CN108414735B - A method for immunoassay of biological macromolecules mediated by triple extension-RNA amplification - Google Patents

Abstract

Translated fromChinese

本发明公开了一种基于三次延伸‑RNA扩增介导的生物大分子免疫分析方法，属于免疫学领域。该方法将生物大分子与识别生物大分子的物质偶联的oligo1和oligo2接触，oligo1一端为标签序列，另一端序列（长约6nt）可以和oligo2末端互补配对；oligo2，一端为T7启动子序列，另一端序列（长约6nt）可以和oligo1末端互补配对；在Klenow酶的作用下进行延伸，形成dsDNA，在T7聚合酶的作用下转录出小RNA分子；利用小RNA搭桥对两个互不重叠的扩增引物进行双向延伸检测小RNA，由此判断待检生物大分子的存在与否。该法灵敏度高、对仪器的要求低，可用于生物医学指标的超敏检测。

The invention discloses a biological macromolecular immunoassay method mediated by three-time extension-RNA amplification, and belongs to the field of immunology. In this method, biological macromolecules are contacted with oligo1 and oligo2 coupled with substances that recognize biological macromolecules. One end of oligo1 is a tag sequence, and the other end sequence (about 6 nt) can be complementary to the end of oligo2; oligo2, one end is a T7 promoter sequence , the other end sequence (about 6nt in length) can be complementary to the end of oligo1; it is extended under the action of Klenow enzyme to form dsDNA, and small RNA molecules are transcribed under the action of T7 polymerase; The overlapping amplification primers are bidirectionally extended to detect small RNAs, thereby judging the presence or absence of the biological macromolecules to be detected. The method has high sensitivity and low requirements on instruments, and can be used for hypersensitive detection of biomedical indicators.

Description

Biomacromolecule immunoassay method based on three-time extension-RNA amplification mediation

Technical Field

The invention belongs to the field of immunology, and particularly relates to a biomacromolecule immunoassay method based on triple extension-RNA amplification mediation.

Background

Immunological methods such as enzyme-linked immunosorbent assay (ELISA) have been widely used in life science research and clinical diagnosis for decades. ELISA-based products have the characteristics of simplicity of use, high versatility and relatively low cost. However, these common immunological methods have limitations like any other techniques, and the most important problems are that their detection sensitivity is not high and it is difficult to detect ultra-low amount of proteins, which are important for early diagnosis of diseases such as coronary artery disease, ovarian tumor, and senile dementia. Therefore, it is necessary to establish an ultra-sensitive biomacromolecule detection technology.

Immune-polymerase chain reaction (iPCR) has become a powerful technology, and the sensitivity is 100-10000 times higher than that of the conventional ELISA method when the target protein is detected. This methodology sensitivity enhancement is achieved by PCR amplification of antibody-conjugated nucleotides. However, there are many disadvantages in this design, i.e., the designed nucleotides coupled to the antibody are long, such as the pair of nucleotide fragments used in the Proximity Ligation (PLA) and extension (PEA) technologies, which are about 40-60 nucleotides long, and the design of the long nucleotides is convenient for the next PCR molecular amplification. The disadvantage of this longer probe design is that the complex of the nucleotide-conjugated antibody is difficult to separate from the free nucleotides, conventional separation methods such as HPLC separation and purification require a certain amount of antibody for effective separation, and when the amount of labeled antibody is small, it is still difficult to achieve effective separation.

Disclosure of Invention

In view of the above problems, the present invention provides a biomacromolecule hypersensitive immunoassay method based on triple extension-RNA amplification mediation, namely immune-TEBRA (triple extension-based RNAamplification). The method has the characteristics of high sensitivity, small antibody dosage and low requirement on instruments, can be used for the hypersensitive detection of biomedical indexes, and can also be used for establishing a new drug screening platform based on the monitoring of the concentration of biomarkers.

In order to solve the problems in the prior art, the invention is realized by the following technical scheme:

in a first aspect of the present invention, there is provided a biomacromolecule immunoassay method based on three times of extension-RNA amplification mediation, comprising the following steps:

1) design of related probes and primers:

(1) design of the first and second proximity probes: oligo1, 20-30nt DNA in total length, with a tag sequence ZC at one end and a complementary matching sequence (5-6 nt in length) at the other end with the end of oligo 2; oligo2, DNA with a total length of about 20-30nt, wherein one end is a T7 promoter sequence, and the other end (with a length of 5-6 nt) is complementarily paired with the end of Oligo 1;

oligo1 and Oligo2 are coupled with a substance recognizing biological macromolecules respectively to form a substance recognizing biological macromolecules-Oligo 1, a substance recognizing biological macromolecules-Oligo 2, and substances marking Oligo1 and Oligo2 are antibodies binding with biological macromolecules or biotin with affinity; because the pair of oligos is designed to be short in length (about 20-30nt), the oligo coupled with the biomacromolecule-recognizing substance can be separated from the free oligos by a simple centrifugal column chromatography after coupling;

(2) amplification primer R (named oligo 3): oligo3 has two parts, the 3 'end is complementary paired with the sequence of half of the transcribed small RNA, i.e., it hybridizes to half of the molecule at the 3' end of the transcribed small RNA by 10 or 11 nucleotides in length; the other end is a tag sequence (named as ZC1, ZC2) and a T7 promoter sequence; designing a capture probe and a detection probe according to a tag sequence for qualitative analysis of a subsequent amplification product, wherein the tag sequence is designed according to needs and has 1-2 strips and the length of 16-20 nt; the T7 promoter sequence is the T7 polymerase recognition site for subsequent transcription by T7 polymerase;

(3) stabilizing sequence 1: to increase the stability of oligo3 and the transcribed small RNA hybrid, stable sequence 1 was introduced, which binds to the tag sequence of oligo3, the complementary pairing of the T7 promoter;

(4) amplification primer F (named oligo 4): similar to the characteristics of oligo3, the cDNA has two parts, one end of which is the same as half of the molecular sequence (10-11 nucleotides) of the 5' end of the transcribed small RNA and can be combined with the cDNA sequence of the full-length transcribed small RNA in a complementary pairing way; the other end contains a tag sequence (named as ZC3), and the tag sequence can be used for coating probes or designing detection probes in the subsequent qualitative detection of an amplification product;

(5) stabilizing sequence 2: in order to increase the stability of the binding of oligo4 to the newly synthesized oligo 3-RNA-cDNA, a stabilizing sequence 2 was introduced, characterized by complementary paired binding to the tag sequence ZC 3;

2) biomacromolecule detection

(1) In a single reaction tube, a substance-oligo 1 (the substance for labeling oligo1 can be an antibody binding to the biological macromolecule, or biotin with affinity ability, etc.) for recognizing the biological macromolecule and a substance-oligo 2 (the substance for labeling oligo2 can be an antibody binding to the biological macromolecule, or biotin with affinity ability, etc.) for recognizing the biological macromolecule are simultaneously bound to the target biological macromolecule, because of the proximity (proximity), 6 nucleotide molecules at the tail of oligo1 and oligo2 are complementarily paired and bound, and then, the extension is carried out under the action of Klenow enzyme to form a dsDNA, which is the first sequence extension;

(2) the dsDNA formed in the step (1) contains a T7 promoter, and small RNA molecules with the size of 20-25 nt are transcribed under the action of T7 polymerase;

(3) and (3) combining the oligo3 with a half sequence at the end of the small RNA 3' transcribed in the step (2) to form a small RNA-oligo3 complex, wherein in order to increase the stability of the complex, the stable sequence 1 is combined to the complex by the principle of base complementary pairing, and finally the oligo3, the stable sequence 1 and the small RNA form a stable oligo 3/stable sequence 1-RNA complex by the fusion effect (stacking effect), and the complex obtains the full-length cDNA of the small RNA molecule under the action of reverse transcriptase, namely, the oligo 3-RNA-cDNA: an RNA complex, which is a second sequence extension;

(4) step (3) oligo 3-RNA-cDNA: under the action of RNaseH, RNA in the RNA complex can be degraded to form oligo 3-RNA-cDNA;

(5) complementary pairing and combination of oligo4 and oligo 3-RNA-cDNA to form oligo 3-RNA-cDNA-oligo 4 complex; to increase the stability of the complex, the stable sequence 2 is bound to the complex by the base complementary pairing principle, and finally Oligo4, the stable sequence 2, the stable sequence 1 and Oligo3 synthesized Oligo 3-RNA-cDNA form a stable complex by the fusion effect (stacking effect), i.e., Oligo 3-RNA-cDNA-Oligo 4/stable sequence 2 complex. The complex obtains double-stranded DNA containing a T7 promoter sequence, a tag sequence and the full length of a small RNA molecule under the action of reverse transcriptase, namely oligo 3-RNA-cDNA-oligo 4 double-stranded DNA, which is the third sequence extension;

(6) the sequence structure of the oligo 3-RNA-cDNA-oligo 4 double-stranded DNA formed in (5) is T7 promoter-ZC 2-ZC 1-RNA-cDNA-ZC 3, and the double-stranded DNA can be amplified by a TMA or NASBA method to obtain an RNA molecule with the sequence of ZC2-ZC 1-RNA-cDNA-ZC 3;

(7) detecting the presence, absence and/or amount of the amplified product of the RNA, thereby determining the presence, absence and/or amount of the biomacromolecule to be detected.

In another preferred example, the detection result in the step 2) and the step 7) is a qualitative or quantitative result.

In another preferred example, the method for detecting the amplification product of the RNA in step 2) (7) comprises molecular beacon, plate hybridization-signal amplification, or nucleic acid colloidal gold detection.

In another preferred example, the method for detecting the amplification product of the RNA in the step 2) (7) is plate hybridization-signal amplification detection, and particularly, a capture probe is designed according to a tag sequence (such as ZC3), and the capture probe can be combined with ZC3 in a complementary pairing manner, so that the amplified RNA molecule can be immobilized in a microplate or on other solid phase carriers; and designing a detection probe according to a tag sequence (such as ZC1 and ZC2), wherein the detection probe can be combined with the bases of ZC1 and ZC2 in a complementary pairing mode, the detection probe can be labeled with biotin, the biotin is further labeled on an amplified RNA molecule, streptavidin-HRP (Strep-HRP) and a chemiluminescent substrate can be sequentially added subsequently, and finally, the existence or quantity of an amplification product is detected by a chemiluminescent method, so that the detection condition of the biomacromolecule to be detected is judged.

The biomacromolecule mentioned above refers to substances that can recognize each other by specific interaction, such as antibodies and antigens, receptors and ligands, enzymes and substrates, lectins and polysaccharides.

The principle of the invention for detecting biomacromolecules is shown in figure 1.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. oligo1 and oligo2 were designed to be short in length, and labeled to recognize biomacromolecules, which could be easily separated from free oligos.

2. The detection sensitivity is higher due to the three times of sequence extension, and the sensitivity of the test detection of the biomacromolecule can reach the level of 0.75 pg/ml.

3. The amplification in the whole process is constant-temperature amplification, the requirement on an instrument is low, the detection can be realized by one constant-temperature device, and the popularization of the technology is facilitated.

Drawings

FIG. 1 shows a schematic flow diagram of the process of the present invention.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions, e.g., Sa mb r ook et al: the conditions described in the laboratory Manual (N e w Y o r k: C o l d S p r i N g H a r o r laboratory Press,1989), or according to the manufacturer' S recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1 activation of antibodies and proximity probes (oligo1 and oligo2)

Before the antibody is labeled with the proximity probe, the antibody and the proximity probe are subjected to corresponding activation treatment, which is specifically as follows:

1) activation treatment of antibody

(1) The antibody was dissolved in PBS buffer (pH8) and the antibody concentration was diluted to 1 mg/ml.

(2) Mu.l of antibody (1mg/ml) + 5. mu.l of TCO-PEG4-NHS (10mM) were mixed well and reacted at room temperature for 30 min.

(3) Adding Tris-HCl buffer solution (1M, Ph8.0) into the system, and stopping the reaction until the final concentration of Tris-HCl is 50-100 mM.

(4) Incubate at room temperature for 5 min.

(5) The product was passed through a column (7K pore size desalting column) to remove free TCO-PEG4-NHS and non-activated antibody, greater than 7K was eluted and collected, and less than 7K remained in the column. The resulting product is the activated antibody.

2) Activation treatment of proximity probes

(1) The proximity probes were dissolved to 100. mu.M with PBS buffer (pH 8).

(2) Mu.l of oligo (100. mu.M) + 5. mu.l of PEG4-NHS (10mM) were mixed well and reacted at room temperature for 30 min.

(3) Adding Tris-HCl buffer solution (1M, pH8.0) into the system, and stopping the reaction, wherein the addition amount is 50-100 mM of the final concentration of Tris-HCl.

(4) Incubate at room temperature for 5 min.

(5) The product was passed through a column (BioRad P-30Columns, 40,000MW limit, eluent PBS) to remove the unactivated oligo and free PEG 4-NHS. More than 40K will be collected by elution and less than 40K will remain in the column. The resulting product is the activated oligo.

Example 2 linking of proximity probes to antibodies after activation

1) Mu.l of activated oligo1 and 50. mu.l of activated antibody were dissolved in PBS buffer (pH8) to 200. mu.l and reacted at room temperature for 1 hour.

2) Purification of the labeled antibody: the purification was performed by Bio-Spin 30Tris Columns (buffer exchanged for PBS), and oligo of less than 30bp was retained in the column after column purification, thereby removing unlabeled oligo.

3) The marker was purified again using a 50K Milipore filter.

4) The final product is labeled with the antibody of the proximity probe (oligo1, oligo 2).

Example 3 detection of Vascular Endothelial Growth Factor (VEGF)

1) VEGF polyclonal antibodies (purchased from Thermo Fisher, LotNumber: P802) were labeled with oligo1 (sequence ACCCGATGGATAGGTCGGTGAA-ACGCAT) and oligo2 (sequence TAATACGACTCACTATAGGGAGA-ATGCGT) in which oligo2 carries the T7 promoter sequence and oligo1 and oligo2 can be linked by 6 base complementary pairings at the respective 3' ends.

2) Detection of VEGF

(1) Formation of double strands of antibody oligo1 and antibody oligo2

VEGF (purchased from Thermo Fisher, Lot Number: PHC9391) was diluted in a 2-fold concentration gradient and subjected to a first sequence extension as follows to give a duplex of oligo1 and oligo 2:

incubate at 37 ℃ for 40 min.

(2) RNA transcription

Transcription was performed according to the following system (transcription system purchased to Thermo Fisher):

1 hour at 37 ℃.

(3) RNA amplification

And (3) amplifying the transcribed small RNA in the step (2) to realize second and third sequence extension, wherein the design of related primers and probes is shown in the table 1.

TABLE 1 second and third sequence extension, related primer and probe design

Small RNAs were amplified according to the following system:

(2) small RNA products of medium transcription: 2 μ l

Buffer (containing oligo3 and its stabilizing sequence,

oligo4 and its stabilizing sequence): 17 μ l

1 μ l of the amplification enzyme mixture (AMV & T7 polymerase & RNaseH)

The reaction was carried out at 42 ℃ for 90min and then examined.

(4) Detecting and analyzing the product in (3) by plate hybridization

Detecting the transcribed small RNA product by a microplate hybridization method, designing a capture probe and a detection probe (a detection probe 1 and a detection probe 2) in a detection system according to the ZC sequence information of the RNA to form a detection probe-RNA product-capture probe compound, wherein the compound can be captured by a section of probe coated in a micropore and is fixed in the micropore. And sequentially adding a section of biotin-labeled probe capable of being combined with the detection probe, streptavidin-HRP enzyme and a chemiluminescent substrate into the micropores, and finally performing luminescence detection. In particular, the method comprises the following steps of,

a. immobilized hybridization of amplification products

Incubate at 42 ℃ for 60 min.

b. The well was discarded and patted dry on clean absorbent paper. Add 200. mu.L of pre-warmed hybridization wash to each well for a total of 3 washes.

c. Add 200. mu.L of blocking solution to each well and shake for 1 min at room temperature.

d. The liquid in the wells was discarded, and 100. mu.L of streptavidin-HRP enzyme conjugate was added to each well and shaken at room temperature for 30 minutes.

e. The well was discarded and patted dry on clean absorbent paper. Add 200. mu.L of pre-warmed hybridization wash to each well for a total of 3 washes.

f. Preparing a substrate solution, wherein a substrate A, a substrate B and a substrate diluent (Tris buffer solution, pH8.5) are mixed according to the proportion of 1: 1: 8 and mixing uniformly.

g. 95 μ L of substrate solution was added to each well and after 5 minutes of incubation, the Relative Light Units (RLU) were measured on a chemiluminescence detector.

h. The experimental results are as follows:

content of VEGF	Dilution ratio	RLU value
			125pg/ml	1:1600	3506542
62.5pg/ml	1:3200	1813211
			31pg/ml	1:6400	1023212
15.5pg/ml	1:12800	609832
			7.5pg/ml	1:25600	423216
3.5pg/ml	1:51200	205432
			1.5pg/ml	1:102400	108844
0.75pg/ml	1:204800	66543
			0.36pg/ml	1:409600	44332
Blank	/	10355

The detection result shows that the detection sensitivity can reach the level of 0.75pg/ml when the detection method detects VEGF.

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Claims (6)

Translated fromChinese

1.一种基于三次延伸-RNA扩增介导的生物大分子免疫分析方法，其特征在于，包括以下步骤：1. a biological macromolecular immunoassay method based on three extension-RNA amplification mediation, is characterized in that, comprises the following steps:1）相关探针及引物的设计：1) Design of related probes and primers:（1）第一和第二邻近探针的设计：oligo1，总长为20-30nt的DNA，一端为标签序列ZC，另一端长为5~6nt序列可以和oligo2末端互补配对；Oligo2，总长为20-30nt的DNA，一端为T7启动子序列，另一端长为5~6nt序列和oligo1末端互补配对；(1) Design of the first and second proximity probes: oligo1, a DNA with a total length of 20-30 nt, with a tag sequence ZC at one end and a sequence of 5-6 nt at the other end, which can be complementary to the end of oligo2; Oligo2, with a total length of 20 -30nt DNA, one end is T7 promoter sequence, the other end is 5~6nt long sequence and oligo1 end complementary pairing;Oligo1和oligo2分别与识别生物大分子的物质偶联，形成识别生物大分子的物质-oligo1、识别生物大分子的物质-oligo2，标记oligo1和oligo2的物质是结合生物大分子的抗体、或具有亲和能力的生物素；由于设计的这对oligo长度较短，为20-30nt，偶联后可以通过简单的离心柱层析法即实现偶联了识别生物大分子的物质的oligo与游离oligo分开；Oligo1 and oligo2 are respectively coupled with substances that recognize biological macromolecules to form substances that recognize biological macromolecules-oligo1 and substances that recognize biological macromolecules-oligo2. The substances labeled oligo1 and oligo2 are antibodies that bind to biological macromolecules, or have an affinity for and the ability of biotin; due to the short length of the designed pair of oligos, 20-30nt, the oligo and free oligo can be separated by a simple spin column chromatography after coupling. ;（2）扩增引物R，命名为oligo3：oligo3有两个部分，3’端与转录的小RNA一半的序列互补配对，即通过10或11核苷酸长度与转录的小RNA的3’端的半个分子杂交；另一端是标签序列ZC1和/或ZC2及T7启动子序列；根据标签序列设计捕获探针和检测探针，用于对后续扩增产物的定性分析，标签序列根据需要进行设计，为1-2条，长度为16~20nt；T7启动子序列是T7 聚合酶识别位点，用于后续的T7聚合酶转录；(2) Amplification primer R, named oligo3: oligo3 has two parts, the 3' end is complementary to the sequence of half of the transcribed small RNA, that is, it is 10 or 11 nucleotides in length with the 3' end of the transcribed small RNA. Half-molecule hybridization; the other end is the tag sequence ZC1 and/or ZC2 and T7 promoter sequence; the capture probe and detection probe are designed according to the tag sequence for qualitative analysis of subsequent amplification products, and the tag sequence is designed as needed , 1-2, with a length of 16~20nt; the T7 promoter sequence is the recognition site of T7 polymerase, which is used for subsequent T7 polymerase transcription;（3）稳定序列1：为了增加oligo3和转录的小RNA杂交体的稳定性，引入稳定序列1，其与标签序列ZC1和/或ZC2以及T7启动子互补配对结合；(3) Stabilizing sequence 1: In order to increase the stability of oligo3 and transcribed small RNA hybrids, a stabilizing sequence 1 was introduced, which is complementary to the tag sequence ZC1 and/or ZC2 and the T7 promoter;（4）扩增引物F，命名为oligo4：与oligo3特征相似，有两个部分，一端与转录的小RNA的5’端的半个分子序列相同，长度为10~11核苷酸，可与转录的小RNA全长的cDNA序列互补配对结合；另一端含有一个标签序列ZC3，该标签序列可以用于后续扩增产物定性检测时捕获探针或检测探针的设计；(4) Amplification primer F, named oligo4: Similar to oligo3, it has two parts, one end is the same as the half molecular sequence of the 5' end of the transcribed small RNA, and the length is 10-11 nucleotides, which can be used with transcription The full-length cDNA sequence of the small RNA is complementary paired; the other end contains a tag sequence ZC3, which can be used for the design of capture probes or detection probes in the qualitative detection of subsequent amplification products;（5）稳定序列2：为了增加oligo4和新合成的oligo3-RNA~cDNA结合的稳定性，引入稳定序列2，其特征为可以和标签序列ZC3互补配对结合；(5) Stable sequence 2: In order to increase the stability of the binding between oligo4 and the newly synthesized oligo3-RNA~cDNA, a stable sequence 2 was introduced, which is characterized by being able to bind to the tag sequence ZC3 in a complementary pairing;2）生物大分子检测2) Detection of biological macromolecules（1）在一个单项反应的试管中识别生物大分子的物质-oligo1和识别生物大分子的物质-oligo2同时结合靶标生物大分子，由于邻位的原因，oligo1和oligo2尾部的6个核苷酸分子会互补配对结合，然后在Klenow 酶的作用下进行延伸，形成一个dsDNA，此为第一次序列延伸；(1) The biomacromolecule-recognizing substance-oligo1 and the biomacromolecule-recognizing substance-oligo2 simultaneously bind the target biomacromolecule in a single reaction test tube. Due to the ortho position, the 6 nucleotides at the tails of oligo1 and oligo2 The molecule will be complementary paired and then extended under the action of Klenow enzyme to form a dsDNA, which is the first sequence extension;（2）步骤（1）形成的dsDNA由于含有T7启动子，在T7聚合酶的作用下转录出小RNA分子，大小为20~25nt；(2) The dsDNA formed in step (1) contains a T7 promoter, and is transcribed into a small RNA molecule with a size of 20-25nt under the action of T7 polymerase;（3）oligo3和步骤（2）转录的小RNA 3’端一半的序列结合，形成小RNA-oligo3复合物，为增加该复合物的稳定性，稳定序列1通过碱基互补配对的原则结合到该复合物上来，最终oligo3、稳定序列1以及小RNA通过融合效应形成一个稳定的oligo3/稳定序列1-RNA复合物，该复合物在逆转录酶的作用下得到小RNA分子全长的cDNA，即为oligo3-RNA~cDNA：RNA复合物，此为第二次序列延伸；(3) oligo3 and the sequence of the 3'-end half of the small RNA transcribed in step (2) are combined to form a small RNA-oligo3 complex. In order to increase the stability of the complex, the stable sequence 1 is combined with the principle of base complementary pairing. The complex comes up, and finally oligo3, stable sequence 1 and small RNA form a stable oligo3/stable sequence 1-RNA complex through fusion effect, and the complex obtains the full-length cDNA of the small RNA molecule under the action of reverse transcriptase, It is oligo3-RNA~cDNA:RNA complex, which is the second sequence extension;（4）步骤（3）oligo3-RNA~cDNA：RNA复合物在RNaseH的作用下，复合物中的RNA会被降解，形成oligo3-RNA~cDNA；(4) Step (3) oligo3-RNA~cDNA: Under the action of RNaseH, the RNA in the complex will be degraded to form oligo3-RNA~cDNA;（5）oligo4会和oligo3-RNA~cDNA互补配对结合，形成oligo3-RNA~cDNA-oligo4复合物；为增加该复合物的稳定性，稳定序列2通过碱基互补配对的原则结合到该复合物上来，最终Oligo4、稳定序列2、稳定序列1以及oligo3合成的oligo3-RNA~cDNA通过融合效应形成一个稳定的复合物，即为oligo3-RNA~cDNA-oligo4/稳定序列2复合物；该复合物在逆转录酶的作用下得到含有T7启动子序列、标签序列以及小RNA分子全长的双链DNA，即oligo3-RNA~cDNA-oligo4双链DNA，此为第三次序列延伸；(5) oligo4 will combine with oligo3-RNA~cDNA complementary pairing to form an oligo3-RNA~cDNA-oligo4 complex; in order to increase the stability of the complex, stable sequence 2 is bound to the complex by the principle of base complementary pairing Finally, the oligo3-RNA~cDNA synthesized by Oligo4, stable sequence 2, stable sequence 1 and oligo3 forms a stable complex through fusion effect, that is, the oligo3-RNA~cDNA-oligo4/stable sequence 2 complex; this complex Under the action of reverse transcriptase, double-stranded DNA containing T7 promoter sequence, tag sequence and full-length small RNA molecule is obtained, that is, oligo3-RNA~cDNA-oligo4 double-stranded DNA, which is the third sequence extension;（6）在（5）中形成的oligo3-RNA~cDNA-oligo4双链DNA序列结构为T7 启动子-ZC2-ZC1-RNA~cDNA-ZC3，该双链DNA通过TMA 或 NASBA方法进行扩增，得到一段序列为ZC2-ZC1-RNA~cDNA-ZC3的RNA分子；(6) The oligo3-RNA~cDNA-oligo4 double-stranded DNA sequence structure formed in (5) is T7 promoter-ZC2-ZC1-RNA~cDNA-ZC3, the double-stranded DNA is amplified by TMA or NASBA method, An RNA molecule whose sequence is ZC2-ZC1-RNA~cDNA-ZC3 is obtained;（7）检测所述RNA 的扩增产物的存在与否和/或数量，由此判断待检生物大分子的存在与否和/或数量。(7) Detecting the presence and/or quantity of the amplification product of the RNA, thereby judging the presence or absence and/or quantity of the biological macromolecule to be tested.2.如权利要求1所述的方法，其特征在于，步骤2）（7）中的检测结果为定性或定量结果。2 . The method of claim 1 , wherein the detection result in step 2) (7) is a qualitative or quantitative result. 3 .3.如权利要求1所述的方法，其特征在于，步骤2）（7）中检测所述RNA 的扩增产物的方法包括分子信标、平板杂交-信号放大、或者核酸胶体金检测。3 . The method of claim 1 , wherein the method for detecting the amplification product of the RNA in step 2) (7) comprises molecular beacons, plate hybridization-signal amplification, or nucleic acid colloidal gold detection. 4 .4.如权利要求3所述的方法，其特征在于，步骤2）（7）中检测所述RNA 的扩增产物的方法为平板杂交-信号放大检测，具体为根据标签序列ZC3设计捕获探针，该捕获探针和ZC3互补配对结合，进而将扩增的RNA分子固定在微孔板中或其他固相载体上；再根据标签序列ZC1和/或ZC2设计检测探针，该检测探针和ZC1和/或ZC2碱基互补配对结合，检测探针标记上生物素，进而将生物素标记到扩增出的RNA分子上，后续依次添加链霉亲和素-HRP酶、化学发光底物，最终通过化学发光的方法检测扩增产物的有无或数量，进而判断待检生物大分子的检出情况。4. The method of claim 3, wherein the method for detecting the amplification product of the RNA in step 2) (7) is plate hybridization-signal amplification detection, specifically designing a capture probe according to the tag sequence ZC3 , the capture probe and ZC3 are complementary paired, and then the amplified RNA molecules are immobilized in a microplate or on other solid-phase carriers; then a detection probe is designed according to the tag sequence ZC1 and/or ZC2, and the detection probe and ZC1 and/or ZC2 are combined with base complementary pairing, the detection probe is labeled with biotin, and then biotin is labeled on the amplified RNA molecule, followed by adding streptavidin-HRP enzyme and chemiluminescent substrate in sequence, Finally, the presence or quantity of the amplification product is detected by the method of chemiluminescence, and then the detection situation of the biological macromolecule to be detected is judged.5.如权利要求1-4任一项所述的方法，其特征在于，所述的生物大分子是指通过特异性作用而相互识别的物质。5. The method of any one of claims 1-4, wherein the biological macromolecules refer to substances that recognize each other through specific interactions.6.如权利要求5所述的方法，其特征在于，所述的通过特异性作用而相互识别的物质为抗体与抗原、受体与配体、酶与底物、凝集素与多糖中的任意一种。6. The method according to claim 5, characterized in that, the substances recognized by specific interaction are any of antibodies and antigens, receptors and ligands, enzymes and substrates, lectins and polysaccharides. A sort of.

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