CN116200492A

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Info

Publication number: CN116200492A
Application number: CN202211389048.7A
Authority: CN
Inventors: 王凯; 徐逸丽; 陈燃
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-11-08
Filing date: 2022-11-08
Publication date: 2023-06-02
Anticipated expiration: 2042-11-08
Also published as: CN116200492B; NL2034340B1; ZA202301656B

Abstract

Translated fromChinese

本发明提供了一种基于探针锚定双链的小核酸检测试剂盒及其应用，本发明属于核酸检测技术领域。一种基于探针锚定双链的小核酸检测试剂盒，包括主探针、辅探针、锚探针和荧光探针、靶标小核酸的扩增引物对和磁珠样本释放剂；主探针依次包括辅探针结合区、靶标小核酸结合区和锚探针结合区；辅探针结合区包括扩增靶标小核酸的上游引物互补区和荧光探针互补区；锚探针结合区与扩增靶标小核酸的下游引物互补；所述锚探针或主探针的5’末端标记有生物素；磁珠样本释放剂包括链霉亲和素修饰磁珠。利用所述试剂盒对小核酸进行检测，有利于排除干扰，控制本底，改善小核酸扩增检测的灵敏度、特异性和组合性，较现有技术具有明显优势。

The invention provides a small nucleic acid detection kit based on a probe-anchored double-strand and an application thereof, and the invention belongs to the technical field of nucleic acid detection. A small nucleic acid detection kit based on a probe-anchored double-strand, including a main probe, an auxiliary probe, an anchor probe and a fluorescent probe, a primer pair for amplification of a target small nucleic acid, and a magnetic bead sample release agent; the main probe The needle includes an auxiliary probe binding region, a target small nucleic acid binding region and an anchor probe binding region in turn; the auxiliary probe binding region includes an upstream primer complementary region and a fluorescent probe complementary region for amplifying the target small nucleic acid; the anchor probe binding region and the The downstream primers for amplifying the target small nucleic acid are complementary; the 5' end of the anchor probe or the main probe is labeled with biotin; the magnetic bead sample release agent includes streptavidin modified magnetic beads. Using the kit to detect small nucleic acids is beneficial to eliminate interference, control the background, improve the sensitivity, specificity and combination of small nucleic acid amplification detection, and has obvious advantages over the prior art.

Description

Translated fromChinese

一种基于探针锚定双链的小核酸检测试剂盒及其应用A small nucleic acid detection kit based on probe anchoring double strands and its application

技术领域Technical Field

本发明属于核酸检测技术领域，具体涉及一种基于探针锚定双链的小核酸检测试剂盒及其应用。The present invention belongs to the technical field of nucleic acid detection, and in particular relates to a small nucleic acid detection kit based on probe-anchored double strands and an application thereof.

背景技术Background Art

小核酸(miRNA，微小核糖核酸)是一类长约20～24个核苷酸(nt)的单链非编码小分子RNA，广泛存在于人体组织、细胞、血液、体液内，具有重要的基因调节功能，其表达异常与各种疾病尤其肿瘤的发生发展转移密切相关。细胞外小核酸(cell-free miRNA，cfmiRNA)又称循环小核酸(circulating miRNA,ctmiRNA)，是一类通过细胞主动分泌或组织损伤和细胞破裂等被动渗漏方式释放进入、并以被外泌体或微囊泡包裹或与蛋白质结合的形式、稳定地存在于血液及其他体液中的小核酸，适合成为疾病检测的分子标记物。血液异常富集的肿瘤来源的cfmiRNA可发生在肿瘤早期阶段，源自组织原位肿瘤细胞的表达和分泌，而循环肿瘤细胞(CTC)和游离DNA(ctDNA)通常则是肿瘤晚期阶段肿瘤细胞直接转移进入血液的结果，因此，cfmiRNA检测比CTC和ctDNA检测能达到更高的灵敏度，也优于肿瘤检测常规肿瘤标志物和自身抗体等蛋白质标志物。Small RNA (miRNA, micro RNA) is a type of single-stranded non-coding small molecule RNA with a length of about 20 to 24 nucleotides (nt). It is widely present in human tissues, cells, blood, and body fluids. It has important gene regulation functions. Its abnormal expression is closely related to the occurrence, development and metastasis of various diseases, especially tumors. Extracellular small nucleic acids (cell-free miRNA, cfmiRNA), also known as circulating small nucleic acids (circulating miRNA, ctmiRNA), are a type of small nucleic acids that are released into the body through active cell secretion or passive leakage such as tissue damage and cell rupture, and are stably present in the blood and other body fluids in the form of being encapsulated by exosomes or microvesicles or bound to proteins. They are suitable as molecular markers for disease detection. Abnormally enriched tumor-derived cfmiRNA in the blood can occur in the early stages of the tumor and originate from the expression and secretion of tumor cells in situ in the tissue, while circulating tumor cells (CTC) and free DNA (ctDNA) are usually the result of direct metastasis of tumor cells into the blood in the late stages of the tumor. Therefore, cfmiRNA detection can achieve higher sensitivity than CTC and ctDNA detection, and is also superior to conventional tumor markers and protein markers such as autoantibodies for tumor detection.

cfmiRNA的扩增检测方法能够比其它方法达到更好的灵敏度和准确度，是最常用的主流方法，通常基于实时荧光定量PCR扩增，主要过程是利用靶标小核酸制备PCR模板，再进行荧光定量PCR扩增，扩增结果Ct值与靶标小核酸拷贝数存在反比关系，Ct值越小，靶标小核酸拷贝数越多，无Ct值，则指示没有检测到靶标小核酸。从靶标小核酸制备PCR模板的方法主要有2大类，第1大类为逆转录法，包含PolyA加尾逆转录法、加尾长引物逆转录法、茎环引物逆转录法(茎环法)和锁核苷酸(LNA)引物逆转录法等，主流为茎环法。这类方法由于小核酸总体长度只有约22nt，可用于逆转录的部分和用于上游引物的部分分别仅有约11nt，因此，难以排除样本中大量存在的非靶标核酸的干扰而难以保证逆转录和PCR扩增的特异性，导致本底较高、重复性较差，而且不兼容通用引物PCR，也较难进行多重PCR，检测组合性能低。第2大类为主探针法。前期本团队发明利用靶标RNA杂交捕获主探针制备PCR模板再进行扩增检测的方法，包括：(1)《一种RNA的免洗涤主探针PCR检测方法》(ZL201310205271.6)是一种用于RNA的PCR检测方法，能使用通用引物，但其直接应用于小核酸检测，容易受前体及基因组DNA的杂交干扰，尚不能很好地解决本底较高、重复性较差等问题。(2)《一种微小核酸组合扩增检测方法及试剂盒》(申请号：202011041856.5)是一种用于小核酸的PCR检测方法，利用单链主探针杂交捕获多个靶标小核酸，通过绿豆核酸酶酶切去除过量单链主探针，纯化得到与靶标小核酸形成双链的主探针，再进行PCR扩增检测，指示靶标小核酸的存在。该方法有利于排除干扰，改善检测组合性能，但具体在扩大应用中发现，该方法主要存在2点不足：(1)不利于遗传异质性较高的疾病如肿瘤的检测。该方法需要样本中同时存在多种靶标小核酸，而遗传异质性程度较高即在不同患者个体中同时出现多种靶标小核酸的几率不高。(2)不利于核酸含量较高样本的检测。绿豆核酸酶对单链核酸无选择性，当样本中核酸含量较高时，体系中的绿豆核酸酶被样本中大量存在的单链核酸饱和而不能及时清除单链主探针，造成严重的本底扩增。The amplification detection method of cfmiRNA can achieve better sensitivity and accuracy than other methods. It is the most commonly used mainstream method, usually based on real-time fluorescence quantitative PCR amplification. The main process is to use the target small nucleic acid to prepare the PCR template, and then perform fluorescence quantitative PCR amplification. The Ct value of the amplification result is inversely proportional to the number of copies of the target small nucleic acid. The smaller the Ct value, the more copies of the target small nucleic acid. No Ct value indicates that the target small nucleic acid has not been detected. There are two main methods for preparing PCR templates from target small nucleic acids. The first category is reverse transcription, including PolyA tailing reverse transcription, tailing long primer reverse transcription, stem-loop primer reverse transcription (stem-loop method) and locked nucleotide (LNA) primer reverse transcription, etc. The mainstream is the stem-loop method. In this type of method, since the overall length of small nucleic acids is only about 22 nt, the part that can be used for reverse transcription and the part that can be used for upstream primers are only about 11 nt respectively. Therefore, it is difficult to eliminate the interference of a large number of non-target nucleic acids in the sample and it is difficult to ensure the specificity of reverse transcription and PCR amplification, resulting in high background and poor repeatability. In addition, it is not compatible with universal primer PCR, and it is also difficult to perform multiple PCR, and the detection combination performance is low. The second category is the main probe method. In the early stage, our team invented a method for preparing PCR templates by hybridizing and capturing the main probe of target RNA and then amplifying and detecting, including: (1) "A Wash-free Main Probe PCR Detection Method for RNA" (ZL201310205271.6) is a PCR detection method for RNA that can use universal primers, but it is directly applied to small nucleic acid detection and is easily interfered by hybridization of precursors and genomic DNA. It cannot solve the problems of high background and poor repeatability. (2) "A method and kit for combined amplification detection of micronucleic acids" (application number: 202011041856.5) is a PCR detection method for small nucleic acids, which uses single-stranded main probe hybridization to capture multiple target small nucleic acids, removes excess single-stranded main probes through mung bean nuclease digestion, purifies the main probe that forms a double strand with the target small nucleic acid, and then performs PCR amplification detection to indicate the presence of the target small nucleic acid. This method is conducive to eliminating interference and improving the performance of the detection combination, but it is found in the specific application that this method has two main shortcomings: (1) It is not conducive to the detection of diseases with high genetic heterogeneity, such as tumors. This method requires the simultaneous presence of multiple target small nucleic acids in the sample, and the high degree of genetic heterogeneity, that is, the probability of multiple target small nucleic acids appearing simultaneously in different patient individuals, is not high. (2) It is not conducive to the detection of samples with high nucleic acid content. Mung bean nuclease has no selectivity for single-stranded nucleic acids. When the nucleic acid content in the sample is high, the mung bean nuclease in the system is saturated with a large amount of single-stranded nucleic acids in the sample and cannot remove the single-stranded main probe in time, resulting in serious background amplification.

发明内容Summary of the invention

有鉴于此，本发明的目的在于提供一种基于探针锚定双链的小核酸检测试剂盒及其应用，具有操作简单、重复性和组合性好，能够抑制本底扩增，改善肿瘤检测敏感性的特点。In view of this, the purpose of the present invention is to provide a small nucleic acid detection kit based on probe-anchored double-stranded nucleic acid and its application, which has the characteristics of simple operation, good repeatability and combination, and can inhibit background amplification and improve tumor detection sensitivity.

本发明提供了一种基于探针锚定双链的小核酸检测试剂盒，包括探针组、靶标小核酸的扩增引物对和磁珠样本释放剂；The present invention provides a small nucleic acid detection kit based on probe-anchored double-stranded DNA, comprising a probe set, an amplification primer pair of a target small nucleic acid, and a magnetic bead sample release agent;

所述探针组包括主探针、辅探针、锚探针和荧光探针；所述主探针依次包括辅探针结合区、靶标小核酸结合区和锚探针结合区；所述主探针的长度与辅探针、靶标小核酸和锚探针的总长度一致；The probe set includes a main probe, an auxiliary probe, an anchor probe and a fluorescent probe; the main probe includes an auxiliary probe binding region, a target small nucleic acid binding region and an anchor probe binding region in sequence; the length of the main probe is consistent with the total length of the auxiliary probe, the target small nucleic acid and the anchor probe;

所述辅探针结合区包括扩增靶标小核酸的上游引物互补区和荧光探针互补区，所述辅探针结合区的长度与扩增靶标小核酸的上游引物和荧光探针的总长度一致；The auxiliary probe binding region includes an upstream primer complementary region and a fluorescent probe complementary region for amplifying the target small nucleic acid, and the length of the auxiliary probe binding region is consistent with the total length of the upstream primer and the fluorescent probe for amplifying the target small nucleic acid;

所述锚探针和锚探针结合区的长度一致，所述锚探针结合区与扩增靶标小核酸的下游引物互补；The anchor probe and the anchor probe binding region have the same length, and the anchor probe binding region is complementary to a downstream primer for amplifying a target small nucleic acid;

所述锚探针或主探针的5’末端标记有生物素；The 5' end of the anchor probe or main probe is labeled with biotin;

所述磁珠样本释放剂包括40～80mM氯化锌、40～80mM乙酸钠、40～80mM半胱氨酸、质量百分含量10％～18％碳酸亚乙酯、体积百分含量10％～30％甲酰胺、质量百分含量4％～10％十二烷基硫酸钠、90～200μg/ml链霉亲和素修饰磁珠。The magnetic bead sample release agent includes 40-80mM zinc chloride, 40-80mM sodium acetate, 40-80mM cysteine, 10%-18% ethylene carbonate by weight, 10%-30% formamide by volume, 4%-10% sodium dodecyl sulfate by weight, and 90-200μg/ml streptavidin-modified magnetic beads.

优选的，所述的主探针的3’末端碱基为腺嘌呤。Preferably, the 3' terminal base of the main probe is adenine.

优选的，所述锚探针结合区的核苷酸序列为SEQ ID NO:3；Preferably, the nucleotide sequence of the anchor probe binding region is SEQ ID NO: 3;

所述锚探针的核苷酸序列为SEQ ID NO:4。The nucleotide sequence of the anchor probe is SEQ ID NO:4.

优选的，所述扩增靶标小核酸的下游引物核苷酸序列为SEQ ID NO:5。Preferably, the nucleotide sequence of the downstream primer for amplifying the target small nucleic acid is SEQ ID NO:5.

优选的，所述辅探针结合区的核苷酸序列为SEQ ID NO:1；Preferably, the nucleotide sequence of the auxiliary probe binding region is SEQ ID NO: 1;

所述辅探针的核苷酸序列为SEQ ID NO:2。The nucleotide sequence of the auxiliary probe is SEQ ID NO:2.

优选的，所述扩增靶标小核酸的上游引物的核苷酸序列为SEQ ID NO:6；Preferably, the nucleotide sequence of the upstream primer for amplifying the target small nucleic acid is SEQ ID NO: 6;

所述荧光探针的核苷酸序列为SEQ ID NO:7。The nucleotide sequence of the fluorescent probe is SEQ ID NO:7.

优选的，所述试剂盒还包括洗涤液、绿豆核酸酶、PCR扩增缓冲液、1×阳性对照；Preferably, the kit further comprises a washing solution, mung bean nuclease, a PCR amplification buffer, and a 1× positive control;

所述洗涤液为含5～20mM 3-吗啉丙磺酸、40～80mM半胱氨酸、30～50mM乙酸钠、体积百分含量0.05％～0.1％吐温20的水溶液。The washing solution is an aqueous solution containing 5-20 mM 3-morpholinepropanesulfonic acid, 40-80 mM cysteine, 30-50 mM sodium acetate and 0.05%-0.1% Tween 20 in volume percentage.

本发明提供了所述基于探针锚定双链的小核酸检测试剂盒在非疾病诊断目的的小核酸检测中的应用。The present invention provides application of the small nucleic acid detection kit based on probe anchoring double strands in small nucleic acid detection for non-disease diagnosis purposes.

优选的，所述小核酸包括以下至少一种mirRNA：miR-191、miR-93、miR-16、miR-3662和miR-181家族；Preferably, the small nucleic acid comprises at least one of the following mirRNAs: miR-191, miR-93, miR-16, miR-3662 and miR-181 families;

所述miR-181家族包括miR-181a-5p、miR-181b-5p、miR-181c-5p和miR-181d-5p中的至少一种。The miR-181 family includes at least one of miR-181a-5p, miR-181b-5p, miR-181c-5p and miR-181d-5p.

本发明提供了一种采用所述试剂盒非疾病诊断目的的小核酸检测方法，包括以下步骤：The present invention provides a method for detecting small nucleic acids for non-disease diagnosis purposes using the kit, comprising the following steps:

将待测样本、主探针、辅探针、锚探针和磁珠样本释放剂混合，孵育，磁场条件下去除上清，洗涤，加入绿豆核酸酶溶液进行酶解，磁场条件下去除上清，洗涤，加入PCR扩增缓冲液、荧光探针、靶标小核酸扩增引物对进行荧光定量PCR扩增反应，检测结果Ct值≤35说明待测样本中含有靶标小核酸，否则不含有。The sample to be tested, the main probe, the auxiliary probe, the anchor probe and the magnetic bead sample releaser are mixed, incubated, the supernatant is removed under magnetic field conditions, washed, and mung bean nuclease solution is added for enzymatic hydrolysis. The supernatant is removed under magnetic field conditions, washed, and PCR amplification buffer, fluorescent probe, and target small nucleic acid amplification primer pairs are added to perform fluorescent quantitative PCR amplification reaction. The detection result Ct value ≤35 indicates that the sample to be tested contains the target small nucleic acid, otherwise it does not contain it.

本发明提供了一种基于探针锚定双链的小核酸检测试剂盒，包括探针组、靶标小核酸的扩增引物对和磁珠样本释放剂。在检测时，主探针、辅探针、锚探针与样本混合保温，主探针可以同时杂交结合辅探针、锚探针和靶标小核酸，形成完整双链，并通过锚探针或主探针末端标记的生物素结合于链霉亲和素磁珠上，吸去上清并洗涤后，样本中过量的核酸被洗去，再加入绿豆核酸酶体系进行酶切反应，没有结合靶标小核酸的主探针处于单链或部分状态而被降解，捕获结合靶标小核酸的主探针在双链中保持完整，再次洗涤之后，加入荧光定量PCR缓冲液进行扩增反应，根据Ct值指示靶标小核酸的存在。与现有技术公开的小核酸检测方法相比，本发明提供的试剂盒使用通用的辅探针、锚探针与主探针杂交捕获单靶标小核酸形成双链，不依赖于多靶标小核酸的同时存在，并在绿豆核酸酶酶切步骤之前引入生物素-链霉亲和素磁珠提取和洗涤，能去除样本中竞争绿豆核酸酶的其它核酸，便于单链主探针被降解清除而消除本底，从而保证检测结果的准确性和可靠性。The present invention provides a small nucleic acid detection kit based on probe anchored double strands, comprising a probe group, an amplification primer pair of a target small nucleic acid and a magnetic bead sample releaser. During detection, the main probe, auxiliary probe, anchor probe and sample are mixed and incubated, the main probe can hybridize and bind to the auxiliary probe, anchor probe and target small nucleic acid at the same time to form a complete double strand, and bind to the streptavidin magnetic beads through the biotin labeled at the end of the anchor probe or the main probe, after the supernatant is sucked off and washed, the excess nucleic acid in the sample is washed away, and then the mung bean nuclease system is added to perform enzyme digestion reaction, the main probe that is not bound to the target small nucleic acid is in a single-stranded or partial state and is degraded, and the main probe that captures the target small nucleic acid remains intact in the double strand, after washing again, a fluorescent quantitative PCR buffer is added to perform an amplification reaction, and the presence of the target small nucleic acid is indicated according to the Ct value. Compared with the small nucleic acid detection methods disclosed in the prior art, the kit provided by the present invention uses universal auxiliary probes, anchor probes and main probes to hybridize and capture single-target small nucleic acids to form double strands, is independent of the simultaneous presence of multiple target small nucleic acids, and introduces biotin-streptavidin magnetic beads for extraction and washing before the mung bean nuclease cleavage step, which can remove other nucleic acids in the sample that compete with the mung bean nuclease, facilitate the degradation and removal of the single-stranded main probe and eliminate the background, thereby ensuring the accuracy and reliability of the detection results.

本发明提供了一种采用所述试剂盒非疾病诊断目的的小核酸检测方法，本发明通过直接裂解提取RNA，同步进行探针特异杂交和生物素-链霉亲和素磁珠捕获主探针和靶标小核酸，不仅能够排除各种绿豆核酸酶抑制物，而且能排除大量的非靶标核酸，大大降低绿豆核酸酶负载，能充分发挥绿豆核酸酶酶切活性，清除探针本底，同时，结合靶标小核酸的主探针与辅探针和锚探针形成完整匹配双链，保留在体系中得到纯化PCR模板，有利于提高PCR扩增效率，因此，本发明既能改善小核酸扩增检测的特异性，也能改善其灵敏度。我们实验验证表明本发明能排除过量核酸干扰，能识别切割低至1nt的错配，能识别小核酸家族内相似小核酸，不仅优于传统茎环法，也优于申请号202011041856.5方法。本发明灵敏度能达到低至100拷贝/ml，比申请号202011041856.5方法或茎环法提高了10倍至100倍。本发明使用通用引物和通用荧光探针，一个反应可以检测8种以上的小核酸，大大改善了组合检测性能，有利于遗传异质性疾病尤其是肿瘤的检测。The present invention provides a small nucleic acid detection method for non-disease diagnosis purposes using the kit. The present invention directly cleaves and extracts RNA, simultaneously performs probe-specific hybridization and captures the main probe and target small nucleic acid with biotin-streptavidin magnetic beads, which can not only exclude various mung bean nuclease inhibitors, but also exclude a large number of non-target nucleic acids, greatly reduce the mung bean nuclease load, give full play to the mung bean nuclease enzyme activity, and remove the probe background. At the same time, the main probe combined with the target small nucleic acid forms a complete matching double-stranded chain with the auxiliary probe and the anchor probe, which is retained in the system to obtain a purified PCR template, which is beneficial to improve the PCR amplification efficiency. Therefore, the present invention can improve the specificity of small nucleic acid amplification detection and its sensitivity. Our experimental verification shows that the present invention can exclude excessive nucleic acid interference, can identify mismatches as low as 1nt, and can identify similar small nucleic acids within the small nucleic acid family. It is not only better than the traditional stem-loop method, but also better than the method of application number 202011041856.5. The sensitivity of the present invention can be as low as 100 copies/ml, which is 10 to 100 times higher than the method of application number 202011041856.5 or the stem-loop method. The present invention uses universal primers and universal fluorescent probes, and one reaction can detect more than 8 small nucleic acids, which greatly improves the performance of combined detection and is conducive to the detection of genetically heterogeneous diseases, especially tumors.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为本发明提供的技术方案的流程原理图。FIG1 is a flow diagram of the technical solution provided by the present invention.

图2为miR-181家族序列同源性分析结果。FIG. 2 shows the results of homology analysis of miR-181 family sequences.

具体实施方式DETAILED DESCRIPTION

本发明提供了一种基于探针锚定双链的小核酸检测试剂盒，包括探针组、靶标小核酸的扩增引物对和磁珠样本释放剂。The invention provides a small nucleic acid detection kit based on probe-anchored double-stranded DNA, comprising a probe group, an amplification primer pair of target small nucleic acid and a magnetic bead sample release agent.

在本发明中，所述探针组包括主探针、辅探针、锚探针和荧光探针。所述主探针依次包括辅探针结合区、靶标小核酸结合区和锚探针结合区。其中辅探针结合区和锚探针结合区为通用区，而靶标小核酸结合区是可变区域，根据靶标小核酸序列的不同而相应改变。所述主探针的长度与辅探针、靶标小核酸和锚探针的总长度一致。所述辅探针与所述辅探针结合区的序列100％互补匹配。所述靶标小核酸结合区与靶标小核酸的序列100％互补匹配。所述锚探针结合区与锚探针的序列100％互补匹配。In the present invention, the probe group includes a main probe, an auxiliary probe, an anchor probe and a fluorescent probe. The main probe includes an auxiliary probe binding region, a target small nucleic acid binding region and an anchor probe binding region in sequence. The auxiliary probe binding region and the anchor probe binding region are universal regions, while the target small nucleic acid binding region is a variable region, which changes accordingly according to the different target small nucleic acid sequences. The length of the main probe is consistent with the total length of the auxiliary probe, the target small nucleic acid and the anchor probe. The auxiliary probe is 100% complementary to the sequence of the auxiliary probe binding region. The target small nucleic acid binding region is 100% complementary to the sequence of the target small nucleic acid. The anchor probe binding region is 100% complementary to the sequence of the anchor probe.

在本发明实施例中，针对小核酸mirRNA：miR-191、miR-93、miR-16、miR-3662设计了4组主探针。所述miR-191、miR-93、miR-16、miR-3662的核苷酸序列如SEQ ID NO:8～SEQID NO:11，对应的主探针的核苷酸序列如SEQ ID NO:12～SEQ ID NO:15。In the embodiment of the present invention, 4 sets of main probes are designed for small nucleic acid mirRNA: miR-191, miR-93, miR-16, miR-3662. The nucleotide sequences of miR-191, miR-93, miR-16, miR-3662 are as shown in SEQ ID NO:8 to SEQ ID NO:11, and the nucleotide sequences of the corresponding main probes are as shown in SEQ ID NO:12 to SEQ ID NO:15.

在本发明中，所述辅探针结合区包括扩增靶标小核酸的上游引物互补区和荧光探针互补区，所述辅探针结合区的长度与扩增靶标小核酸的上游引物和荧光探针的总长度一致。所述扩增靶标小核酸的上游引物互补区与扩增靶标小核酸的上游引物的序列100％互补匹配。In the present invention, the auxiliary probe binding region includes an upstream primer complementary region and a fluorescent probe complementary region for amplifying a target small nucleic acid, and the length of the auxiliary probe binding region is consistent with the total length of the upstream primer and the fluorescent probe for amplifying a target small nucleic acid. The upstream primer complementary region for amplifying a target small nucleic acid is 100% complementary to the sequence of the upstream primer for amplifying a target small nucleic acid.

在本发明中，所述锚探针和锚探针结合区的长度一致，所述锚探针结合区与扩增靶标小核酸的下游引物的序列100％互补匹配。所述锚探针或主探针的5’末端标记有生物素。In the present invention, the anchor probe and the anchor probe binding region have the same length, and the anchor probe binding region is 100% complementary to the sequence of the downstream primer for amplifying the target small nucleic acid. The 5' end of the anchor probe or main probe is labeled with biotin.

在本发明中，设计探针和引物时，设计原则优选为上游引物同源部分、荧光探针同源部分和锚探针结合区长度分别在20～30nt之间，序列无二级结构，无4个以上的连续单核苷酸；上游引物同源部分和锚探针结合区Tm值在55℃～60℃之间，荧光探针同源部分Tm值在65℃～70℃之间。In the present invention, when designing probes and primers, the design principle is preferably that the lengths of the upstream primer homologous part, the fluorescent probe homologous part and the anchor probe binding region are respectively between 20 and 30 nt, the sequences have no secondary structure, and there are no more than 4 consecutive single nucleotides; the Tm values of the upstream primer homologous part and the anchor probe binding region are between 55°C and 60°C, and the Tm value of the fluorescent probe homologous part is between 65°C and 70°C.

在本发明实施例中，所述的主探针的3’末端碱基优选为腺嘌呤。所述锚探针结合区的核苷酸序列优选为SEQ ID NO:3；所述锚探针的核苷酸序列优选为SEQ ID NO:4。所述扩增靶标小核酸的下游引物核苷酸序列优选为SEQ ID NO:5。所述辅探针结合区的核苷酸序列优选为SEQ ID NO:1；所述辅探针的核苷酸序列优选为SEQ ID NO:2。所述扩增靶标小核酸的上游引物的核苷酸序列优选为SEQ ID NO:6；所述荧光探针的核苷酸序列优选为SEQID NO:7。In an embodiment of the present invention, the 3' terminal base of the main probe is preferably adenine. The nucleotide sequence of the anchor probe binding region is preferably SEQ ID NO:3; the nucleotide sequence of the anchor probe is preferably SEQ ID NO:4. The nucleotide sequence of the downstream primer for amplifying the target small nucleic acid is preferably SEQ ID NO:5. The nucleotide sequence of the auxiliary probe binding region is preferably SEQ ID NO:1; the nucleotide sequence of the auxiliary probe is preferably SEQ ID NO:2. The nucleotide sequence of the upstream primer for amplifying the target small nucleic acid is preferably SEQ ID NO:6; the nucleotide sequence of the fluorescent probe is preferably SEQ ID NO:7.

在本发明中，所述磁珠样本释放剂包括40～80mM氯化锌、40～80mM乙酸钠、40～80mM半胱氨酸、质量百分含量10％～18％碳酸亚乙酯、体积百分含量10％～30％甲酰胺、质量百分含量4％～10％十二烷基硫酸钠、90～200μg/ml链霉亲和素修饰磁珠。所述磁珠样本释放剂中，乙酸钠、氯化锌、半胱氨酸、碳酸亚乙酯、甘油，提供合适的离子强度、pH值和核酸杂交条件。十二烷基硫酸钠、氯化锌、半胱氨酸提供样本裂解、蛋白质变性和RNase酶抑制作用。链霉亲和素磁珠特异吸附锚探针及其与主探针形成的双链。In the present invention, the magnetic bead sample releaser includes 40-80mM zinc chloride, 40-80mM sodium acetate, 40-80mM cysteine, 10%-18% ethylene carbonate by mass, 10%-30% formamide by volume, 4%-10% sodium dodecyl sulfate by mass, and 90-200μg/ml streptavidin modified magnetic beads. In the magnetic bead sample releaser, sodium acetate, zinc chloride, cysteine, ethylene carbonate, and glycerol provide suitable ionic strength, pH value, and nucleic acid hybridization conditions. Sodium dodecyl sulfate, zinc chloride, and cysteine provide sample lysis, protein denaturation, and RNase enzyme inhibition. Streptavidin magnetic beads specifically adsorb anchor probes and the double strands formed with the main probe.

在本发明中，所述试剂盒优选还包括洗涤液、绿豆核酸酶、PCR扩增缓冲液、1×阳性对照。所述洗涤液为含5～20mM 3-吗啉丙磺酸、40～80mM半胱氨酸、30～50mM乙酸钠、0.05％～0.1％(v/v)吐温20的水溶液。PCR扩增缓冲液为含热启动DNA聚合酶、dNTP的荧光定量PCR常规组分的水溶液。本发明对所述PCR扩增缓冲液的来源没有特殊限制，采用本领域所熟知的PCR扩增缓冲液即可，在本发明实施例中，所述PCR扩增缓冲液购自生工生物工程(上海)股份有限公司。In the present invention, the kit preferably also includes a washing solution, mung bean nuclease, a PCR amplification buffer, and a 1× positive control. The washing solution is an aqueous solution containing 5-20 mM 3-morpholinepropanesulfonic acid, 40-80 mM cysteine, 30-50 mM sodium acetate, and 0.05%-0.1% (v/v) Tween 20. The PCR amplification buffer is an aqueous solution containing conventional components of fluorescent quantitative PCR of hot-start DNA polymerase and dNTP. The present invention has no special restrictions on the source of the PCR amplification buffer, and the PCR amplification buffer known in the art can be used. In the embodiment of the present invention, the PCR amplification buffer is purchased from Sangon Biotech (Shanghai) Co., Ltd.

在本发明实施例中，所述小核酸优选包括以下至少一种mirRNA：miR-191、miR-93、miR-16、miR-3662和miR-181家族。所述miR-191、miR-93、miR-16、miR-3662的核苷酸序列如SEQ ID NO:8～SEQ ID NO:11。所述miR-181家族包括miR-181a-5p、miR-181b-5p、miR-181c-5p和miR-181d-5p中的至少一种。所述miR-181a-5p、miR-181b-5p、miR-181c-5p和miR-181d-5p的核苷酸序列如SEQ ID NO:20～SEQ ID NO:23。In an embodiment of the present invention, the small nucleic acid preferably includes at least one of the following mirRNAs: miR-191, miR-93, miR-16, miR-3662 and miR-181 family. The nucleotide sequences of miR-191, miR-93, miR-16 and miR-3662 are as shown in SEQ ID NO:8 to SEQ ID NO:11. The miR-181 family includes at least one of miR-181a-5p, miR-181b-5p, miR-181c-5p and miR-181d-5p. The nucleotide sequences of miR-181a-5p, miR-181b-5p, miR-181c-5p and miR-181d-5p are as shown in SEQ ID NO:20 to SEQ ID NO:23.

在本发明中，所述主探针的工作浓度优选为110～130nM，更优选为120nM。辅探针的工作浓度优选为110～130nM，更优选为120nM。锚探针的工作浓度优选为110～130nM，更优选为120nM。所述待测样本优选包括血浆样本或从组织液或细胞培养液。待测样本和磁珠样本释放剂的体积比为9～10:4，更优选为9.5:4。In the present invention, the working concentration of the main probe is preferably 110-130nM, more preferably 120nM. The working concentration of the auxiliary probe is preferably 110-130nM, more preferably 120nM. The working concentration of the anchor probe is preferably 110-130nM, more preferably 120nM. The sample to be tested preferably includes a plasma sample or a tissue fluid or a cell culture fluid. The volume ratio of the sample to be tested and the magnetic bead sample release agent is 9-10:4, more preferably 9.5:4.

在本发明中，所述孵育的条件优选为28～35℃保温8～12min，更优选为30℃下保温10min。所述绿豆核酸酶溶液的浓度优选为0.15～0.25U/μl，更优选为0.2U/μl。所述绿豆核酸酶溶液的溶剂为pH5.0的50mM乙酸钠缓冲液中含有30mM氯化钠、1mM氯化锌的水溶液。所述绿豆核酸酶溶液的添加体积为1/2体积的待测样本。In the present invention, the incubation condition is preferably 28-35°C for 8-12 min, more preferably 30°C for 10 min. The concentration of the mung bean nuclease solution is preferably 0.15-0.25 U/μl, more preferably 0.2 U/μl. The solvent of the mung bean nuclease solution is an aqueous solution containing 30 mM sodium chloride and 1 mM zinc chloride in 50 mM sodium acetate buffer at pH 5.0. The added volume of the mung bean nuclease solution is 1/2 volume of the sample to be tested.

在本发明中，荧光定量PCR扩增反应的程序为95℃10分钟，再40个循环的93℃10秒、65℃40秒，荧光信号采集在65℃。所述待测样本和PCR扩增缓冲液的体积比优选为9～10:3，更优选为9.5:3。In the present invention, the procedure of the fluorescence quantitative PCR amplification reaction is 95° C. for 10 minutes, followed by 40 cycles of 93° C. for 10 seconds and 65° C. for 40 seconds, and the fluorescence signal is collected at 65° C. The volume ratio of the sample to be tested to the PCR amplification buffer is preferably 9 to 10:3, more preferably 9.5:3.

在本发明中，本发明提供的方法具有较强的抗干扰能力，较高的检测灵敏度(样本细胞浓度为0.1个/μl，小核酸10拷贝/ml)，比申请号202011041856.5方法更具优势。本发明方法能达到分辨同一家族中不同种小核酸的高特异性，检测肺癌血液阳性率为100％，具体见表1。In the present invention, the method provided by the present invention has strong anti-interference ability and high detection sensitivity (sample cell concentration is 0.1/μl, small nucleic acid 10 copies/ml), which is more advantageous than the method of application number 202011041856.5. The method of the present invention can achieve high specificity in distinguishing different types of small nucleic acids in the same family, and the positive rate of lung cancer blood detection is 100%, as shown in Table 1.

表1不同方法检测小核酸的结果对比Table 1 Comparison of results of different methods for detecting small nucleic acids

下面结合实施例对本发明提供的一种基于探针锚定双链的小核酸检测试剂盒及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。The following is a detailed description of a small nucleic acid detection kit based on probe-anchored double-stranded nucleic acid and its application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

序列及对应编号见表2。The sequences and corresponding numbers are shown in Table 2.

表2编号及序列信息Table 2 Number and sequence information

实施例1Example 1

利用本发明方法和通用荧光探针检测细胞培养液上清小核酸Detection of small nucleic acids in cell culture supernatant using the method of the present invention and universal fluorescent probe

以A549肺癌细胞和肺癌相关小核酸miR-411为例，首先设计合成用于检测的主探针，miR-411主探针TS0411，序列为：5’-GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCAGGTTAGTGGACCGTGTTACATACCTCCACGTGACCCTGACGTA-3’，其中，5’端与3’端部分(下划线所示)为通用序列，5’端与上游通用引物及通用荧光探针完全同源，3’端与通用锚探针完全互补，中间部分(黑体所示)为靶标小核酸结合区，与靶标小核酸miR-16完全互补。辅探针(SEQ IDNO:2)、锚探针(SEQ ID NO:4)、PCR引物(SEQ ID NO:5和SEQ ID NO:6)、荧光探针(SEQ IDNO:7)均为通用。荧光探针5’端标记荧光基团FAM，3’端标记淬灭基团BHQ1。体系配制准备如前述。取A549细胞培养液上清100μl或纯化水100μl(阴性对照)，12000rpm离心1min，吸取上清90μl，转移至含有40μl磁珠样本释放剂的管中，混匀，30℃保温10min；以磁铁吸附，吸去上清，加入900μl洗涤液混匀，再以磁铁吸附，吸去上清，加入50μl绿豆核酸酶液，30℃保温1min；再以磁铁吸附，吸去上清，加入900μl洗涤液混匀，再以磁铁吸附，吸去上清，加入30μlPCR液，吸打混匀并转移至PCR管中，在ABI7500 PCR热循环仪上，以PCR程序：95℃10min，再40个循环的93℃10秒、65℃40秒，荧光信号采集在65℃(FAM通道)，进行荧光定量PCR反应，A549细胞培养液上清样本结果为阳性(有扩增，即有Ct值35)指示样本中miR-411的含量达到可检测水平。阴性对照结果为阴性(无扩增，即无Ct值)指示操作过程正常，无污染。Taking A549 lung cancer cells and lung cancer-related small nucleic acid miR-411 as examples, the main probe for detection was first designed and synthesized, the miR-411 main probe TS0411, the sequence of which is: 5'-GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCA GGTTAGTGGACCGTGTTACATACCTCCACGTGACCCTGACGTA -3', wherein the 5' end and the 3' end part (underlined) are universal sequences, the 5' end is completely homologous to the upstream universal primer and the universal fluorescent probe, the 3' end is completely complementary to the universal anchor probe, and the middle part (in bold) is the target small nucleic acid binding region, which is completely complementary to the target small nucleic acid miR-16. The auxiliary probe (SEQ ID NO: 2), anchor probe (SEQ ID NO: 4), PCR primers (SEQ ID NO: 5 and SEQ ID NO: 6), and fluorescent probe (SEQ ID NO: 7) are all universal. The 5' end of the fluorescent probe is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group BHQ1. The system was prepared as described above. Take 100 μl of A549 cell culture supernatant or 100 μl of purified water (negative control), centrifuge at 12000 rpm for 1 min, aspirate 90 μl of supernatant, transfer to a tube containing 40 μl of magnetic bead sample release agent, mix, and keep warm at 30°C for 10 min; adsorb with a magnet, remove the supernatant, add 900 μl of washing solution and mix, adsorb with a magnet again, remove the supernatant, add 50 μl of mung bean nuclease solution, and keep warm at 30°C for 1 min; adsorb with a magnet again, remove the supernatant, add 900 μl of washing solution and mix, adsorb with a magnet again, remove the supernatant, add 30 μl of PCR solution, mix by pipetting and transfer to a PCR tube, and incubate at ABI7500 On a PCR thermal cycler, the PCR program was as follows: 95°C for 10 min, followed by 40 cycles of 93°C for 10 sec, 65°C for 40 sec, and the fluorescence signal was collected at 65°C (FAM channel). The fluorescence quantitative PCR reaction was performed, and the result of the A549 cell culture supernatant sample was positive (amplification, i.e., Ct value 35), indicating that the content of miR-411 in the sample reached a detectable level. The negative control result was negative (no amplification, i.e., no Ct value), indicating that the operation process was normal and there was no contamination.

实施例2Example 2

利用本发明方法和特异荧光探针检测细胞培养液上清小核酸Detection of small nucleic acids in cell culture supernatant using the method of the present invention and specific fluorescent probe

在需要同步扩增检测并区分不同靶标小核酸的应用场景，本发明方法可以使用特异荧光探针，以A549肺癌细胞和miR-411、miR-16为例，首先设计合成用于检测的主探针，miR-411的主探针TS0411同实施例1，miR-16的主探针TS0016见序列表。辅探针、锚探针、PCR引物为通用，序列如前述。荧光探针为靶标小核酸特异，miR-411特异荧光探针(SP0411)序列为：5’-ATGGCAGGTTAGTGGACCGTGTTACATA-3’，5’端标记荧光基团FAM，3’端标记淬灭基团BHQ1，miR-16特异荧光探针(SP0016)序列为：5’-ATGGCACGC CAATATTTACGTGCTGCTA-3’,5’端标记荧光基团HEX，3’端标记淬灭基团BHQ1，其中，黑体部分为与靶标小核酸完全互补部分，上游下划线部分为部分通用序列，以增加探针Tm值。体系配制准备如前述。取A549细胞培养液上清100μl或纯化水100μl(阴性对照)，12000rpm离心1min，吸取上清90μl，转移至含有40μl磁珠样本释放剂的管中，混匀，30℃保温10min；以磁铁吸附，吸去上清，加入900μl洗涤液混匀，再以磁铁吸附，吸去上清，加入50μl绿豆核酸酶液，30℃保温1min；再以磁铁吸附，吸去上清，加入900μl洗涤液混匀，再以磁铁吸附，吸去上清，加入30μlPCR液，吸打混匀并转移至PCR管中，在ABI7500 PCR热循环仪上，以PCR程序：95℃10min，再40个循环的93℃10秒、65℃40秒，荧光信号采集在65℃(FAM/HEX双通道)，进行荧光定量PCR反应，A549细胞培养液上清样本FAM通道和HEX通道结果均为阳性(有扩增，即有Ct值)指示样本中miR-411和miR-16的含量均达到可检测水平。阴性对照FAM通道和HEX通道结果均为阴性(无扩增，即无Ct值)指示操作过程正常，无污染。In the application scenario where synchronous amplification detection and differentiation of different target small nucleic acids are required, the method of the present invention can use specific fluorescent probes. Taking A549 lung cancer cells and miR-411 and miR-16 as examples, the main probes for detection are first designed and synthesized. The main probe TS0411 of miR-411 is the same as in Example 1, and the main probe TS0016 of miR-16 is shown in the sequence table. The auxiliary probe, anchor probe, and PCR primer are universal, and the sequences are as described above. The fluorescent probe is specific to the target small nucleic acid. The sequence of the miR-411 specific fluorescent probe (SP0411) is: 5'-ATGGCA GGTTAGTGGACCGTGTTACATA-3', the 5' end is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group BHQ1. The sequence of the miR-16 specific fluorescent probe (SP0016) is: 5'-ATGGCA CGC CAATATTTACGTGCTGCTA-3', the 5' end is labeled with the fluorescent group HEX, and the 3' end is labeled with the quenching group BHQ1. Among them, the bold part is the part that is completely complementary to the target small nucleic acid, and the upstream underlined part is a part of the universal sequence to increase the probe Tm value. The system preparation is as described above. Take 100 μl of A549 cell culture supernatant or 100 μl of purified water (negative control), centrifuge at 12000 rpm for 1 min, aspirate 90 μl of supernatant, transfer to a tube containing 40 μl of magnetic bead sample release agent, mix, and keep warm at 30°C for 10 min; adsorb with a magnet, aspirate the supernatant, add 900 μl of washing solution and mix, adsorb with a magnet again, aspirate the supernatant, add 50 μl of mung bean nuclease solution, and keep warm at 30°C for 1 min; adsorb with a magnet again, aspirate the supernatant, add 900 μl of washing solution and mix, adsorb with a magnet again, aspirate the supernatant, add 30 μl of PCR solution, aspirate and mix, and transfer to a PCR tube, and incubate at ABI7500 On the PCR thermal cycler, the PCR program was as follows: 95°C for 10 min, followed by 40 cycles of 93°C for 10 s and 65°C for 40 s, and the fluorescence signal was collected at 65°C (FAM/HEX dual channels). The results of the FAM channel and HEX channel of the A549 cell culture supernatant sample were both positive (amplification, i.e., Ct value), indicating that the contents of miR-411 and miR-16 in the sample reached a detectable level. The results of the negative control FAM channel and HEX channel were both negative (no amplification, i.e., no Ct value), indicating that the operation process was normal and there was no contamination.

实施例3Example 3

利用本发明方法和通用荧光染料检测细胞培养液上清小核酸Detection of small nucleic acids in cell culture supernatant using the method of the present invention and universal fluorescent dyes

以A549肺癌细胞和miR-411为例，主探针、辅探针、锚探针、PCR引物、同实施例1，不使用荧光探针，代之为在PCR液中添加SYBR Green I至0.4×。其余体系配制准备如前述。取A549细胞培养液上清100μl或纯化水100μl(阴性对照)，12000rpm离心1min，吸取上清900μl，转移至含有40μl磁珠样本释放剂的管中，混匀，30℃保温10min；以磁铁吸附，吸去上清，加入900μl洗涤液混匀，再以磁铁吸附，吸去上清，加入50μl绿豆核酸酶液，30℃保温1min；再以磁铁吸附，吸去上清，加入900μl洗涤液混匀，再以磁铁吸附，吸去上清，加入30μl PCR液，吸打混匀并转移至PCR管中，在宏石SLAN-96p PCR热循环仪上，以PCR程序：95℃10min，再40个循环的93℃10秒、65℃30秒、74℃34秒，荧光信号采集在74℃(SYBR通道)，进行荧光定量PCR反应，A549细胞培养液上清样本结果为阳性(有扩增，即有Ct值)指示样本中miR-411的含量达到可检测水平。阴性对照结果为阴性(无扩增，即无Ct值)指示操作过程正常，无污染。Taking A549 lung cancer cells and miR-411 as an example, the main probe, auxiliary probe, anchor probe, and PCR primers are the same as in Example 1, but the fluorescent probe is not used, and SYBR Green I is added to 0.4× in the PCR solution. The rest of the system is prepared as described above. Take 100 μl of A549 cell culture supernatant or 100 μl of purified water (negative control), centrifuge at 12000 rpm for 1 min, aspirate 900 μl of supernatant, transfer to a tube containing 40 μl magnetic bead sample release agent, mix, and keep warm at 30°C for 10 min; adsorb with a magnet, aspirate the supernatant, add 900 μl washing solution and mix, adsorb with a magnet again, aspirate the supernatant, add 50 μl mung bean nuclease solution, and keep warm at 30°C for 1 min; adsorb with a magnet again, aspirate the supernatant, add 900 μl washing solution and mix, adsorb with a magnet again, aspirate the supernatant, add 30 μl PCR solution, aspirate and mix, and transfer to a PCR tube, and incubate at Macrostone SLAN-96p On the PCR thermal cycler, the PCR program was: 95°C for 10 min, then 40 cycles of 93°C for 10 seconds, 65°C for 30 seconds, and 74°C for 34 seconds. The fluorescence signal was collected at 74°C (SYBR channel). The fluorescence quantitative PCR reaction was performed. The result of the A549 cell culture supernatant sample was positive (amplification, that is, Ct value), indicating that the content of miR-411 in the sample reached a detectable level. The negative control result was negative (no amplification, that is, no Ct value), indicating that the operation process was normal and there was no contamination.

实施例4Example 4

本发明试剂盒的组成Composition of the kit of the present invention

以按该试剂盒常规规格8反应/包装生产95套为例。按通常95％的得率计划量100套，各组分及所需量见下表3，进行物料准备(表4)，按说明书正文试剂盒配方配制分装。Take the production of 95 sets of the kit according to the conventional specification of 8 reactions/package as an example. According to the usual 95% yield, 100 sets are planned. The components and required quantities are shown in Table 3 below. Materials are prepared (Table 4) and the kit is prepared and packaged according to the kit formula in the manual.

表3 100套计划量组分表Table 3 100 sets of planned quantity groups

表4物料清单Table 4 Bill of Materials

实施例5Example 5

抗干扰实验Anti-interference experiment

本发明设计合成了与miR-93、miR-191、miR-3662、miR-16等4个人体miRNA的主探针，分别记为：TS0093、TS0191、TS3662、TS0016，并且，人工合成了序列分别与这4个miRNA同源的寡核苷酸：OLI0093、OLI0191、OLI3662、OLI0016，具体序列信息见表1。The present invention designs and synthesizes the main probes for four human miRNAs, namely miR-93, miR-191, miR-3662, and miR-16, which are respectively denoted as TS0093, TS0191, TS3662, and TS0016. In addition, the present invention artificially synthesizes oligonucleotides whose sequences are homologous to the four miRNAs, namely OLI0093, OLI0191, OLI3662, and OLI0016. The specific sequence information is shown in Table 1.

利用DH5α大肠杆菌培养液和同源寡核苷酸对本发明方法与申请号202011041856.5方法进行抗干扰比较实验。阴性样本NL01、NH01分别为20μl和200μl 10⁵/μl的大肠杆菌培养液，阳性样本PL01、PH01分别为20μl和200μl 10⁵/μl大肠杆菌培养液添加了10³拷贝的上述4个同源寡核苷酸，各进行3个重复。在用本发明方法进行实验时，我们向样本补充PBS缓冲液使得样本体积达到100μl，再按前文所述的步骤进行检测操作。在用申请号202011041856.5方法进行实验时，我们按该方法直接向样本加入10倍体积的含探针的处理液1，并循该方法步骤继续操作。The anti-interference comparison experiment of the method of the present invention and the method of application number 202011041856.5 was carried out using DH5α Escherichia coli culture fluid and homologous oligonucleotides. The negative samples NL01 and NH01 were 20μl and 200μl 10⁵ /μl Escherichia coli culture fluid, respectively, and the positive samples PL01 and PH01 were 20μl and 200μl 10⁵ /μl Escherichia coli culture fluid, respectively, with 10³ copies of the above 4 homologous oligonucleotides added, and 3 repetitions were performed each. When conducting experiments using the method of the present invention, we supplemented the sample with PBS buffer so that the sample volume reached 100μl, and then performed the detection operation according to the steps described above. When conducting experiments using the method of application number 202011041856.5, we directly added 10 times the volume of the probe-containing treatment solution 1 to the sample according to the method, and continued the operation according to the steps of the method.

结果显示，当大肠杆菌含量为20×10⁵时，2种方法都能得到“No Ct”的无本底扩增的阴性结果，同时也能给出Ct平均值(Cta)分别为27.67和28.52的良好扩增的阳性结果；当大肠杆菌含量提高10倍达到200×10⁵时，本发明方法仍能分别得到“No Ct”的无本底扩增的阴性结果和27.66的良好扩增的阳性结果，但申请号202011041856.5方法已不能得到无本底扩增的阴性结果，同时，其扩增结果波动加大，显示受到严重干扰。具体数据如下表所示(表5)：The results showed that when the E. coli content was 20×10⁵ , both methods could obtain a negative result of "No Ct" for background-free amplification, and also give a positive result of good amplification with an average Ct value (Cta) of 27.67 and 28.52 respectively; when the E. coli content was increased by 10 times to 200×10⁵ , the method of the present invention could still obtain a negative result of "No Ct" for background-free amplification and a positive result of good amplification of 27.66, respectively, but the method of application number 202011041856.5 could no longer obtain a negative result of background-free amplification, and at the same time, the fluctuation of its amplification results increased, indicating that it was seriously interfered. The specific data are shown in the following table (Table 5):

表5本发明方法(PADSA)与申请号202011041856.5方法(MCAP)实验比对Table 5 Experimental comparison between the method of the present invention (PADSA) and the method of application No. 202011041856.5 (MCAP)

注：Ct1-Ct3，为3次平行实验结果，Cta为平均值。Note: Ct1-Ct3 are the results of 3 parallel experiments, and Cta is the average value.

进一步利用大肠杆菌总RNA和同源寡核苷酸对本发明方法与申请号202011041856.5方法进行抗干扰比对实验。采用碱裂解法从DH5α大肠杆菌提取纯化制备了大肠杆菌总RNA，并以超纯水调整至所需浓度。阴性样本NL02、NH02分别为20μl浓度0.1μg/μl和1.0μg/μl的大肠杆菌总RNA，阳性样本PL02、PH02分别为20μl浓度0.1μg/μl和1.0μg/μl的大肠杆菌总RNA添加了10³拷贝的上述4个同源寡核苷酸，各进行3个重复。在用本发明方法进行实验时，我们向样本补充PBS缓冲液使得样本体积达到100μl，再按前文所述的步骤进行检测操作。在用申请号202011041856.5方法进行实验时，我们按该方法直接向样本加入10倍体积的含探针的处理液1，并循该方法步骤继续操作。结果显示，当大肠杆菌总RNA浓度为0.1μg/μl时，2种方法都能得到“No Ct”的无本底扩增的阴性结果，同时也能给出Ct平均值(Cta)分别为27.61和28.20的良好扩增的阳性结果；当大肠杆菌RNA浓度提高10倍达到1.0μg/μl时，本发明方法仍能分别得到“No Ct”的无本底扩增的阴性结果和27.69的良好扩增的阳性结果，但申请号202011041856.5方法已不能得到无本底扩增的阴性结果，同时，其扩增结果波动加大，显示受到严重干扰。具体数据如下表6所示。Escherichia coli total RNA and homologous oligonucleotides were further used to conduct anti-interference comparison experiments between the method of the present invention and the method of application number 202011041856.5. Escherichia coli total RNA was extracted and purified from DH5α Escherichia coli using alkaline lysis method, and adjusted to the required concentration with ultrapure water. The negative samples NL02 and NH02 were 20μl of Escherichia coli total RNA with concentrations of 0.1μg/μl and 1.0μg/μl, respectively, and the positive samples PL02 and PH02 were 20μl of Escherichia coli total RNA with concentrations of 0.1μg/μl and 1.0μg/μl, respectively. 10³ copies of the above 4 homologous oligonucleotides were added, and 3 replicates were performed each. When conducting experiments using the method of the present invention, we supplemented the sample with PBS buffer so that the sample volume reached 100μl, and then performed the detection operation according to the steps described above. When conducting experiments using the method of application number 202011041856.5, we directly added 10 times the volume of the treatment solution 1 containing the probe to the sample according to the method, and continued the operation according to the steps of the method. The results show that when the total RNA concentration of E. coli is 0.1 μg/μl, both methods can obtain a negative result of "No Ct" for background-free amplification, and can also give a positive result of good amplification with an average Ct value (Cta) of 27.61 and 28.20 respectively; when the RNA concentration of E. coli is increased by 10 times to 1.0 μg/μl, the method of the present invention can still obtain a negative result of background-free amplification of "No Ct" and a positive result of good amplification of 27.69, respectively, but the method of application number 202011041856.5 can no longer obtain a negative result of background-free amplification, and at the same time, the fluctuation of its amplification results increases, indicating that it is seriously interfered. The specific data are shown in Table 6 below.

表6本发明方法(PADSA)与申请号202011041856.5方法(MCAP)实验比对Table 6 Experimental comparison between the method of the present invention (PADSA) and the method of application No. 202011041856.5 (MCAP)

实施例6Example 6

利用肺癌A549细胞比对对本发明方法与申请号202011041856.5方法用于细胞检测的灵敏度。Lung cancer A549 cells were used to compare the sensitivity of the method of the present invention and the method of application number 202011041856.5 for cell detection.

取新鲜收获的A549细胞并以PBS缓冲液将细胞浓度调整至100个/μl，取20μl细胞悬浮液，以PBS缓冲液进行10倍梯度稀释，得到浓度分别为10个/μl、1个/μl、0.1个/μl的细胞悬浮液样本各100μl，分别记为PA010、PA001、PA0.1。我们各取样10μl，每样本3个重复，应用本发明方法进行miR-191、miR-93、miR-16、miR-3662这4个靶标小核酸的组合检测，同时进行申请号202011041856.5方法比对。在用申请号202011041856.5方法进行实验时，我们按该方法直接向样本加入10倍体积的含探针的处理液1，并循该方法步骤继续操作。结果显示，当样本细胞浓度为10个/μl和1个/μl时，2种方法都能得到有扩增的阳性结果，本发明方法得到更小的Ct值，表明能达到更好的扩增。当样本细胞浓度为0.1个/μl时，本发明方法仍能得到良好扩增的阳性结果，但比对方法已不能得到扩增。因此，可见本发明方法比申请号202011041856.5方法能达到更好的细胞检测灵敏度(100个/ml)。具体数据如下表7所示。Take freshly harvested A549 cells and adjust the cell concentration to 100 cells/μl with PBS buffer, take 20μl of cell suspension, dilute 10 times with PBS buffer, and obtain 100μl of cell suspension samples with concentrations of 10/μl, 1/μl, and 0.1/μl, respectively, recorded as PA010, PA001, and PA0.1. We took 10μl of each sample, repeated 3 times for each sample, and applied the method of the present invention to perform combined detection of the four target small nucleic acids miR-191, miR-93, miR-16, and miR-3662, and compared the method of application number 202011041856.5 at the same time. When conducting experiments using the method of application number 202011041856.5, we directly added 10 times the volume of the probe-containing treatment solution 1 to the sample according to the method, and continued to operate according to the steps of the method. The results show that when the sample cell concentration is 10/μl and 1/μl, both methods can obtain positive results with amplification, and the method of the present invention obtains a smaller Ct value, indicating that better amplification can be achieved. When the sample cell concentration is 0.1/μl, the method of the present invention can still obtain a positive result of good amplification, but the comparison method can no longer obtain amplification. Therefore, it can be seen that the method of the present invention can achieve better cell detection sensitivity (100/ml) than the method of application number 202011041856.5. The specific data are shown in Table 7 below.

表7本发明方法(PADSA)与申请号202011041856.5方法(MCAP)实验比对Table 7 Experimental comparison between the method of the present invention (PADSA) and the method of application No. 202011041856.5 (MCAP)

注：NC为纯化水阴性对照。Ct1-Ct3，为3次平行实验结果，Cta为平均值。Note: NC is the negative control of purified water. Ct1-Ct3 are the results of 3 parallel experiments, and Cta is the average value.

实施例7Example 7

我们将A549细胞在生长密度接近100％的条件下连续培养3天，然后收集培养液上清，以10μl/份分装得到A549培养液上清样本，记为EC010。利用本发明方法和申请号202011041856.5方法进行miR-191、miR-93、miR-16、miR-3662这4个靶标小核酸的组合检测，各进行3个重复。在用本发明方法进行实验时，我们向样本补充PBS缓冲液使得样本体积达到100μl，再按前文所述的步骤进行检测操作。在用申请号202011041856.5方法进行实验时，我们按该方法直接向样本加入10倍体积的含探针的处理液1，并循该方法步骤继续操作。结果显示，2种方法都能得到有扩增的阳性结果，本发明方法得到更小的Ct值，表明能达到更好的扩增，即能达到更好的细胞外小核酸检测灵敏度。具体数据如下表8所示。We cultured A549 cells for 3 consecutive days at a growth density close to 100%, then collected the culture supernatant, and aliquoted it at 10 μl/portion to obtain the A549 culture supernatant sample, which was recorded as EC010. The combined detection of the four target small nucleic acids miR-191, miR-93, miR-16, and miR-3662 was performed using the method of the present invention and the method of application number 202011041856.5, and 3 repetitions were performed for each. When the method of the present invention was used to perform the experiment, we supplemented the sample with PBS buffer so that the sample volume reached 100 μl, and then performed the detection operation according to the steps described above. When the method of application number 202011041856.5 was used to perform the experiment, we directly added 10 times the volume of the probe-containing treatment solution 1 to the sample according to the method, and continued to operate according to the steps of the method. The results showed that both methods could obtain positive results with amplification, and the method of the present invention obtained a smaller Ct value, indicating that better amplification could be achieved, that is, better sensitivity for detecting extracellular small nucleic acids could be achieved. The specific data are shown in Table 8 below.

表8本发明方法(PADSA)与申请号202011041856.5方法(MCAP)实验比对Table 8 Experimental comparison between the method of the present invention (PADSA) and the method of application No. 202011041856.5 (MCAP)

注：NC为阴性对照，PA010为10个/μlA549细胞阳性对照。Ct1-Ct3，为3次平行实验结果，Cta为平均值。Note: NC is the negative control, PA010 is the positive control of 10 cells/μl A549 cells. Ct1-Ct3 are the results of 3 parallel experiments, and Cta is the average value.

实施例8Example 8

利用miR-16同源寡核苷酸OLI0016对本发明方法进行灵敏度测试。The sensitivity of the method of the present invention was tested using miR-16 homologous oligonucleotide OLI0016.

以0.1μg/μl大肠杆菌总RNA溶液对10³拷贝/ml的前述OLI0016样本进行10倍梯度稀释，分别得到1ml 100拷贝/ml(LC0016100)、10拷贝/ml(LC0016010)、1拷贝/ml(LC0016001)的阳性稀释样本，然后按前文所述的步骤进行检测操作，各进行3个重复，结果显示阳性稀释样本LC0016100和LC0016010均有明显扩增，LC0016001或有扩增，但波动大，已接近灵敏度极限。据此，我们推断本发明方法灵敏度可以稳定达到10拷贝/ml。具体数据如下表9所示。The aforementioned OLI0016 sample of 10³ copies/ml was diluted 10 times with 0.1 μg/μl E. coli total RNA solution, and 1 ml of 100 copies/ml (LC0016100), 10 copies/ml (LC0016010), and 1 copy/ml (LC0016001) positive dilution samples were obtained, and then the detection operation was performed according to the steps described above, and 3 repetitions were performed respectively. The results showed that the positive dilution samples LC0016100 and LC0016010 were significantly amplified, and LC0016001 was amplified, but the fluctuation was large and close to the sensitivity limit. Based on this, we infer that the sensitivity of the method of the present invention can stably reach 10 copies/ml. The specific data are shown in Table 9 below.

表9本发明方法(PADSA)的灵敏度测试Table 9 Sensitivity test of the method of the present invention (PADSA)

注：NCR01为大肠杆菌阴性对照。Ct1-Ct3，为3次平行实验结果，Cta为平均值，若平行实验同时出现有Ct值和无Ct值，则不计算平均值。Note: NCR01 is a negative control for E. coli. Ct1-Ct3 are the results of three parallel experiments, and Cta is the average value. If parallel experiments have both Ct values and no Ct values, the average value will not be calculated.

实施例9Example 9

进一步通过对人miR-181小核酸家族同源寡核苷酸的检测来考察本发明方法的特异性。The specificity of the method of the present invention was further investigated by detecting homologous oligonucleotides of the human miR-181 small nucleic acid family.

人miR-181家族主要有四个不同的5p成熟形式：miR-181a-5p、miR-181b-5p、miR-181c-5p、miR-181d-5p，序列非常相似，相互之间仅有1-5个核苷酸差异(参见附图2)，但在不同疾病病理中表达与调控作用不同，例如：越来越多的证据表明miR-181a和b在胃癌中表达上调，可能促进胃癌发生发展侵袭转移，而miR-181c在胃癌组织中低表达，可能具有抑制胃癌的作用，miR-181d在胃癌中的表达和作用尚不明确。因此，小核酸扩增检测方法不仅应该达到高灵敏性，也应该达到能够区分同一家族中不同种小核酸的高特异性。人工合成了与4种miR-181完全同源的4种寡核苷酸，分别记为：OLI181a、OLI181b、OLI181c、OLI181d，及相应的主探针，分别记为：TS181a、TS181b、TS181c、TS181d(序列具体信息参见序列表),寡核苷酸样本浓度调整至10⁵拷贝/ml，本发明方法操作同前文所述。结果显示寡核苷酸样本与对应的主探针组合均能得到明显扩增，而交叉组合均无扩增，显示本发明方法能达到分辨同一家族中不同种小核酸的高特异性。具体数据如下表10所示。The human miR-181 family mainly has four different 5p mature forms: miR-181a-5p, miR-181b-5p, miR-181c-5p, and miR-181d-5p. The sequences are very similar, with only 1-5 nucleotide differences between them (see Figure 2), but their expression and regulatory effects are different in different disease pathologies. For example, more and more evidence shows that miR-181a and b are upregulated in gastric cancer, which may promote the occurrence, development, invasion and metastasis of gastric cancer, while miR-181c is lowly expressed in gastric cancer tissues and may have an inhibitory effect on gastric cancer. The expression and role of miR-181d in gastric cancer are still unclear. Therefore, the small nucleic acid amplification detection method should not only achieve high sensitivity, but also high specificity that can distinguish different types of small nucleic acids in the same family. Four oligonucleotides completely homologous to four miR-181 were artificially synthesized, and they were respectively recorded as: OLI181a, OLI181b, OLI181c, OLI181d, and the corresponding main probes were respectively recorded as: TS181a, TS181b, TS181c, TS181d (see the sequence table for specific sequence information). The concentration of the oligonucleotide sample was adjusted to 10⁵ copies/ml, and the method of the present invention was operated as described above. The results showed that the combination of the oligonucleotide sample and the corresponding main probe could be significantly amplified, while the cross combination had no amplification, indicating that the method of the present invention can achieve high specificity in distinguishing different types of small nucleic acids in the same family. The specific data are shown in Table 10 below.

表10本发明方法(PADSA)对人miR-181小核酸家族同源寡核苷酸的检测Table 10 Detection of human miR-181 small nucleic acid family homologous oligonucleotides by the method of the present invention (PADSA)

注：Ct1-Ct3，为3次平行实验结果。Note: Ct1-Ct3 are the results of 3 parallel experiments.

实施例10Example 10

小核酸组合检测即同时检测多个靶标小核酸中的某个或多个在样本中有否表达是遗传异质性较高的疾病如肿瘤的检测达到高性能的关键。进一步利用上述8个寡核苷酸(OLI0093、OLI0191、OLI3662、OLI0016、OLI181a、OLI181b、OLI181c、OLI181d)来考察本发明方法的组合性能。Small nucleic acid combination detection, i.e., simultaneous detection of whether one or more of multiple target small nucleic acids are expressed in a sample, is the key to high performance in the detection of diseases with high genetic heterogeneity, such as tumors. The above-mentioned eight oligonucleotides (OLI0093, OLI0191, OLI3662, OLI0016, OLI181a, OLI181b, OLI181c, OLI181d) were further used to investigate the combination performance of the method of the present invention.

将8个主探针(TS0093、TS0191、TS3662、TS0016、TS181a、TS181b、TS181c、TS181d)加入到磁珠样本释放剂中，浓度均为15nM。其余试剂组成同前，检测操作同前。各寡核苷酸样本浓度调整至10⁴拷贝/ml，8寡核苷酸混合样本(OL1TO8)浓度为8×10⁴拷贝/ml。结果显示各寡核苷酸样本及寡核苷酸混合样本均能得到明显扩增，显示本发明方法能达到分辨同一家族中不同种小核酸的高特异性。具体数据如下表11所示。Eight main probes (TS0093, TS0191, TS3662, TS0016, TS181a, TS181b, TS181c, TS181d) were added to the magnetic bead sample release agent, all at a concentration of 15 nM. The composition of the remaining reagents was the same as before, and the detection operation was the same as before. The concentration of each oligonucleotide sample was adjusted to 10⁴ copies/ml, and the concentration of the 8 oligonucleotide mixed sample (OL1TO8) was 8×10⁴ copies/ml. The results showed that each oligonucleotide sample and the oligonucleotide mixed sample could be significantly amplified, indicating that the method of the present invention can achieve high specificity in distinguishing different types of small nucleic acids in the same family. The specific data are shown in Table 11 below.

表11本发明方法对8种寡核苷酸的组合检测Table 11 Combination detection of 8 oligonucleotides by the method of the present invention

注：NC为阴性对照，Ct1-Ct3，为3次平行实验结果，Cta为平均值。Note: NC is the negative control, Ct1-Ct3 are the results of 3 parallel experiments, and Cta is the average value.

实施例11Embodiment 11

miR-885-5p、miR-34b和miR-3662是3个经多篇文献报道验证为肺癌组织高表达而癌旁组织不表达的小核酸。我们合成了这3个靶标小核酸的主探针(序列具体信息见序列表)，分别记为：TS0885、TS034b、TS3662。MiR-885-5p, miR-34b and miR-3662 are three small nucleic acids that have been verified by multiple literature reports to be highly expressed in lung cancer tissues but not expressed in adjacent tissues. We synthesized the main probes for these three target small nucleic acids (see the sequence list for detailed sequence information), which are recorded as: TS0885, TS034b, and TS3662.

收集了10例确诊肺癌患者和10例健康志愿者各2ml肝素钠抗凝全血样本，同时进行纯化水阴性对照和A549细胞阳性对照，以5000rpm离心3min后，吸取约95μl上清转移至含有40μl磁珠样本释放剂的管中，混匀，30℃保温10min；以磁铁吸附后，吸去上清，加入950μl洗涤液混匀，再以磁铁吸附，吸去上清，加入50μl的绿豆核酸酶液，30℃保温1min；再以磁铁吸附，吸去上清，加入950μl洗涤液混匀，再以磁铁吸附，吸去上清，加入30μl的PCR液，95℃保温10min后，12000rpm短暂离心后，吸取上清30μl转移至PCR管中，以前述PCR程序进行荧光定量PCR反应，检测结果：阴性对照无扩增，阳性对照Ct值为22.21，显示实验质控合格；10例肺癌患者血液样品的Ct值在24.29～30.43之间，均为阳性，显示以本发明方法对这3个靶标小核酸组合检测肺癌血液阳性率为100％；同时，10例健康志愿者血液样品均无扩增，显示以本发明方法对这3个靶标小核酸组合检测血液阴性符合率为100％。2 ml of sodium heparin anticoagulated whole blood samples were collected from 10 confirmed lung cancer patients and 10 healthy volunteers, and purified water negative control and A549 cell positive control were performed at the same time. After centrifugation at 5000 rpm for 3 min, about 95 μl of supernatant was transferred to a tube containing 40 μl of magnetic bead sample release agent, mixed, and kept at 30°C for 10 min; after adsorption with a magnet, the supernatant was aspirated, 950 μl of washing solution was added and mixed, and then adsorbed with a magnet again, the supernatant was aspirated, 50 μl of mung bean nuclease solution was added, and the mixture was kept at 30°C for 1 min; then the supernatant was aspirated, 950 μl of washing solution was added and mixed, and then adsorbed with a magnet again, the supernatant was aspirated, and 30 μl of PC was added. R solution was incubated at 95°C for 10 minutes and briefly centrifuged at 12000rpm. 30μl of the supernatant was transferred to a PCR tube and the fluorescent quantitative PCR reaction was performed using the above-mentioned PCR program. The test results showed that the negative control had no amplification and the positive control Ct value was 22.21, indicating that the experimental quality control was qualified; the Ct values of the blood samples of 10 lung cancer patients were between 24.29 and 30.43, all of which were positive, indicating that the positive rate of the blood for lung cancer detection using the method of the present invention for the combination of these three target small nucleic acids was 100%; at the same time, the blood samples of 10 healthy volunteers had no amplification, indicating that the negative compliance rate of the blood for the combination of these three target small nucleic acids using the method of the present invention was 100%.

实施例12Example 12

利用本发明试剂盒检测人体血液中的3个肺癌特异小核酸Detection of three lung cancer-specific small nucleic acids in human blood using the kit of the present invention

利用所生产的试剂盒对miR-885-5p、miR-34b和miR-3662等3个肺癌特异高表达的小核酸进行扩增检测。我们合成了这3个靶标小核酸的主探针(序列具体信息见序列表)：TS0885、TS034b、TS3662，以等比例混合，使得总浓度为1.2μM(各0.4μM)，吸取40μl加入到试剂盒提供的300μl 1.1×磁珠样本释放剂中，混匀后，按40μl/管分装至2ml离心管中，并向10×洗涤液加入15.3ml纯化水，其余组分的具体组成及检测操作与前文相同。我们收集了10例确诊肺癌患者和10例健康志愿者各2ml肝素钠抗凝全血样本，同时进行纯化水阴性对照和A549细胞阳性对照，以5000rpm离心3min后，吸取约100μl上清转移至含有40μl磁珠样本释放剂的管中，混匀，30℃保温10min；以磁铁吸附后，吸去上清，加入950μl洗涤液混匀，再以磁铁吸附，吸去上清，加入50μl的绿豆核酸酶液，30℃保温1min；再以磁铁吸附，吸去上清，加入950μl洗涤液混匀，再以磁铁吸附，吸去上清，加入30μl的PCR液，吸打混匀并转移至PCR管中，在ABI7500 PCR仪上，以PCR程序：95℃10min，再40个循环的93℃10秒、65℃40秒，荧光信号采集在65℃(FAM通道)，进行荧光定量PCR反应；电脑自动阈值进行结果分析判断：样本有Ct值，判断为有扩增，Ct值＜35，判断为有显著扩增，为阳性；无Ct值，判断为无扩增，为阴性；Ct值在35～40之间，为灰色区域，若可重复，判断为弱阳性。本例检测结果：阴性对照无扩增，阳性对照Ct值为23.03，显示实验质控合格；10例肺癌患者血液样品的Ct值在25.13～33.19之间，均为阳性，显示以本发明试剂盒对这3个靶标小核酸组合检测肺癌血液阳性率为100％；同时，10例健康志愿者血液样品均无扩增，显示以本发明试剂盒对这3个靶标小核酸组合检测血液阴性符合率为100％。The kit produced was used to amplify and detect three small nucleic acids that are highly expressed in lung cancer, such as miR-885-5p, miR-34b, and miR-3662. We synthesized the main probes of these three target small nucleic acids (see the sequence table for specific sequence information): TS0885, TS034b, and TS3662, mixed them in equal proportions to make the total concentration 1.2μM (0.4μM each), and took 40μl and added it to 300μl 1.1× magnetic bead sample release agent provided by the kit. After mixing, it was divided into 2ml centrifuge tubes at 40μl/tube, and 15.3ml purified water was added to the 10× washing solution. The specific composition and detection operation of the remaining components were the same as above. We collected 2 ml of sodium heparin anticoagulated whole blood samples from 10 confirmed lung cancer patients and 10 healthy volunteers, and performed purified water negative control and A549 cell positive control at the same time. After centrifugation at 5000 rpm for 3 min, about 100 μl of supernatant was transferred to a tube containing 40 μl of magnetic bead sample release agent, mixed, and kept at 30°C for 10 min; after adsorption with a magnet, the supernatant was removed, 950 μl of washing solution was added and mixed, and then adsorbed with a magnet again, the supernatant was removed, 50 μl of mung bean nuclease solution was added, and kept at 30°C for 1 min; then adsorbed with a magnet again, the supernatant was removed, 950 μl of washing solution was added and mixed, and then adsorbed with a magnet again, the supernatant was removed, 30 μl of PCR solution was added, pipetted and mixed, and transferred to a PCR tube, and then incubated at ABI7500 On the PCR instrument, the PCR program is as follows: 95°C for 10 min, then 40 cycles of 93°C for 10 seconds and 65°C for 40 seconds, and the fluorescence signal is collected at 65°C (FAM channel) to perform fluorescence quantitative PCR reaction; the computer automatically analyzes the results based on the threshold: if the sample has a Ct value, it is judged to be amplified, and if the Ct value is less than 35, it is judged to be significantly amplified, which is positive; if there is no Ct value, it is judged to be non-amplified, which is negative; if the Ct value is between 35 and 40, it is a gray area, and if it can be repeated, it is judged to be weakly positive. The test results in this case: the negative control has no amplification, and the positive control Ct value is 23.03, indicating that the experimental quality control is qualified; the Ct values of the blood samples of 10 lung cancer patients are between 25.13 and 33.19, all of which are positive, indicating that the positive rate of the present invention kit for detecting lung cancer blood for these three target small nucleic acid combinations is 100%; at the same time, the blood samples of 10 healthy volunteers have no amplification, indicating that the negative compliance rate of the present invention kit for detecting these three target small nucleic acid combinations is 100%.

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The small nucleic acid detection kit based on the probe anchored double strand is characterized by comprising a probe set, an amplification primer pair of target small nucleic acid and a magnetic bead sample releasing agent;

the probe set comprises a main probe, an auxiliary probe, an anchor probe and a fluorescent probe; the main probe sequentially comprises an auxiliary probe binding region, a target small nucleic acid binding region and an anchor probe binding region; the length of the main probe is consistent with the total length of the auxiliary probe, the target small nucleic acid and the anchor probe;

the auxiliary probe binding region comprises an upstream primer complementary region for amplifying the target small nucleic acid and a fluorescent probe complementary region, and the length of the auxiliary probe binding region is consistent with the total length of the upstream primer for amplifying the target small nucleic acid and the fluorescent probe;

the length of the anchor probe is consistent with that of the anchor probe binding region, and the anchor probe binding region is complementary with a downstream primer for amplifying the target small nucleic acid;

the 5' -end of the anchor probe or the main probe is marked with biotin;

the magnetic bead sample release agent comprises 40-80 mM zinc chloride, 40-80 mM sodium acetate, 40-80 mM cysteine, 10-18% ethylene carbonate, 10-30% formamide, 4-10% sodium dodecyl sulfate and 90-200 mug/ml streptavidin modified magnetic beads.

2. The probe-anchored duplex-based small nucleic acid detection kit of claim 1, wherein the 3' -terminal base of the main probe is adenine.

3. The probe-anchored double-strand based small nucleic acid detection kit according to claim 1 or 2, wherein the nucleotide sequence of the anchor probe binding region is SEQ ID No. 3;

the nucleotide sequence of the anchor probe is SEQ ID NO. 4.

4. The probe anchored duplex based small nucleic acid detection kit of claim 3, wherein the downstream primer nucleotide sequence for amplifying the target small nucleic acid is SEQ ID No. 5.

5. The probe anchored duplex based small nucleic acid detection kit of claim 1, wherein the nucleotide sequence of said co-probe binding region is SEQ ID No. 1;

the nucleotide sequence of the auxiliary probe is SEQ ID NO. 2.

6. The probe anchored duplex based small nucleic acid detection kit of claim 5, wherein the nucleotide sequence of the upstream primer for amplifying the target small nucleic acid is SEQ ID No. 6;

the nucleotide sequence of the fluorescent probe is SEQ ID NO. 7.

7. The probe-anchored double-strand based small nucleic acid detection kit according to any one of claims 1, 2, 4 to 6, wherein said kit further comprises a wash solution, mung bean nuclease, PCR amplification buffer, 1 x positive control;

the washing liquid is an aqueous solution containing 5-20 mM 3-morpholinopropane sulfonic acid, 40-80 mM cysteine, 30-50 mM sodium acetate and 0.05-0.1% Tween 20 by volume percent.

8. Use of a probe-anchored double-stranded based small nucleic acid detection kit according to any one of claims 1to 7 for the detection of small nucleic acids for non-disease diagnostic purposes.

9. The use of claim 8, wherein the small nucleic acid comprises at least one of the following mirrRNA: miR-191, miR-93, miR-16, miR-3662 and miR-181 family;

the miR-181 family comprises at least one of miR-181a-5p, miR-181b-5p, miR-181c-5p and miR-181d-5 p.

10. A method for detecting a small nucleic acid for non-disease diagnosis using the kit according to any one of claims 1to 7, comprising the steps of:

mixing a sample to be detected, a main probe, an auxiliary probe, an anchor probe and a magnetic bead sample releasing agent, incubating, removing the supernatant under the magnetic field condition, washing, adding mung bean nuclease solution for enzymolysis, removing the supernatant under the magnetic field condition, washing, adding a PCR amplification buffer solution, a fluorescent probe and a target small nucleic acid amplification primer pair for fluorescent quantitative PCR amplification reaction, wherein the Ct value of the detection result is less than or equal to 35, which indicates that the sample to be detected contains the target small nucleic acid, otherwise, the sample to be detected does not contain the target small nucleic acid.