CN111763713A - Method and kit for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes - Google Patents

Method and kit for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes Download PDF

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CN111763713A
CN111763713A CN202010207009.5A CN202010207009A CN111763713A CN 111763713 A CN111763713 A CN 111763713A CN 202010207009 A CN202010207009 A CN 202010207009A CN 111763713 A CN111763713 A CN 111763713A
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常津
陈明慧
宫晓群
王汉杰
罗冉
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Abstract

The invention belongs to the field of miRNA detection, and particularly relates to a method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purposes. Comprises the following steps of 1) preparing colloidal gold nanoparticles with uniform particle size by a hydrothermal method; 2) activating sulfhydryl group h-DNA by using TCEP, and reacting with colloidal gold to form a gold-sulfur bond for preparing a colloidal gold nucleic acid probe; 3) complementary pairing is carried out on the colloidal gold nucleic acid probe and the miRNA to be detected, and double-strand specific nuclease is added to realize target recycling and signal amplification; after the reaction is finished, adding a stop solution to inactivate the enzyme to obtain a reaction solution; 4) assembling and detecting the nucleic acid test strip: adding the reaction solution generated in the step 3) into the assembled nucleic acid test strip, and observing the detection result. The invention can obviously improve the detection sensitivity of miRNA.

Description

非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测 miRNA-21的方法及试剂盒Non-diagnostic purpose based on target isothermal cyclic amplification and nucleic acid test strip technology detection Methods and kits for miRNA-21

技术领域technical field

本发明属于miRNA检测领域,具体涉及一种非诊断目的基于靶标等温循环 扩增及核酸试纸条技术检测miRNA-21的方法。The invention belongs to the field of miRNA detection, and in particular relates to a method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes.

背景技术Background technique

微小RNA-21(miRNA-21)是一类内源性非编码RNA小分子,miRNA-21的显 着异常表达与各种疾病,特别是人类肿瘤的发生及发展密切相关。有关miRNA-21 在癌症中的功能已经有大量的文献研究,多项研究表明癌症患者的外周血miRNA 表达谱发生特异性改变,越来越多证据表明miRNA-21可作为一种新颖的分子标 志物应用于癌症诊断和预后评估.传统的基于miRNA-21的检测如基因测序、荧 光定量PCR等,但是这些检测方式毫无例外都需要大型的仪器配合,不仅费时、 费力,而且只适合医院等一些检测机构,无法造福于每个家庭以实现便携、惠 民大众化。因此miRNA-21的早期简便检测已经成为现今的研究热点。MicroRNA-21 (miRNA-21) is a class of endogenous non-coding RNA small molecules, and the significant abnormal expression of miRNA-21 is closely related to the occurrence and development of various diseases, especially human tumors. There have been a lot of literature studies on the function of miRNA-21 in cancer. Many studies have shown that the miRNA expression profile in peripheral blood of cancer patients is specifically altered. More and more evidences suggest that miRNA-21 can be used as a novel molecular marker. It can be used in cancer diagnosis and prognosis evaluation. Traditional miRNA-21-based detections such as gene sequencing, fluorescence quantitative PCR, etc., but these detection methods all require large-scale instruments without exception, which are not only time-consuming and labor-intensive, but also only suitable for hospitals, etc. Some testing institutions cannot benefit every family to achieve portability and popularization. Therefore, the early and simple detection of miRNA-21 has become a research hotspot.

靶标等温循环扩增技术通常是指基于酶促反应的核酸体外等温扩增技术, 主要利用核酸酶剪切DNA识别位点的能力,在等温条件下,促进目标物的结合 与释放,从而产生相应的信号实现信号累积及扩增。其中双链特异性核酸酶(DSN) 是一种最常见的适用于miRNA检测的核酸酶,DSN能高效识别并酶切完全互补配 对的DNA双链或者DNA/RNA杂交双链中的DNA链,而对单链DNA和单/双链RNA 几乎没有作用的核酸酶,此外,这种降解作用仅针对于至少12个完全匹配的核 苷酸序列,因此它具有高度特异性。Target isothermal cyclic amplification technology usually refers to the in vitro isothermal amplification of nucleic acid based on enzymatic reaction, which mainly uses the ability of nucleases to cleave DNA recognition sites, and promotes the binding and release of targets under isothermal conditions, thereby producing corresponding The signal achieves signal accumulation and amplification. Among them, double-strand specific nuclease (DSN) is one of the most common nucleases suitable for miRNA detection. DSN can efficiently identify and digest DNA double-stranded DNA that is completely complementary paired or DNA/RNA hybrid double-stranded DNA strands. While nucleases have little effect on single-stranded DNA and single/double-stranded RNA, in addition, this degradation is only directed against at least 12 perfectly matched nucleotide sequences, so it is highly specific.

胶体金核酸试纸条是一种对复杂样品中目标物质进行检测的固相免疫分析方法,其主要原理如下:利用胶体金标记一种短的DNA序列,与目标物质核酸反 应形成复合物,由于毛细管作用在固相层析膜上涌动,被检测线上的DNA序列 捕获,形成肉眼可见的T线。一定条件下,T线信号强弱与目标核酸浓度正相关, 将胶体金免疫层析检测试纸条联合胶体金读取仪的方法,根据反射光度法原理, 可以快速、准确、简单的对相关物质进行定量或者半定量检测,胶体金核酸试 纸条已成为一种核酸即时检验(Point-of-care testing,POCT)方法。The colloidal gold nucleic acid test strip is a solid-phase immunoassay method for the detection of target substances in complex samples. Capillary action surges on the solid-phase chromatography membrane and is captured by the DNA sequence on the detection line, forming a T-line visible to the naked eye. Under certain conditions, the strength of the T-line signal is positively correlated with the concentration of the target nucleic acid. The method of combining the colloidal gold immunochromatographic detection test strip with the colloidal gold reader, according to the principle of reflection photometry, can quickly, accurately and simply measure the correlation. For quantitative or semi-quantitative detection of substances, colloidal gold nucleic acid test strips have become a point-of-care testing (POCT) method.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于克服现有技术中的缺陷,提供一种非诊断目的基于靶标 等温循环扩增及核酸试纸条技术检测miRNA-21的方法。The object of the present invention is to overcome the defects in the prior art, and provide a non-diagnostic method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology.

本发明为实现上述目的,采用以下技术方案:The present invention adopts the following technical solutions to achieve the above object:

一种非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测miRNA-21的 方法,包括下述步骤:A method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes, comprising the following steps:

1)使用水热法制备均一粒径的胶体金纳米粒子;1) use the hydrothermal method to prepare the colloidal gold nanoparticles of uniform particle size;

2)使用TCEP活化巯基h-DNA后,与胶体金反应形成金硫键用于制备胶体金 核酸探针;其中,巯基h-DNA为 5’-SH-TTTTTAGCTTATCAACTACTGATCAACATCAGTCTGATAAGCTA-3’;2) after using TCEP to activate sulfhydryl h-DNA, react with colloidal gold to form a gold sulfide bond for the preparation of colloidal gold nucleic acid probe; Wherein, sulfhydryl h-DNA is 5'-SH-TTTTTAGCTTATCAACTACTGATCAACATCAGTCTGATAAGCTA-3';

3)将胶体金核酸探针和待检miRNA互补配对,加入双链特异性核酸酶,实 现靶标循环回收及信号放大;反应结束后,加入终止液,使酶失活,得到反应 液;3) complementary pairing of the colloidal gold nucleic acid probe and the miRNA to be detected, adding a double-stranded specific nuclease to achieve target recycling and signal amplification; after the reaction is completed, adding a stop solution to inactivate the enzyme to obtain a reaction solution;

4)核酸试纸条的组装和检测:将上步骤3)产生的反应液加入到组装好的 核酸试纸条上,观察检测结果;所述的核酸试纸条上包含有检测垫;所述检测 垫上包含有检测线,所述的检测线上保护有sd—DNA,浓度为5-20μM;sd—DNA 的碱基序列号为5’-biotin-AAAACATGGTTACCGATCCAAGTTCAGTAGTTGATA-3’。4) Assembly and detection of nucleic acid test strips: add the reaction solution generated in the above step 3) to the assembled nucleic acid test strips, and observe the detection results; the nucleic acid test strips contain detection pads; the nucleic acid test strips contain detection pads; The detection pad contains a detection line, and the detection line is protected with sd-DNA at a concentration of 5-20 μM; the base sequence number of the sd-DNA is 5'-biotin-AAAACATGGTTACCGATCCAAGTTCAGTAGTTGATA-3'.

2.根据权利要求1所述的非诊断目的基于靶标等温循环扩增及核酸试纸条 技术检测miRNA-21的方法,其特征在于,步骤1)的具体步骤为:2. the non-diagnostic purpose according to claim 1 detects the method for miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology, it is characterized in that, the concrete steps of step 1) are:

将柠檬酸钠溶液加入到反应器,在磁力搅拌条件下加热至沸腾,第一次加 入氯金酸溶液;氯金酸与柠檬酸钠摩尔配比为1:10-20;溶液变为浅粉色后停止 加热,溶液降温到90-95℃,加入与第一次加入量等量的氯金酸溶液反应 20-30min,再次加入与第一次加入量等量的氯金酸溶液反应得到金种溶液;The sodium citrate solution was added to the reactor, heated to boiling under magnetic stirring, and the chloroauric acid solution was added for the first time; the molar ratio of chloroauric acid and sodium citrate was 1:10-20; the solution turned light pink Then stop heating, the solution is cooled to 90-95 ℃, add the same amount of chloroauric acid solution added for the first time to react for 20-30min, and then add the same amount of chloroauric acid solution added for the first time to react to obtain gold seeds solution;

将金种溶液和另外准备的柠檬酸钠溶液混合作为下一步反应的生长液,生 长液加热到90-95℃后加入氯金酸溶液反应20-30min,再次加入等量氯金酸溶 液得到下一步反应的大粒径胶体金纳米粒子溶液;其中,金种子溶液:氯金酸: 柠檬酸钠摩尔配比为1:1:13.75;制备得到的胶体金纳米粒子溶液粒径为 13-40nm。The gold seed solution and the separately prepared sodium citrate solution are mixed as the growth solution for the next step of the reaction, the growth solution is heated to 90-95 ° C, and then the chloroauric acid solution is added to react for 20-30min, and the same amount of chloroauric acid solution is added again to obtain the following: A large-diameter colloidal gold nanoparticle solution for one-step reaction; wherein, the molar ratio of gold seed solution: chloroauric acid: sodium citrate is 1:1:13.75; the diameter of the prepared colloidal gold nanoparticle solution is 13-40 nm.

步骤2)的具体步骤为10-50μL的浓度为10-50μM的巯基茎环h-DNA探针, 加入10-50μL的浓度为10-50mM的三(2-羧乙基)膦TCEP溶液,活化1-3h后, 加入1-5mL均一粒径纳米粒子,旋转培养反应16-24小时;之后加入4-10μL 的1mg/mL的鼠源单克隆第一抗体Ab1(北京博奥森生物科技,bsm-2027M),反 应1-3h;再加入0.1-0.5mL的浓度为3M的氯化钠溶液,置于4℃反应过夜后, 在8000-15000转速条件下离心15-30min,除去上清,用0.1-0.5mL的0.01M磷 酸缓冲溶液PBS重悬沉淀,得到的则为胶体金核酸探针。The specific steps of step 2) are as follows: 10-50 μL of a thiol stem-loop h-DNA probe with a concentration of 10-50 μM, adding 10-50 μL of a tris(2-carboxyethyl) phosphine TCEP solution with a concentration of 10-50 mM, and activated After 1-3h, add 1-5mL of uniform particle size nanoparticles, and rotate for 16-24 hours; then add 4-10μL of 1mg/mL mouse monoclonal primary antibody Ab1 (Beijing Boaosen Biotechnology, bsm -2027M), react for 1-3h; then add 0.1-0.5mL of sodium chloride solution with a concentration of 3M, put it at 4°C to react overnight, centrifuge at 8000-15000 revolutions for 15-30min, remove the supernatant, and use Resuspend the pellet in 0.1-0.5mL of 0.01M phosphate buffered solution PBS to obtain a colloidal gold nucleic acid probe.

步骤3)的具体步骤为取3-7μL胶体金核酸探针和待检miRNA按照体积比 为1-3:1比例混合于磷酸缓冲液中,加入0.1-0.6U双链特异性核酸酶及10×双 链特异性核酸酶缓冲液,涡旋仪上低速旋转使其混匀,然后将上述混合溶液置 于金属浴中,35-60℃下反应20-60分钟,实现靶标循环回收及信号放大。反应 结束后,加入终止液,45℃反应5分钟,使酶失活。The specific steps of step 3) are to mix 3-7 μL of colloidal gold nucleic acid probe and miRNA to be detected in phosphate buffer solution according to the volume ratio of 1-3:1, add 0.1-0.6U of double-stranded specific nuclease and 10 ×Double-strand specific nuclease buffer, rotate at low speed on a vortexer to mix evenly, then place the above mixed solution in a metal bath and react at 35-60°C for 20-60 minutes to achieve target recycling and signal amplification . After the reaction was completed, a stop solution was added and the reaction was carried out at 45°C for 5 minutes to inactivate the enzyme.

步骤4)中核酸试纸条包括试纸条卡壳以及设置在所述的试纸条卡壳内的顺 序设置的样本垫、胶金垫、检测垫、以及吸收垫;其中样本垫的长度为1.1-2cm 之间;胶金垫的长度为0.3-0.8cm;检测垫的长度为2.5-4.5cm;吸收垫的长度 为1.1-2cm。In step 4), the nucleic acid test strip includes a test strip jam and sequentially arranged sample pads, glue gold pads, detection pads, and absorption pads arranged in the test strip jam; wherein the length of the sample pad is 1.1- Between 2cm; the length of the glue gold pad is 0.3-0.8cm; the length of the detection pad is 2.5-4.5cm; the length of the absorption pad is 1.1-2cm.

所述的检测垫包含检测线和质控线,其中质控线上包含了抗Ab1((北京博 奥森生物科技,bsm-2027M))的第二抗体Ab2(赛默飞科技,A32723),浓度为 0.8-2mg。The detection pad comprises a detection line and a quality control line, wherein the quality control line contains the secondary antibody Ab2 (Thermo Fisher Scientific, A32723) of anti-Ab1 ((Beijing Boaosen Biotechnology, bsm-2027M)), The concentration is 0.8-2mg.

步骤4)的具体步骤为井步骤3)得到的反应液加入到试纸条,试纸条信号 收集时间为5-15min,通过相应时间内的生检测线上胶体金信号的累积与相应 miRNA浓度的关系,建立此发明检测技术的标准曲线。The specific step of step 4) is to add the reaction solution obtained in step 3) to the test strip, and the signal collection time of the test strip is 5-15min. relationship to establish the standard curve of the detection technology of this invention.

本发明还包括一种所述的检测miRNA-21的试剂盒,包括胶体金核酸探针、 核酸试纸条以及双链特异性核酸酶。The present invention also includes a kit for detecting miRNA-21, comprising a colloidal gold nucleic acid probe, a nucleic acid test strip and a double-strand specific nuclease.

与现有技术相比,本发明的有益效果是:Compared with the prior art, the beneficial effects of the present invention are:

1.显著提高miRNA的检测灵敏度。主要通过放大检测信号,降低背景噪音 两方面的有机结合,即以基于DSN辅助靶标循化回收及信号富集扩增策略,提 供更多待检测的d-DNA,用于核酸试纸条的检测;通过胶体金核酸探针聚集作为 检测信号,胶体金信号无需额外光激发的特点克服了背景噪音的干扰;从而显 著提高了检测的灵敏度。1. Significantly improve the detection sensitivity of miRNA. Mainly through the organic combination of amplifying the detection signal and reducing the background noise, that is, based on the DSN-assisted target recycling and signal enrichment amplification strategy, to provide more d-DNA to be detected for the detection of nucleic acid test strips ; Through the aggregation of colloidal gold nucleic acid probes as the detection signal, the colloidal gold signal does not need additional light excitation and overcomes the interference of background noise; thereby significantly improving the detection sensitivity.

2.实现样品中miRNA的快速、灵敏直接检测,针对样品,由于DSN酶的高 校特异性以及核酸试纸条的快速性,显著提高了样本中miRNA的检测时间,将 miRNA检测时间缩短到1小时左右。2. Realize the rapid, sensitive and direct detection of miRNA in the sample. For samples, due to the university specificity of DSN enzyme and the rapidity of nucleic acid test strips, the detection time of miRNA in the sample is significantly improved, and the miRNA detection time is shortened to 1 hour about.

3.实现样品中miRNA的高效特异性检测,通过分子茎环探针与DSN酶的协 同作用,可以从待测样品中高特异性的检测出目标miRNA,实现高特异性检测。3. Realize the efficient and specific detection of miRNA in the sample. Through the synergistic effect of the molecular stem-loop probe and DSN enzyme, the target miRNA can be detected with high specificity from the sample to be tested, and high-specificity detection can be achieved.

附图说明Description of drawings

图1为非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测miRNA-21的方法的检测原理示意图;Figure 1 is a schematic diagram of the detection principle of a method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes;

图2为基于DSN辅助靶标等温循环扩增的琼脂糖凝胶电泳表征结果图;Figure 2 is a graph showing the results of agarose gel electrophoresis characterization results based on isothermal cyclic amplification of DSN-assisted targets;

图3为在阴性条件下使用不同h-DNA的显色情况,用于本方法h-DNA的选择;Figure 3 shows the color development of different h-DNA under negative conditions, which is used for the selection of h-DNA in this method;

图4为非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测miRNA的灵敏 度实验图。Figure 4 is a graph showing the sensitivity of miRNA detection based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes.

具体实施方式Detailed ways

为了使本技术领域的技术人员更好地理解本发明的技术方案,下面结合附 图和最佳实施例对本发明作进一步的详细说明。In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention will be described in further detail below in conjunction with the accompanying drawings and the best embodiments.

本发明的试剂盒检测miRNA-21原理如图1所示,首先,使用水热法制备均 一粒径的胶体金纳米粒子,接着将胶体金纳米粒子与巯基化的h-DNA分子茎环 探针反应,制备高特异性的胶体金核酸探针。在有目标物存在的情况下,首先, 胶体金核酸探针上的h-DNA可以高效特异性的和临床样本中的目标miRNA-21结 合;接着,通过DSN的能高效识别并酶切完全互补配对的DNA双链或者DNA/RNA 杂交双链中的DNA链的能力,将目标miRNA-21释放出来,h-DNA被切割生成胶体 金d-DNA;核酸试纸条上的检测线通过生物素链霉亲和素固定sd-DNA,质控线 上固定羊抗鼠抗体;存在miRNA-21时生成的d-DNA最后与核酸试纸条上检测线 上的sd-DNA互补配对,产生检测信号,同时,核酸探针流过质控线时都会产生 对应的质控信号。本方法的可行性验证如图2所示,当目标miRNA-21和DSN同 时存在时,可以看到h-DNA被切割,条带变弱。当不存在目标物的情况下,胶 体金核酸探针无法从样本中结合到目标物质,因此后续反应无法进行,不产生 核酸试纸条胶体金信号。本方法的检测灵敏度曲线结果如图4所示,其灵敏度 可以达到nM级别。本试剂盒中所用到的引物及DNA、RNA序列均由上海生工合 成。The principle of the kit of the present invention for detecting miRNA-21 is shown in Figure 1. First, colloidal gold nanoparticles with uniform particle size are prepared by hydrothermal method, and then the colloidal gold nanoparticles are combined with thiolated h-DNA molecular stem-loop probes. reaction to prepare a highly specific colloidal gold nucleic acid probe. In the presence of the target, first, h-DNA on the colloidal gold nucleic acid probe can efficiently and specifically bind to the target miRNA-21 in clinical samples; then, it can efficiently recognize and digest complete complementation through DSN The ability of paired DNA double strands or DNA/RNA hybridization double strands to release the target miRNA-21, h-DNA is cleaved to generate colloidal gold d-DNA; the detection line on the nucleic acid test strip passes through biotin Streptavidin was used to immobilize sd-DNA, and goat anti-mouse antibody was immobilized on the quality control line; the d-DNA generated in the presence of miRNA-21 was finally complementary to the sd-DNA on the detection line on the nucleic acid test strip to generate a detection signal , and at the same time, when the nucleic acid probe flows through the quality control line, a corresponding quality control signal will be generated. The feasibility verification of this method is shown in Figure 2. When the target miRNA-21 and DSN are present at the same time, it can be seen that h-DNA is cleaved and the band becomes weaker. In the absence of the target substance, the colloidal gold nucleic acid probe cannot bind to the target substance from the sample, so the subsequent reaction cannot proceed, and the colloidal gold signal of the nucleic acid test strip will not be generated. The detection sensitivity curve results of this method are shown in Figure 4, and its sensitivity can reach the nM level. The primers, DNA and RNA sequences used in this kit were synthesized by Shanghai Shenggong.

特异性h-DNA的设计:通过对目标miRNA的序列分析,根据配对Tm温度值 和△G能量值的差异,创造性的设计特异性的h-DNA茎环探针。miRNA-21配对 Tm温度值为57℃,△G能量值为-12.3,因此设计的hDNA探针Tm值高于57℃, △G能量值应高于-12.3,h-DNA探针可以高效特异的结合目标miRNA。由于DSN 的选择切割性,如图3所示,设计了三种茎环探针,分别为h-DNA1、h-DNA2以 及h-DNA3;其中用到的核酸序列如表1示出:表1Design of specific h-DNA: creatively design specific h-DNA stem-loop probes according to the difference of the paired Tm temperature value and ΔG energy value through the sequence analysis of the target miRNA. The Tm temperature value of miRNA-21 pairing is 57°C, and the ΔG energy value is -12.3. Therefore, the designed hDNA probe has a Tm value higher than 57°C and a ΔG energy value higher than -12.3. The h-DNA probe can be highly specific and specific. binding target miRNA. Due to the selective cleavage of DSN, as shown in Figure 3, three stem-loop probes were designed, namely h-DNA1, h-DNA2 and h-DNA3; the nucleic acid sequences used are shown in Table 1: Table 1

Figure BDA0002421466440000051
Figure BDA0002421466440000051

实施例1:一种非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测 miRNA-21的方法,包括下述步骤:Embodiment 1: a kind of non-diagnostic purpose is based on the method of target isothermal cyclic amplification and nucleic acid test strip technology detection miRNA-21, comprises the following steps:

1)使用水热法制备均一粒径的胶体金纳米粒子;将柠檬酸钠溶液加入到反 应器,在磁力搅拌条件下加热至沸腾,第一次加入氯金酸溶液;此次氯金酸与柠 檬酸钠摩尔配比为1:15;溶液变为浅粉色后停止加热,溶液降温到90-95℃, 加入与第一次加入量等量的氯金酸溶液反应20-30min,再次加入与第一次加入 量等量的氯金酸溶液反应得到金种溶液;1) Use the hydrothermal method to prepare colloidal gold nanoparticles of uniform particle size; add sodium citrate solution to the reactor, heat to boiling under magnetic stirring conditions, and add chloroauric acid solution for the first time; The molar ratio of sodium citrate is 1:15; after the solution becomes light pink, the heating is stopped, the solution is cooled to 90-95°C, and the same amount of chloroauric acid solution as the first added amount is added to react for 20-30min, and then added with The first time adding the same amount of chloroauric acid solution reaction to obtain gold seed solution;

将金种溶液和另外准备的柠檬酸钠溶液混合作为下一步反应的生长液,生 长液加热到90-95℃后加入氯金酸溶液反应20-30min,再次加入等量氯金酸溶 液得到下一步反应的大粒径胶体金纳米粒子溶液;其中,金种子溶液:氯金酸: 柠檬酸钠摩尔配比为1:1:13.75;制备得到的胶体金纳米粒子溶液粒径为 13-40nm。The gold seed solution and the separately prepared sodium citrate solution are mixed as the growth solution for the next step of the reaction, the growth solution is heated to 90-95 ° C, and then the chloroauric acid solution is added to react for 20-30min, and the same amount of chloroauric acid solution is added again to obtain the following: A large-diameter colloidal gold nanoparticle solution for one-step reaction; wherein, the molar ratio of gold seed solution: chloroauric acid: sodium citrate is 1:1:13.75; the diameter of the prepared colloidal gold nanoparticle solution is 13-40 nm.

2)使用TCEP活化巯基h-DNA1后,与胶体金反应形成金硫键用于制备胶体 金核酸探针;20μL的浓度为10-50μM的巯基茎环h-DNA探针,加入20μL的浓 度为20mM的三(2-羧乙基)膦TCEP溶液,活化1-3h后,加入2mL均一粒径纳米 粒子,旋转培养反应16-24小时;之后加入6μL的1mg/mL的鼠源单克隆第一 抗体Ab1,反应1-3h;再加入0.2mL的浓度为3M的氯化钠溶液,置于4℃反应 过夜后,在8000-15000转速条件下离心15-30min,除去上清,用0.3mL的0.01M 磷酸缓冲溶液PBS重悬沉淀,得到的则为胶体金核酸探针。2) After using TCEP to activate sulfhydryl h-DNA1, it reacts with colloidal gold to form gold-sulfur bonds for the preparation of colloidal gold nucleic acid probes; 20 μL of 10-50 μM sulfhydryl stem-loop h-DNA probe is added, and 20 μL of the concentration is 20mM tris(2-carboxyethyl)phosphine TCEP solution, after activation for 1-3h, add 2mL of uniform particle size nanoparticles, and rotate for 16-24 hours; then add 6μL of 1mg/mL mouse monoclonal first Antibody Ab1, react for 1-3h; then add 0.2mL of 3M sodium chloride solution, put it at 4°C for overnight reaction, centrifuge at 8000-15000 revolutions for 15-30min, remove the supernatant, and use 0.3mL of The precipitate was resuspended in 0.01M phosphate buffered solution PBS to obtain colloidal gold nucleic acid probes.

3)将胶体金核酸探针和待检miRNA互补配对,加入双链特异性核酸酶,实 现靶标循环回收及信号放大;反应结束后,加入终止液,使酶失活,得到反应 液;具体为取5μL胶体金核酸探针和待检miRNA按比例混合于磷酸缓冲液中, 加入0.3U双链特异性核酸酶及10×双链特异性核酸酶缓冲液,胶体金核酸探 针:待检miRNA:双链特异性核酸酶体积比为1:1:1;涡旋仪上低速旋转使其 混匀,然后将上述混合溶液置于金属浴中,50℃下反应50分钟,实现靶标循环 回收及信号放大。反应结束后,加入终止液,45℃反应5分钟,使酶失活。其中,分别配制待检miRNA的浓度分别为0M、0.5x10-9M、1x10-9M、2x10-9M、4x10-9 M、10x10-9M。3) The colloidal gold nucleic acid probe and the miRNA to be detected are complementary paired, and double-stranded specific nuclease is added to realize target recycling and signal amplification; after the reaction is completed, a stop solution is added to inactivate the enzyme to obtain a reaction solution; Take 5μL of colloidal gold nucleic acid probe and the miRNA to be tested and mix them in phosphate buffer in proportion, add 0.3U double-strand specific nuclease and 10× double-strand specific nuclease buffer, colloidal gold nucleic acid probe: miRNA to be tested : The volume ratio of double-strand specific nuclease is 1:1:1; rotate at low speed on a vortexer to make it evenly mixed, then place the above mixed solution in a metal bath and react at 50°C for 50 minutes to achieve target recycling and recovery. Signal amplification. After the reaction was completed, a stop solution was added, and the reaction was carried out at 45°C for 5 minutes to inactivate the enzyme. The concentrations of the miRNAs to be tested are respectively prepared at 0M, 0.5x10-9M , 1x10-9M , 2x10-9M , 4x10-9M , and 10x10-9M , respectively.

4)核酸试纸条的组装和检测:将上步骤3)产生的反应液加入到组装好的 核酸试纸条上,观察检测结果;所述的核酸试纸条上包含有检测垫;核酸试纸 条包括试纸条卡壳以及设置在所述的试纸条卡壳内的顺序设置的样本垫、胶金 垫、检测垫、以及吸收垫;其中样本垫的长度为1.1-2cm之间;胶金垫的长度 为0.3-0.8cm;检测垫的长度为2.5-4.5cm;吸收垫的长度为1.1-2cm。所述的检 测垫包含检测线和质控线,其中检测线上包含了检测用脱氧核糖核苷酸 (sd-DNA),浓度为5-20μM;质控线上包含了抗Ab1的第二抗体Ab2((赛默飞科 技,A32723),浓度为0.15mg-1.0mg/mL。4) Assembly and detection of nucleic acid test strips: add the reaction solution generated in the above step 3) to the assembled nucleic acid test strips, and observe the detection results; the nucleic acid test strips contain detection pads; the nucleic acid test strips contain detection pads; The paper strip includes a test strip jam shell and a sample pad, a glue gold pad, a detection pad, and an absorption pad arranged in sequence in the test strip jam shell; wherein the length of the sample pad is between 1.1-2 cm; the glue gold The length of the pad is 0.3-0.8 cm; the length of the detection pad is 2.5-4.5 cm; the length of the absorbent pad is 1.1-2 cm. The detection pad comprises a detection line and a quality control line, wherein the detection line contains deoxyribonucleotide (sd-DNA) for detection at a concentration of 5-20 μM; the quality control line contains a secondary antibody against Ab1 Ab2 ((Thermo Scientific, A32723), the concentration is 0.15mg-1.0mg/mL.

步骤4)的具体步骤为井步骤3)得到的反应液加入到组装好的核酸试纸条 上,试纸条信号收集时间为10min,通过相应时间内的生检测线上胶体金信号的 累积与相应miRNA浓度的关系,建立此发明检测技术的标准曲线。结果如图4 所示,核酸试纸条胶体金显色情况与miRNA-21浓度关系成正比,浓度越大,显 色强度越大,因此本发明可应用于样品中miRNA-21的检测The specific step of step 4) is to add the reaction solution obtained in step 3) to the assembled nucleic acid test strip, and the collection time of the test strip signal is 10 min. The relationship between the corresponding miRNA concentrations was established to establish a standard curve for the detection technology of the invention. The results are shown in Figure 4, the color development of nucleic acid test strip colloidal gold is proportional to the concentration of miRNA-21, the greater the concentration, the greater the color intensity, so the present invention can be applied to the detection of miRNA-21 in samples

对比例1以及对比例2步骤2)中分别使用h-DNA2以及h-DNA3胶体金反应 形成金硫键用于制备胶体金核酸探针;结果表明其不能特异性的结合目标 miRNA。In step 2) of comparative example 1 and comparative example 2, h-DNA2 and h-DNA3 colloidal gold were respectively used to form gold-sulfur bonds for the preparation of colloidal gold nucleic acid probes; the results showed that they could not specifically bind to the target miRNA.

以上内容仅为本发明的较佳实施例,对于本领域的普通技术人员,依据本 发明的思想,在具体实施方式及应用范围上均会有改变之处,本说明书内容不 应理解为对本发明的限制。The above contents are only preferred embodiments of the present invention. For those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific embodiments and application scope. limits.

Claims (8)

1. A method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purposes is characterized by comprising the following steps:
1) preparing colloidal gold nanoparticles with uniform particle size by using a hydrothermal method;
2) activating sulfhydryl group h-DNA by using TCEP, and reacting with colloidal gold to form a gold-sulfur bond for preparing a colloidal gold nucleic acid probe; wherein, the sulfhydryl-H-DNA is 5 '-SH-TTTTTAGCTTATCAACTACTGATCAACATCAGTCTGATAAGCTA-3';
3) complementary pairing is carried out on the colloidal gold nucleic acid probe and the miRNA to be detected, and double-strand specific nuclease is added to realize target recycling and signal amplification; after the reaction is finished, adding a stop solution to inactivate the enzyme to obtain a reaction solution;
4) assembling and detecting the nucleic acid test strip: adding the reaction solution generated in the step 3) to the assembled nucleic acid test strip, and observing the detection result; the nucleic acid test strip comprises a detection pad; the detection pad comprises a detection line, and sd-DNA is protected on the detection line, and the concentration is 5-20 mu M; the base sequence of sd-DNA is 5 '-biotin-AAAACATGGTTACCGATCCAAGTTCAGTAGTTGATA-3'.
2. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 1, wherein the specific steps of step 1) are as follows:
adding a sodium citrate solution into a reactor, heating to boil under the condition of magnetic stirring, and adding a chloroauric acid solution for the first time; the molar ratio of the chloroauric acid to the sodium citrate is 1: 10-20 parts of; stopping heating after the solution turns into light pink, cooling the solution to 90-95 ℃, adding chloroauric acid solution with the same amount as the first addition amount for reaction for 20-30min, and adding chloroauric acid solution with the same amount as the first addition amount again for reaction to obtain gold seed solution;
mixing the gold seed solution and an additionally prepared sodium citrate solution to serve as a growth solution for the next reaction, heating the growth solution to 90-95 ℃, adding a chloroauric acid solution to react for 20-30min, and adding an equivalent chloroauric acid solution again to obtain a large-particle-size colloidal gold nanoparticle solution for the next reaction; wherein, the gold seed solution: gold chloride acid: the molar ratio of the sodium citrate is 1:1: 13.75; the grain diameter of the prepared colloidal gold nano particle solution is 13-40 nm.
3. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 1, wherein the specific steps of step 2) are 10-50 μ L of a sulfhydryl stem-loop h-DNA probe with a concentration of 10-50 μ M, 10-50 μ L of a tris (2-carboxyethyl) phosphine TCEP solution with a concentration of 10-50mM is added, 1-5mL of nanoparticles with uniform particle size are added after activation for 1-3h, and the rotation culture reaction is carried out for 16-24 h; then 4-10 μ L of 1mg/mL murine monoclonal primary antibody Ab1 is added for reaction for 1-3 h; then 0.1-0.5mL of 3M sodium chloride solution is added, after reaction at 4 ℃ overnight, the mixture is centrifuged for 15-30min at 8000-.
4. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes as claimed in claim 1, wherein the specific steps of step 3) are to take 3-7 μ L of the colloidal gold nucleic acid probe and miRNA to be detected to mix in phosphate buffer according to the volume ratio of 1-3:1, add 0.1-0.6U of double-stranded specific nuclease and 10 Xdouble-stranded specific nuclease buffer, rotate on a vortex apparatus at low speed to mix them uniformly, then place the mixed solution in a metal bath, react for 20-60 minutes at 35-60 ℃, and realize target recycling and signal amplification. After the reaction, the enzyme was inactivated by adding a stop solution and reacting at 45 ℃ for 5 minutes.
5. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid strip technology for non-diagnostic purposes as claimed in claim 1, wherein the nucleic acid strip in step 4) comprises a strip shell and a sample pad, a gold pad, a detection pad and an absorption pad arranged in sequence in the strip shell; wherein the length of the sample pad is between 1.1-2 cm; the length of the rubber gold pad is 0.3-0.8 cm; the length of the detection pad is 2.5-4.5 cm; the length of the absorption pad is 1.1-2 cm.
6. The method of claim 5 for detecting miRNA-21 for non-diagnostic purposes based on target isothermal amplification and nucleic acid dipstick technology, wherein the detection pad comprises a detection line and a quality control line, wherein the quality control line comprises the second antibody Ab2 of anti Ab1 at a concentration of 0.8-2 mg.
7. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 6, wherein the specific step of step 4) is to add the reaction solution obtained in step 3) to a test strip, the test strip signal collection time is 5-15min, and a standard curve of the detection technology is established according to the relation between the accumulation of colloidal gold signals on a raw detection line and the concentration of the corresponding miRNA in the corresponding time.
8. A kit for detecting miRNA-21 according to any one of claims 1-7, comprising a colloidal gold nucleic acid probe, a nucleic acid strip, and a double-strand specific nuclease.
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