention uses magnetic beads as a carrier to attach target molecules, magnetically separate the detection molecules, and the operation is simple.
  • the nucleic acid ligand of the present invention is non-tumor (N-seq), gastric cancer (G-seq), liver cancer (H-seq), lung cancer (L-seq) serum specific obtained by the two-way thermal cycle reduction SELEX rapid screening method Sex nucleic acid ligand sequence.
  • Fluorescent probes are designed according to ligands respectively, and amplified by PCR to achieve exponential amplification of the target molecular signal.
  • Non-tumor (N-seq) serum nucleic acid ligand can specifically recognize the markers in non-tumor serum, and can be used as a negative control for detecting tumor serum.
  • the present invention is a detection reagent composed of non-tumor (N-seq) serum nucleic acid ligands and gastric cancer (G-seq), liver cancer (H-seq), lung cancer (L-seq) serum specific nucleic acid ligands, which
  • N-seq non-tumor serum nucleic acid ligands
  • G-seq gastric cancer
  • H-seq liver cancer
  • lung cancer L-seq serum specific nucleic acid ligands
  • FIG. 1 is a schematic diagram of the detection of tumor and non-tumor serum by multiple real-time quantitative-PCR in Example 2 of the present invention.
  • kits used in the following embodiments include the following reagents:
  • Magnetic Bead Reagent 5mL, 0.01M Binding buffer with pH 7.4, dissolved 50% of the magnetic bead particles with a particle size of 5 ⁇ 5000nm;
  • Reagent 2 Detection reagent: tumor serum specific nucleic acid ligand: gastric cancer (G-seq), liver cancer (H-seq), lung cancer (L-seq), etc. and non-tumor serum specific nucleic acid ligand: non-tumor (N- seq), using 0.1 ⁇ Binding buffer to dissolve into a mixture of 10 9 molecular copy number;
  • Reagent 3 blocking solution: 10mL including skimmed milk powder and casein;
  • Reagent 4 Washing solution 1: 10 mL, 0.01 M Binding buffer (with 0.17% Tween) at pH 7.4;
  • Reagent 5 Washing solution 2: 10mL 3 ⁇ SSC including citric acid and sodium chloride;
  • Reagent 6 Real-time quantitative-PCR reaction solution 1ml, a PCR system containing a pair of primers and nucleic acid ligand fluorescent probes with different emission wavelengths;
  • Example 1 Real-time quantitative-PCR detection of tumor and non-tumor serum
  • Magnetic beads to capture target molecules Take two equal amounts of 50 ⁇ L each of NHS-based agar magnetic beads in a 1.5 ml EP tube, labeled H1 and H2 respectively, add 50 ⁇ L of serum to be tested to H1 and H2, and place in Incubate at 37 ° C for 1 h, and then block with protein blocking solution at 37 ° C for 1 hour. The washing solution was washed 3 times, 3 minutes / time, and magnetically separated.
  • Binding ligand Take 200 ⁇ L of non-tumor (N-seq), gastric cancer (G-seq), liver cancer (H-seq) and lung cancer nucleic acid ligand (L-seq) detection solutions respectively, and heat at 95 °C for 5min and After cooling in ice water for 5 minutes, add non-tumor (N-seq) to H1 magnetic beads; the mixture of gastric cancer (G-seq), liver cancer (H-seq) and lung cancer (L-seq) is added to H2 magnetic In the beads, combine at 37 ° C for 1 hour, magnetically separate, and discard the supernatant.
  • washing solution 2 0.5ml washing 3 times, 3 minutes / time
  • washing solution 1 0.5ml washing 3 times, 3 minutes / time
  • Preparation of detection template add 15 ⁇ L of 1 ⁇ PCR buffer to H1 and H2 magnetic beads, heat at 95 °C for 5 minutes, and separate the supernatant by magnetic separation.
  • test results result 1, CT value H1 is less than H2, indicating that the serum sample is non-tumor serum; result 2, CT value H1 is greater than H2, indicating that the serum sample is tumor serum.
  • Nucleic acid ligand capture agar magnetic beads are chemically coupled to streptavidin and then combined with avidin and biotinylated ligands to form capture magnetic beads to capture target molecule ligands and detect ligands , Can be the same molecule, or it can be a specific ligand of the target molecule obtained by screening and screening of different nucleic acid libraries; other capture magnetic beads can also be coupled to antibodies or antigens through chemical bonds to form capture magnetic beads; or through chemical bonds to capture target molecules)
  • Binding ligand 200 ⁇ L of mixed detection solution of non-tumor (N-seq), gastric cancer (G-seq), liver cancer (H-seq) and lung cancer nucleic acid ligand (L-seq), heated at 95 °C for 5min and After being placed in ice water and rapidly cooled for 5 min, it was added to the magnetic beads of step (2), combined at 37 ° C for 1 hour, magnetically separated, and the supernatant was discarded.
  • N-seq non-tumor
  • G-seq gastric cancer
  • H-seq liver cancer
  • L-seq lung cancer nucleic acid ligand
  • Multiplex PCR detection take 2 ⁇ L of the supernatant obtained in step (5), add it to 18 ⁇ L of real-time quantitative-PCR reaction solution, referring to the principle shown in Figure 1, the reaction solution contains ligand upstream and downstream primers P7, P11 and four different Wavelength TaqMan probe labeled non-tumor (N-seq), gastric cancer (G-seq), liver cancer (H-seq) and lung cancer (L-seq) nucleic acid ligands, the emission wavelength of the probe is in 4 channels, Perform multiple real-time quantitative-PCR to collect and process data.
  • N-seq non-tumor
  • G-seq gastric cancer
  • H-seq liver cancer
  • L-seq lung cancer
  • test results result 1, CT value: the first channel is less than the second, third and fourth channels, indicating that the serum sample is non-tumor serum; result 2, CT value: the second channel is less than the first, third and fourth channels, indicating the serum
  • the sample may be gastric cancer serum; result 3, CT value: the third channel is less than the first, second, and fourth channels, indicating that the serum sample may be liver cancer serum; result 4, CT value: the fourth channel is less than the first, second, and third channels, This indicates that the serum sample may be lung cancer serum.
  • each tube PCR system can be used to add a reference to detect 3 samples.
  • each group has TaqMan probes with non-tumor nucleic acid ligands as a reference for PCR tube error.
  • the TaqMan probe uses one emission wavelength and is detected in a PCR detection system.
  • nucleic acid libraries can be used to screen ligands for different sample markers as needed.
  • the same detection template can be detected by using different primer PCR systems to obtain marker information and determine the sample type.
  • Example 3 Gene sequencing to detect tumor and non-tumor serum
  • Nucleic acid ligand capture agar magnetic beads are chemically coupled to streptavidin and then combined with avidin and biotinylated ligands to form capture magnetic beads to capture target molecule ligands and detect ligands , Can be the same molecule, or it can be a specific ligand of the target molecule obtained by screening different nucleic acid libraries; other capture magnetic beads can also be coupled to the antibody or antigen through chemical bonds to form the capture magnetic beads; or through chemical bonds to capture the target molecules)
  • Binding ligand configure the nucleic acid ligand to a non-tumor (N-seq), gastric cancer (G-seq), liver cancer (H-seq) and lung cancer nucleic acid ligand (L-seq) with a final concentration of 109 200 ⁇ L of the mixed detection solution was heated at 95 ° C. for 5 min and placed in ice water to rapidly cool for 5 min. Then, it was added to the magnetic beads of (2), combined at 37 ° C. for 1 hour, magnetically separated, and the supernatant was discarded.
  • N-seq non-tumor
  • G-seq gastric cancer
  • H-seq liver cancer
  • L-seq lung cancer nucleic acid ligand
  • washing solution 2 0.5ml washing 3 times, 3 minutes / time
  • washing solution 1 0.5ml washing 3 times, 3 minutes / time
  • step (5) Gene sequencing: The supernatant obtained in step (5) is sequenced and detected by the method of gene second or third generation sequencing, and then each ligand is analyzed.
  • the copy number of the ligand represents the number of specific target molecules, which also represents the nature of the tested serum.

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Abstract

一种复合靶标-肿瘤血清核酸配基检测方法及试剂盒。该方法是对复合样品中标志物靶标的检测方法，是通过特异性靶标的核酸配基组与靶标结合，将靶标志物信号转换成配基核酸信号，利用多重实时定量-PCR进行动态定量检测的方法。该试剂盒包括：捕捉磁珠试剂、检测试剂、封闭液、洗涤液和实时定量-PCR反应液，其中：该捕捉磁珠试剂内溶有粒径为5～5000nm的捕捉磁珠；该封闭液为蛋白封闭液；该检测试剂内溶有肿瘤和非肿瘤血清特异性核酸配基组；该实时定量-PCR反应液包括引物和针对核酸配基的荧光探针。该方法具有快速、灵敏度高、特异性强、多配体同时检测等特点。

Description

一种复合靶标-肿瘤血清核酸配基检测方法及试剂盒 技术领域
本发明涉及生物医学检测领域，具体涉及一种复合靶标-肿瘤血清核酸配基检测方法及试剂盒。
背景技术
随着SELEX技术的发展，核酸配基作为一种特异性强、适用范围广、易于修饰的新型配基分子被应用在疾病的检测和治疗、药物的筛选和应用、食品与环境安全的监测、生物学检测等方面。
核酸信标配基是在核酸配基基础上衍生出的一种新的检测分子，在其配基的5’端连接一段带有荧光标记探针的双链核苷酸序列与一个已知特异性靶分子的核酸配基制备而成的，其既具有核酸配基的识别能力又具有信号储存、传递、放大功能，是一种新型的易于构建、特异性强、灵敏度高、使用范围广的检测分子。但核酸信标配基由于增加信标序列，从而改变配基的核酸分子序列，影响分子空间结构及配基配体的结合力，因此如果不改变核酸配体的结构，又能用实时定量PCR检测将会更方便实用。
磁珠作为一种新型的多功能材料，因其具有很好的生物相容性和表面官能团性使其成为核酸配基检测的理想载体，其在食品、医学、环境和生物分离等方面的应用越来越广泛。
血清是临床工作中最容易获得的标本，而且能够提供大量的机体功能信息。几乎体内所有的细胞都直接或间接地与血液交通，因此任何一种疾病都有可能在血清蛋白中留有痕迹，产生某些特征的变化。
传统检测蛋白质大多用酶联免疫法(ELISA)，捕捉抗体和血清中的抗原结合，再加入连有偶连酶的检测抗体，形成捕捉抗体-抗原-检测抗体“三明治”复合物，最后测偶连酶 活性显示检测结果，但是检测范围受捕捉抗体和抗原反应的Kd值限制，灵敏度不佳。
核酸信标配基检测技术目前有两种具体方法。一种是核酸信标配基介导的免疫-PCR检测，其过程与ELISA法类似，不同点在于检测抗原的不是与捕捉抗体对应的酶标二抗，而是特异性的核酸信标配基与捕捉抗体形成的复合物，利用实时定量PCR检测来完成；另一种是核酸信标配基介导的PCR检测方法，这种方法直接将与靶分子对应的核酸信标配基作为检测分子，与靶分子结合，再洗脱分离后，用实时定量-PCR进行检测。这两种方法都是通过核酸信标配基分子首先对靶分子进行特异性识别，再进行信号传导最后通过实时定量PCR完成信号放大功能从而实现对靶分子的检测。核酸信标配基方法检测靶分子具有快速、灵敏度高、特异性强等优势。
核酸配基实时定量-PCR检测技术是核酸信标配基检测技术的改进型，核酸信标配基是在配基序列两端加上一个双链信标序列，其目的是通过配基结合靶分子将信号通过配基传递给信标序列，再通过实时定量-PCR对信标检测，从而间接实现对靶分子的检测。但是在实施过程中发现，配体作用靶分子往往不一定是一条单链单独完成，而是几条链协同作用完成，同时信标双链也影响配基的空间结构，因此信标序列不仅会影响配基结构，同样也会影响配基间的协同作用，从而影响配基与靶分子结合和检测结果。因此，有必要对该不足加以改进，提升检测技术。
发明内容
为克服现有技术存在的上述不足，本发明提供一种复合靶标-肿瘤血清核酸配基检测方法及试剂盒。本发明通过以下技术方案实现：一种复合靶标-肿瘤血清核酸配基检测试剂盒，包括捕捉磁珠试剂、封闭液、检测试剂、洗涤液和实时定量-PCR反应液，其中：
所述捕捉磁珠试剂内溶有粒径为5～5000nm的捕捉磁珠；
所述封闭液为蛋白封闭液；
所述检测试剂内溶有肿瘤和非肿瘤血清特异性核酸配基组；
所述实时定量-PCR反应液包括引物和针对核酸配基的荧光探针。
上述捕捉磁珠试剂具体可选为5mL、pH值为7.4的0.01M Binding buffer，内溶50％粒径5～5000nm的捕捉磁珠微粒；上述检测试剂为内溶有胃癌(G-seq)、肝癌(H-seq)、肺癌(L-seq)等的肿瘤血清特异性核酸配基组和非肿瘤(N-seq)血清特异性核酸配基，所述检测试剂中的各配基，为10 9分子拷贝数，0.1×Binding buffer混合液；上述洗涤液优选包括第一洗涤液和第二洗涤液，所述第一洗涤液具体可选为10mL、pH值为7.4的0.01M Binding buffer(含0.17％Tween)，所述第二洗涤液具体可选为10mL 3×SSC，内溶有柠檬酸、氯化钠；上述实时定量-PCR反应液具体可选为包含一对引物和不同发射波长核酸配基荧光探针的PCR体系。
优选的，所述的肿瘤和非肿瘤血清特异性核酸配基组均通过双向热循环消减SELEX技术筛选获得。具体可选为用胃癌、肝癌和肺癌血清(分别10例以上血清混合液)分别和非肿瘤血清(10例以上血清混合液)彼此互为消减靶标进行双向(或多项)消减SELEX技术筛选获得的胃癌、肝癌和肺癌血清特异性核酸配基组，以及非肿瘤血清特异性核酸配基组。在检测试剂中每个血清特异性核酸配基含量优选为10 6—10 9分子拷贝数。
优选的，所述的肿瘤和非肿瘤血清特异性核酸配基组中的核酸配基与所述荧光探针一一对应，即荧光探针是针对每一个核酸配基序列设计的一一对应的荧光探针。具体地，荧光探针优选包括MGB探针、TaqMan探针和分子信标等至少一种，长度为5-25bp的序列，其3’端和5’端分别标记有荧光基团和淬灭基团(荧光基团：FAM、HEX、TET；淬灭基团：TAMRA，BHQ等荧光淬灭材料)，可进行辅助实时定量-PCR检测。
优选的，所述捕捉磁珠表面处具有可与靶分子发生偶联的官能团或捕捉分子，所述官能团包括环氧基、羧基、氨基和NHS中的至少一种，通过化学偶联靶分子。所述捕捉分子为抗原、抗体、亲合蛋白和核酸适配体等中的一种或多种，能与靶分子特异结合，通过官 能团结合到磁珠表面，通过免疫结合、蛋白配体配基和核酸配体配基结合等方法与靶分子结合；所述靶分子包括核酸、蛋白、脂类和氨基酸及其它生物分子中的至少一种或多种。
优选的，所述引物为配基引物，其探针为配基序列上的5-25个碱基序列，序列3’和5’端带有淬灭基团和荧光基团。
优选的，所述封闭液包括脱脂奶粉和酪蛋白，或者牛血白蛋白。
本发明提供一种用上述试剂盒进行复合靶标-肿瘤血清核酸配基检测的方法，该方法具体包括以下步骤：
1)制备待检测样品：血液去除血细胞和血脂，分离得到血清；
2)捕获检测靶分子：将捕捉磁珠试剂与待检测样品混合，在37℃孵育1h，形成磁珠-靶分子复合物(如非特异性结合需用封闭液37℃封闭1小时)，第一洗涤液洗3次，3分钟/次，磁分离，取磁珠。
3)结合信标配基：将检测试剂95℃加热5min，再置于冰水中迅速冷却5min，加入磁珠中37℃结合1小时，磁分离，弃上清。
4)洗涤：加入0.5ml第二洗涤液，洗1次，3分钟/次，磁分离；再加入0.5ml第一洗涤液，洗3次，3分钟/次，磁分离，取磁珠。
5)提取配基：在磁珠中加入1×PCR缓冲液，15μL，95℃加热5分钟，磁分离，取上清。
6)配基检测：吸取步骤5)所述上清2μL加入18μL实时定量-PCR反应液中，进行PCR，采集并处理数据(或用基因测序完成多靶配基的定性定量)。
上述的步骤1)，可通过以下方式进行：用静脉穿刺法采取血液于含抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，然后3000转，离心10min，收集上清于-80℃冷冻30min后，12000g，离心30min,除去血脂，分离得待检测血清(或分别经过水:乙腈：血清＝2:0.5:1的比例混合处理，5000r/min低温离心30min,吸取上清，去除高丰度蛋白)。
上述的步骤6)，优选通过以下方式进行：根据需要采用多重实时定量-PCR检测、多库筛选多引物检测、基因测序等方法对捕捉磁珠上特异结合的配基或配基组进行检测。通过多重实时定量-PCR(基因测序等)对核酸配基组的检测，可实现对特异性标志物组的定性定量检测。
核酸配基实时定量-PCR检测方法不需要在配基上增加任何序列，不改变配基空间结构，只是实时定量-PCR检测探针序列为配基序列上的5-25个碱基序列。这样不需要改造配基结构、配基和配体的结合力，又提高了检测的灵敏度。
本发明的优点在于：
1.本发明的检测试剂盒是对复合靶标的检测，通过特异性核酸配基组与血清特异性靶标结合，将复合靶血清标志物蛋白信号转换成核酸信号，即可利用实时定量-PCR进行动态定量检测。该检测方法可使多种靶分子的信号通过核酸配基转换成核酸信号，具有快速、灵敏度高、特异性强、多配体同时检测等特点。
2.本发明以磁珠作为载体附着靶分子，对检测分子进行磁性分离，操作简单。
3.本发明的核酸配基是通过双向热循环消减SELEX快速筛选方法得到的非肿瘤(N-seq)、胃癌(G-seq)、肝癌(H-seq)、肺癌(L-seq)血清特异性核酸配基序列。根据配基分别设计荧光探针，通过PCR扩增，实现靶分子信号的指数级放大。
4.非肿瘤(N-seq)血清核酸配基可以特异性识别非肿瘤血清中的标志物，可作为检测肿瘤血清的阴性对照。
5.本发明是采用非肿瘤(N-seq)血清核酸配基和胃癌(G-seq)、肝癌(H-seq)、肺癌(L-seq)血清特异性核酸配基组成的检测试剂，其优点是通过检测可相对明确的区分血清中具有肿瘤标志物还是非肿瘤标志物。
附图说明
下面结合附图和具体实施例对本发明作进一步详细说明。
图1为本发明实施例2多重实时定量-PCR检测肿瘤和非肿瘤血清的原理示意图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的描述：
作为一种示例，下述各实施例所用试剂盒，包括以下试剂：
试剂1：捕捉磁珠试剂：5mL、pH值为7.4的0.01M Binding buffer，内溶50％粒径5～5000nm的捕捉磁珠微粒；
试剂2：检测试剂：肿瘤血清特异性核酸配基：胃癌(G-seq)、肝癌(H-seq)、肺癌(L-seq)等和非肿瘤血清特异性核酸配基：非肿瘤(N-seq)，分别用0.1×Binding buffer溶解为10 9分子拷贝数的混合液；
试剂3：封闭液：10mL包括脱脂奶粉和酪蛋白；
试剂4：洗涤液1：10mL、pH值为7.4的0.01M Binding buffer(含0.17％Tween)；
试剂5：洗涤液2：10mL 3×SSC包括柠檬酸、氯化钠；
试剂6：实时定量-PCR反应液1ml，包含一对引物和不同发射波长核酸配基荧光探针的PCR体系；
实施例1：实时定量-PCR分别检测肿瘤和非肿瘤血清
本实施例包括以下步骤：
(1)制备待检测样品：按静脉穿刺法采取血液于含抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，然后在3000转，离心10min，收集上清于-80℃冷冻30min后，12000g，离心30min,除去血脂；再经过水:乙腈：血清＝2:0.5:1的比例混合处理，5000r/min低温离心30min,吸取上清，去除高丰度蛋白，得待检测血清。
(2)磁珠捕捉靶分子：取2份等量的NHS基琼脂磁珠各50μL于1.5ml EP管中，分 别标记为H1和H2、向H1、H2中各加入50μL待检测血清，置于37℃孵育1h，再用蛋白封闭液37℃封闭1小时后洗涤液1洗3次，3分钟/次，磁分离。
(3)结合配基：分别取非肿瘤(N-seq)、胃癌(G-seq)、肝癌(H-seq)和肺癌核酸配基(L-seq)检测液各200μL，95℃加热5min并置于冰水中迅速冷却5min后，将非肿瘤(N-seq)加入到H1磁珠中；胃癌(G-seq)、肝癌(H-seq)和肺癌(L-seq)混合液加入到H2磁珠中，37℃结合1小时，磁分离，弃上清。
(4)洗涤：以上两管中分别加入洗涤液2，0.5ml洗涤3次，3分钟/次；再加入洗涤液1，0.5ml洗涤3次，3分钟/次；
(5)制备检测模板：在H1和H2磁珠中加入15μL 1×PCR缓冲液，95℃加热5分钟，磁分离取上清。
(6)检测：分别取步骤(5)H1和H2上清2μL加入至18μL，实时定量-PCR反应体系中(SYBRGreen I)，进行实时定量-PCR，采集并处理数据。
检测结果分析：结果1，CT值H1小于H2说明血清样本属非肿瘤血清；结果2，CT值H1大于H2说明血清样本属肿瘤血清。
实施例2：多重实时定量-PCR检测肿瘤和非肿瘤血清
本实施例包括以下步骤：
(1)制备待检测样品：按静脉穿刺法采取血液于含抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，然后在3000转，离心10min，收集上清于-80℃冷冻30min后，12000g，离心30min,除去血脂；再经过水:乙腈：血清＝2:0.5:1的比例混合处理，5000r/min低温离心30min,吸取上清，去除高丰度蛋白，得待检测血清。
(2)制备磁珠靶分子复合物：取1份的捕捉琼脂磁珠50μL于1.5ml EP管中，加入50μL待检测血清，置于37℃孵育1h，洗涤液1，洗3次，3分钟/次，磁分离。(注：核酸配基捕捉琼脂磁珠是通过化学键偶联链霉亲和素，再通过亲和素与生物素化的配基结合形 成的捕捉磁珠，捕捉靶分子的配基和检测配基，可以是同一分子，也可是不同核酸文库筛选筛选获得的靶分子特异性配基；其他捕捉磁珠也可以通过化学键偶联抗体或抗原形成捕捉磁珠；或通过化学键偶联捕捉靶分子)
(3)结合配基：分别将非肿瘤(N-seq)、胃癌(G-seq)、肝癌(H-seq)和肺癌核酸配基(L-seq)混合检测液200μL，95℃加热5min并置于冰水中迅速冷却5min后，加到步骤(2)的磁珠中，37℃结合1小时，磁分离，弃上清。
(4)洗涤；在管中分别加入洗涤液2，0.5ml洗涤3次，3分钟/次；再加入洗涤液1，0.5ml洗涤3次，3分钟/次；
(5)制备检测模板：在磁珠中，加入15μL 1×PCR缓冲液，95℃加热5分钟，磁分离取上清。
(6)多重PCR检测：取2μL步骤(5)所得上清，加入18μL实时定量-PCR反应液中，参考图1所示原理，反应液含配基上下游引物P7、P11及用四种不同波长TaqMan探针标记的非肿瘤(N-seq)、胃癌(G-seq)、肝癌(H-seq)和肺癌(L-seq)核酸配基，探针的发射波长分别在4个通道中，进行多重实时定量-PCR，采集并处理数据。
检测结果分析：结果1，CT值：第一通道小于第二、三和四通道，说明血清样本属非肿瘤血清；结果2，CT值：第二通道小于第一、三和四通道，说明血清样本可能属胃癌血清；结果3，CT值：第三通道小于第一、二和四通道，说明血清样本可能属肝癌血清；结果4，CT值：第四通道小于第一、二和三通道，说明血清样本可能属肺癌血清。
需要说明的是：
1、检测样本大于实时定量-PCR检测4通道时，可采用每管PCR体系加一个参照，检测3个样本。如：检测十种肿瘤，则可分成4组PCR检测体系，每组中都带有非肿瘤核酸配基的TaqMan探针作为PCR管间误差参照。
2、每种肿瘤如果有2种以上的配基时，可以归到一组，TaqMan探针采用一个发射波 长，在一个PCR检测体系中检测。
3、本实施例根据需要，可对不同的样本标志物采用不同的核酸文库筛选配基。这样在样品检测时，就可以对相同检测模板，采用不同的引物PCR体系检测，获得标志物信息，确定样本类型。
实施例3：基因测序检测肿瘤和非肿瘤血清
本实施例包括以下步骤：
(1)制备待检测样品：按静脉穿刺法采取血液于含抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，然后在3000转，离心10min，收集上清于-80℃冷冻30min后，12000g，离心30min,除去血脂；再经过水:乙腈：血清＝2:0.5:1的比例混合处理，5000r/min低温离心30min,吸取上清，去除高丰度蛋白，得待检测血清。
(2)制备磁珠靶分子复合物：取1份的捕捉琼脂磁珠50μL于1.5ml EP管中，加入50μL待检测血清，置于37℃孵育1h，洗涤液1，洗3次，3分钟/次，磁分离。(注：核酸配基捕捉琼脂磁珠是通过化学键偶联链霉亲和素，再通过亲和素与生物素化的配基结合形成的捕捉磁珠，捕捉靶分子的配基和检测配基，可以是同一分子，也可是不同核酸文库筛选获得的靶分子特异性配体；其他捕捉磁珠也可以通过化学键偶联抗体或抗原形成捕捉磁珠；或通过化学键偶联捕捉靶分子)
(3)结合配基：分别将核酸配基配置终浓度为10 9的非肿瘤(N-seq)、胃癌(G-seq)、肝癌(H-seq)和肺癌核酸配基(L-seq)混合检测液200μL，95℃加热5min并置于冰水中迅速冷却5min后，加到(2)的磁珠中，37℃结合1小时，磁分离，弃上清。
(4)洗涤：在管中分别加入洗涤液2，0.5ml洗涤3次，3分钟/次；再加入洗涤液1，0.5ml洗涤3次，3分钟/次；
(5)制备检测模板：在磁珠中，加入15μL 1×PCR缓冲液，95℃加热5分钟，磁分离取上清。
(6)基因测序：采用基因二代或三代测序的方法对步骤(5)所得上清进行测序检测，然后对每条配基进行分析。总之：配基的拷贝数代表特异性靶分子的多少，也就代表被检血清的性质。
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
上面对本发明专利进行了示例性的描述，显然本发明专利的实现并不受上述方式的限制，只要采用了本发明专利的方法构思和技术方案进行的各种改进，或未经改进将本发明专利的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (9)

  1. 一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，包括捕捉磁珠试剂、封闭液、检测试剂、洗涤液和实时定量-PCR反应液，其中：
    所述捕捉磁珠试剂内溶有粒径为5～5000nm的捕捉磁珠；
    所述封闭液为蛋白封闭液；
    所述检测试剂内溶有肿瘤和非肿瘤血清特异性核酸配基组；
    所述实时定量-PCR反应液包括引物和针对核酸配基的荧光探针。
  2. 根据权利要求1所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，所述的肿瘤和非肿瘤血清特异性核酸配基组均通过双向热循环消减SELEX技术筛选获得。
  3. 根据权利要求1所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，所述的肿瘤和非肿瘤血清特异性核酸配基组中的核酸配基与所述荧光探针是一一对应。
  4. 根据权利要求3所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，所述荧光探针包括MGB探针、TaqMan探针和分子信标中的至少一种，且所述荧光探针是针对核酸配基序列设计。
  5. 根据权利要求1所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，所述捕捉磁珠表面具有可与靶分子发生偶联的官能团或捕捉分子，所述官能团包括环氧基、羧基、氨基和NHS中的至少一种，可通过化学基团偶联所述靶分子；所述捕捉分子为抗原、抗体、亲合蛋白和核酸配基中的一种或多种，可通过免疫结合、蛋白配体配基或核酸配体配基方式结合捕捉所述靶分子；所述靶分子包括核酸、蛋白、脂类、氨基酸及其它生物分子中的至少一种或多种。
  6. 根据权利要求1所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，所述检测试剂内溶有针对非肿瘤血清SELEX消减筛选的胃癌、肝癌和肺癌的肿瘤血清特异性核酸配基和针对肿瘤血清SELEX消减筛选的非肿瘤血清特异性核酸配基。
  7. 根据权利要求1所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于， 所述洗涤液包括第一洗涤液和第二洗涤液，所述第一洗涤液为内溶有Tween的Binding buffer，所述第二洗涤液为内溶有柠檬酸和氯化钠的SSC。
  8. 根据权利要求1所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，所述封闭液包括脱脂奶粉和酪蛋白，或者牛血清白蛋白。
  9. 根据权利要求1所述的一种复合靶标-肿瘤血清核酸配基检测试剂盒，其特征在于，所述引物为配基引物，其探针为配基序列上的5-25个碱基序列，序列3’和5’端带有淬灭基团和荧光基团。
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Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113624724A (zh) * 2020-05-07 2021-11-09 廖世奇 一种适配体分子信标对靶分子的多元检测分析方法
CN111849996A (zh) * 2020-08-07 2020-10-30 燕山大学 一种高特异性识别肺癌血清的寡核酸适配体Seq-32及应用
CN116087497B (zh) * 2021-11-05 2024-08-16 湖南早晨纳米机器人有限公司 一种非线性杂交链式免疫检测试剂及方法
CN115980013B (zh) * 2023-02-22 2025-07-01 东南大学 一种基于荧光共振能量转移的人血清afp-l3%检测试剂盒及其检测方法与应用
WO2025049720A2 (en) * 2023-08-31 2025-03-06 Illumina, Inc. Aptamer dynamic range compression and detection techniques
CN119044488A (zh) * 2024-10-31 2024-11-29 内蒙古大学 一种牛奶中大肠杆菌o157:h7快速检测试剂盒及其检测方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1521272A (zh) * 2003-01-28 2004-08-18 甘肃省医学科学研究院 新型配体检测方法
CN1721852A (zh) * 2004-07-15 2006-01-18 甘肃省医学科学研究院 抗原及配体pcr管式检测试剂盒及其制备方法和应用
CN101130814A (zh) * 2007-08-30 2008-02-27 西北师范大学 核酸配基组芯片及其制备方法
CN101473227A (zh) * 2006-06-22 2009-07-01 奥林巴斯株式会社 目标核酸的检测方法及该检测方法中所使用的容器
US20150140560A1 (en) * 2013-11-04 2015-05-21 The Regents Of The University Of Michigan Asynchronous magnetic bead rotation (ambr) microviscometer for analysis of analytes
CN105018590A (zh) * 2015-01-30 2015-11-04 廖世奇 蛋白配体和基因同时检测试剂盒及应用
CN108778508A (zh) * 2016-01-14 2018-11-09 欧洲分子生物学实验室 配体诱导的细胞表达的微流体分析

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1776474A2 (en) * 2004-07-23 2007-04-25 (Osi) Eyetech, Inc. Detection of oligonuleotides by dual hybridization
CN100507522C (zh) * 2004-12-15 2009-07-01 中国科学院上海应用物理研究所 Dna的荧光检测方法及其试剂盒
CN101131814B (zh) * 2006-08-25 2010-08-11 智宝科技股份有限公司 图像处理方法以及图像显示系统
WO2008044214A1 (en) * 2006-10-12 2008-04-17 Koninklijke Philips Electronics N.V. Fast biosensor with reagent layer
CN102382813A (zh) * 2010-08-30 2012-03-21 中国人民解放军军事医学科学院基础医学研究所 一种针对液相非纯化复合靶标的电泳凝胶阻滞-selex技术方法
CN102230938B (zh) * 2011-06-22 2013-10-30 中国科学院武汉病毒研究所 一种基于免疫磁珠富集的甲型流感病毒检测试剂盒及方法
CN102391372B (zh) * 2011-11-08 2014-07-23 中国人民解放军军事医学科学院基础医学研究所 一种肝癌药物治疗的靶标及其应用
CN102766634B (zh) * 2012-08-09 2013-09-11 武汉大学 一种靶向人高转移肝癌细胞的单链dna适配子及其应用
CN103320445B (zh) * 2013-07-11 2017-07-28 重庆市肿瘤研究所 特异性识别胃癌细胞的dna适配子gca‑5及其应用
CN103756967B (zh) * 2013-12-31 2018-09-21 卢英 抗hla-g的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用
CN104450713B (zh) * 2014-04-11 2017-09-15 中国人民解放军军事医学科学院基础医学研究所 一种特异识别异质性核糖核蛋白A2/B1(hnRNPA2/B1)的寡核苷酸适配体C6‑8的序列和应用
CN103966224B (zh) * 2014-05-12 2016-07-13 朱育盼 一种适配子及其筛选方法和应用
CN104345154B (zh) * 2014-08-22 2016-10-26 北京蛋白质组研究中心 一种检测多肿瘤相关“多肽-蛋白组合式标志物”的双抗体夹心试剂盒
CN104931703A (zh) * 2015-06-29 2015-09-23 上海交通大学 一种检测肿瘤标记物ca72-4的免疫磁珠试纸条及制备方法
CN105158485A (zh) * 2015-08-17 2015-12-16 山东大学 人绒毛膜促性腺激素肿瘤标志物过糖基化修饰的检测试剂盒
CN106093437B (zh) * 2016-08-03 2018-03-20 广州伯信生物科技有限公司 一种DNA pulldown方法及试剂盒
CN107723363A (zh) * 2016-08-11 2018-02-23 博尔诚（北京）科技有限公司 肿瘤标志物的组合检测方法及其应用
CN106353499A (zh) * 2016-09-14 2017-01-25 燕山大学 肺癌血清标志物适配体和基因同时检测试剂盒及应用
CN107893101B (zh) * 2017-12-22 2021-06-15 郑州大学 一种用于肿瘤疾病早期诊断的试剂盒、方法及应用
CN108753941B (zh) * 2018-06-22 2022-03-08 广东顺德工业设计研究院(广东顺德创新设计研究院) 双重标记磁珠及其制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1521272A (zh) * 2003-01-28 2004-08-18 甘肃省医学科学研究院 新型配体检测方法
CN1721852A (zh) * 2004-07-15 2006-01-18 甘肃省医学科学研究院 抗原及配体pcr管式检测试剂盒及其制备方法和应用
CN101473227A (zh) * 2006-06-22 2009-07-01 奥林巴斯株式会社 目标核酸的检测方法及该检测方法中所使用的容器
CN101130814A (zh) * 2007-08-30 2008-02-27 西北师范大学 核酸配基组芯片及其制备方法
US20150140560A1 (en) * 2013-11-04 2015-05-21 The Regents Of The University Of Michigan Asynchronous magnetic bead rotation (ambr) microviscometer for analysis of analytes
CN105018590A (zh) * 2015-01-30 2015-11-04 廖世奇 蛋白配体和基因同时检测试剂盒及应用
CN108778508A (zh) * 2016-01-14 2018-11-09 欧洲分子生物学实验室 配体诱导的细胞表达的微流体分析

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ROGGE, L. ET AL: "Transcript imaging of the development of human T helper cells using oligonucleotide arrays", NATURE GENETICS, vol. 25, 1 May 2000 (2000-05-01), pages 96 - 101, XP055705832, ISSN: 1061-4036, DOI: 10.1038/75671 *

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