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CN-121992080-A - Novel multiplex microRNA detection method based on RNA fluorescent aptamer and exponential amplification reaction

CN121992080ACN 121992080 ACN121992080 ACN 121992080ACN-121992080-A

Abstract

The invention belongs to the technical field of biological detection, and discloses a novel method for detecting multiple microRNAs based on RNA fluorescent aptamer and exponential amplification reaction. The method integrates efficient amplification of EXPAR and specific signal transduction of RNA fluorescent aptamer, and constructs a FLAP-EXPAR detection system. The target microRNA triggers EXPAR reaction, a short DNA trigger chain is exponentially generated, and then RNA fluorescent aptamer is driven to be transcribed, and a transcription product is combined with a specific fluorescent group to generate a target-dependent fluorescent signal. The single tube synchronous detection of multiple microRNAs is realized by designing a dedicated combination of an amplification template, a transcription template, a fluorescent aptamer and a fluorescent group. The platform has high sensitivity, strong specificity and simple operation, has the detection limit on miR-21 as low as 0.12 pM, has the linear range of 1 pM-50 nM, has good stability in complex matrixes such as serum, cell lysate and the like, can be widely applied to clinical scenes such as early diagnosis, prognosis evaluation and the like of cancers, provides an accurate detection tool for tumor molecular typing, and has remarkable clinical conversion value and market application prospect.

Inventors

  • XIE GUOMING
  • HE YU
  • ZHANG YIQI
  • ZHANG LI

Assignees

  • 重庆医科大学国际体外诊断研究院

Dates

Publication Date
20260508
Application Date
20260305

Claims (6)

  1. 1. The novel method for detecting the multiplex microRNA based on the RNA fluorescent aptamer and the exponential amplification reaction is characterized by comprising an amplification template, a transcription template, an enzyme mixed solution and a fluorescent group: (1) The amplification template comprises a target microRNA recognition region and a nicking enzyme recognition site, wherein the sequence structure of the amplification template is X ' -N-X ', X ' is a sequence complementary to the target microRNA, and N is a recognition site of Nt.BsmAI nicking enzyme; (2) The transcription template comprises a complementary recognition sequence, a T7 promoter region and an RNA fluorescent aptamer coding sequence, wherein the sequence structure is X' -T-B, T is the T7 promoter sequence, and B is the RNA fluorescent aptamer coding sequence; (3) The enzyme mixture comprises Klenow fragment polymerase (3 '. Fwdarw.5' exo-), nt.BsmAI nickase, T7 RNA polymerase and ribonuclease inhibitor; (4) The fluorescent group is a small molecular substance specifically combined with the RNA fluorescent aptamer and comprises one or more of DFHBI-1T, malachite green and HBC 620.
  2. 2. The method of claim 1, wherein the RNA fluorogenic aptamer comprises one or more of Broccoli, MGA, pepper, different RNA fluorogenic aptamers corresponding to different target micrornas and specific fluorophores.
  3. 3. A multiplex microRNA detection method based on the detection method of claim 1, comprising the steps of: (1) Preparing a reaction system, namely preparing a reaction mixed solution and an enzyme mixed solution on ice, wherein the reaction mixed solution comprises an amplification template, a target sample, dNTPs, DTT, mgSO 4 , rCutSmart buffer solution and RNase-free water, and the enzyme mixed solution comprises a transcription template, NTP, a fluorescent group, klenow fragment polymerase, nt.BsmAI nickase, T7 RNA polymerase, ribonuclease inhibitor and RNase-free water; (2) One-pot reaction, namely, rapidly mixing the reaction mixed solution with the enzyme mixed solution, and incubating for 90-120 min at 37 ℃; (3) And (3) fluorescence signal detection, namely acquiring fluorescence signals in detection channels corresponding to the fluorescent groups by adopting a real-time fluorescence quantitative PCR instrument, and realizing qualitative and quantitative analysis of the target microRNA according to the change of the fluorescence signals.
  4. 4. The method according to claim 3, wherein the final concentration parameters of the reaction system in the step (1) are amplification template concentration 5 nM, transcription template concentration 20 nM, dNTPs concentration 1 mM, mgSO 4 concentration 15 mM, klenow fragment polymerase concentration 0.05U/. Mu.L, nt.BsmaI nickase concentration 0.2U/. Mu. L, T7 RNA polymerase concentration 1.5U/. Mu.L, ribonuclease inhibitor concentration 1U/. Mu.L, and fluorophore concentration 25. Mu.M.
  5. 5. A method according to claim 3, wherein the incubation time in step (2) is preferably 100 min, which is sufficient to ensure that the EXPAR amplification reaction and the downstream transcription reaction proceed.
  6. 6. The method according to claim 3, wherein the multiplex micrornas in step (3) are detected by assigning specific combinations of "amplified template-transcribed template-RNA fluorogenic aptamer-fluorophore" to different target micrornas, the different combinations corresponding to different fluorescent detection channels, with no apparent signal crosstalk between the channels.

Description

Novel multiplex microRNA detection method based on RNA fluorescent aptamer and exponential amplification reaction Technical Field The invention belongs to the technical field of biomedical detection, and particularly relates to a multiplex microRNA detection method based on an RNA fluorescent aptamer and an exponential amplification reaction (EXPAR), which is particularly suitable for high-sensitivity and high-specificity detection of breast cancer related microRNA. Background MicroRNA (miRNA) is an endogenous non-coding single-stranded RNA molecule with the length of about 18-23 nucleotides, and is widely involved in physiological processes such as cell proliferation, differentiation, apoptosis and the like, and the abnormal expression of MicroRNA (miRNA) is closely related to the occurrence and development of diseases such as cancers and the like, and is an important biomarker for early diagnosis and prognosis evaluation of the cancers. However, miRNA has the characteristics of small molecular weight, high sequence homology, low abundance and poor stability, and brings great challenges to sensitive and specific detection. The traditional detection methods such as Northern blot hybridization, real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR), gene chip technology and the like have the defects of complex operation, high instrument requirement, insufficient specificity, background signal interference and the like. Isothermal amplification technology has the advantages of high amplification efficiency, mild reaction conditions, simple operation and the like, and becomes an important technical means for miRNA detection. Exponential amplification reaction (EXPAR) is a typical isothermal amplification technology, and target nucleic acid exponential amplification is realized through nicking enzyme cleavage and polymerase chain displacement reaction, but a traditional EXPAR detection system adopts SYBR Green I and other nonspecific fluorescent dyes, so that the problems of high background fluorescence and low signal-to-noise ratio exist, and the detection sensitivity and accuracy are seriously affected. The RNA fluorescent aptamer is a short RNA sequence with a specific three-dimensional structure, can be combined with a specific small molecule fluorescent group and activate fluorescence, has target dependence of a fluorescent signal, and can effectively reduce background noise. The RNA fluorescent aptamer is combined with the EXPAR technology, so that inherent defects of a traditional EXPAR detection system are hopeful to be overcome, and high-sensitivity and high-specificity detection of miRNA is realized. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides a multiplex microRNA detection method (shown in figure 1) based on an RNA fluorescent aptamer and an EXPAR, and the method combines the efficient amplification capability of the EXPAR and the specific signal transduction capability of the RNA fluorescent aptamer to construct a FLAP-EXPAR detection system, so that single-tube synchronous detection of various miRNAs can be realized, and the method has the characteristics of high sensitivity, strong specificity and simplicity and convenience in operation. 1. Composition of the detection method The detection method comprises four core parts of an amplification template, a transcription template, enzyme mixed solution and a fluorescent group, and the functions and structures of the four core parts are as follows: the amplification template has a sequence structure of X ' -N-X ', wherein X ' is a recognition sequence complementary with the target miRNA and can specifically bind with the target miRNA, and N is a recognition site of Nt.BsmAI incision enzyme and is used for starting EXPAR amplification reaction. The transcription template has sequence structure of X '-T-B, X' complementary to EXPAR amplification product, T being T7 RNA polymerase recognized promoter sequence, and B being RNA fluorescent aptamer encoding sequence for transcription to produce specific RNA fluorescent aptamer. Enzyme mixture comprising Klenow fragment polymerase, nt.BsmaI nicking enzyme, T7 RNA polymerase and ribonuclease inhibitor. Klenow fragment polymerase is used for DNA chain extension, nt.BsmAI nickase is used for cutting DNA double chain to generate new amplification primer, T7 RNA polymerase drives RNA fluorescent aptamer transcription, and ribonuclease inhibitor prevents RNA fluorescent aptamer from degradation. Fluorescent groups, namely small molecular substances which are specifically combined with the RNA fluorescent aptamer, generate fluorescence only after combination, effectively reduce background noise, and preferably correspond to Broccoli, MGA, pepper RNA fluorescent aptamers respectively, namely DFHBI-1T, malachite green and HBC 620. 2. The detection method comprises the steps of The detection method of the invention is a one-pot r