MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease

An exponential amplification and reaction system technology, applied in the field of microRNA detection, can solve the problems of serious false positives, low abundance, and low detection efficiency, and achieve the effect of improving diagnostic accuracy and sensitivity

Pending Publication Date: 2022-03-29
FUDAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0007] (1) There are many types of miRNAs, low abundance and short fragments, so it is still a technical problem to detect miRNAs in biological samples
EXPAR technology does not require the reverse transcription process, and can directly amplify miRNA for signal amplification and detection, but its false positive phenomenon is serious, and its sensitivity is limited, so it is difficult to realize the application in clinical detection (Angewandte Chemie International Edition 49.32 (2010): 5498-5501 )
EXPAR technology can complete the detection of a certain miRNA in about 30 minutes at a constant temperature of 55°C without the need for reverse transcription, but its amplification specificity is poor, false positives are prone to occur, and it is difficult for EXPAR to distinguish miRNA family members (single base base or polybase differences)
On the other hand, EXPAR cannot realize simultaneous detection of multiple targets in one reaction system
[0008] (2) The occurrence of a disease such as cancer is closely related to the dysregulation of multiple miRNAs, and most of the current methods are limited to the detection of a single miRNA target at a time, the detection efficiency is low, and it is difficult to meet the clinical needs
Currently, there is a lack of methods for multiple detection of miRNA with high specificity and high sensitivity
In addition, there are single / polynucleotide polymorphisms among miRNA family members, and often a specific member miRNA is closely related to certain diseases. The current technology has not yet achieved simple, fast, low-cost, and high-sensitivity Highly specific miRNA typing analysis
[0009] (3) The sensitivity of CRISPR / Cas technology to detect miRNA can reach the cellular level, which is difficult to be used in actual clinical blood sample detection (Anal.Chem.2019, 91, 5278-5285)
CRISPR / Cas technology coupled with catalytic hairpin assembly (Catalytic Hairpin Assembly, CHA) nucleic acid amplification can detect sub-femtomolar (sub-femtomolar) level microRNA, but it is still unable to achieve multiple detection in the same system (Chem.Sci., 2020, 11,7362)
The use of CRISPR / Cas technology for miRNA typing analysis requires a variety of Cas, which is difficult, high cost, and does not have detection advantages

Method used

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  • MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease
  • MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease
  • MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease

Examples

Experimental program
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Effect test

Embodiment 1

[0107] Example 1: High specificity detection capability of TtAgo single base resolution

[0108] This example explores the cleavage specificity of TtAgo. Firstly, miDNA-21 and single base mismatched miDNA-21 (as shown in Table 2), and mock probes (ie, detection probes), as shown in Table 3, were synthesized.

[0109] Among them, miDNA-21 is 100% complementary to the simulated probe, and there is a single base mismatch between miDNA-21 and the simulated probe. Use miDNA-21 to simulate the EXPAR pre-amplification product of miRNA-21, the detection system based on TtAgo nuclease contains miDNA-21 (5μM) or single base mismatched miDNA-21 (5μM), mock probe (1μM) , TtAgo (100nM), Reaction buffer, MnCl 2 (750μM) and ultrapure water. The reaction temperature of the detection system based on TtAgo nuclease is 80° C.; the reaction time is 30 minutes. In the detection system based on TtAgo nuclease, the cleavage product (short-chain nucleic acid) of TtAgo nuclease is verified by 15...

Embodiment 2

[0113] Embodiment 2: the sensitivity of the miRNA detection method based on EXPAR and TtAgo system

[0114] This example explores the sensitivity of the miRNA detection method based on EXPAR and TtAgo nuclease.

[0115] Taking miRNA-21 as an example, a series of concentrations of miRNA (as a nucleic acid sample) (0, 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM) for pre-amplification, the reaction conditions are 55°C, 20min. The detection system based on TtAgo nuclease contains preamplification product (1μL), detection probe (1μM), TtAgo (100nM), Reaction buffer, MnCl 2 (750μM) and ultrapure water. The reaction temperature of the detection system based on TtAgo nuclease is 80° C.; the reaction time is 30 minutes. Test results such as Figure 5 As shown in A, the detection sensitivity reaches 1aM (10 -18 M)( Figure 5 in B).

[0116] In this example, the sensitivity of the miRNA detection method based on EXPAR and TtAgo nuclease was further compared w...

Embodiment 3

[0117] Embodiment 3: The miRNA detection method based on EXPAR and TtAgo nuclease detects multiple miRNA

[0118] This example explores the ability of the miRNA detection method based on EXPAR and TtAgo nuclease of the present invention to detect multiple miRNAs.

[0119] Taking the four target miRNAs of miRNA-21, miRNA-92a, miRNA-31 and miRNA-14 as examples, the detection process is divided into two steps, first based on the EXPAR pre-amplification system described in "Materials and Methods" , in the EXPAR pre-amplification system, add the amplification templates corresponding to four miRNAs and the corresponding four target miRNAs (as nucleic acid samples), the target miRNA concentration is 100pM, these four target miRNAs in the same reaction tube At the same time, pre-amplification was carried out, and the reaction conditions were 55° C. for 20 min. Secondly, the miRNA detection system based on TtAgo nuclease includes detection probes corresponding to four amplicons and EX...

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Abstract

The invention discloses a microRNA (Ribonucleic Acid) detection system and a microRNA detection method based on an exponential amplification reaction and Argonaute nuclease. The invention provides a microRNA (Ribonucleic Acid) detection system based on exponential amplification reaction and Argonaute nuclease, and the microRNA detection system comprises: (a) guide DNA (Deoxyribose Nucleic Acid), which is an amplification product obtained by amplifying a target object microRNA by adopting an exponential amplification reaction system; (b) an Argonaute nuclease, and a method for preparing the same; (c) the detection probe is provided with a fluorophore and a quenching group, and the detection probe comprises a region complementary to an amplification product obtained by amplifying the target microRNA by adopting the index amplification reaction system. According to the microRNA detection system provided by the invention, miRNA detection with single base specificity and high sensitivity as well as multiple miRNA detection and miRNA typing analysis are realized.

Description

technical field [0001] The invention relates to the technical field of microRNA detection, in particular to a microRNA detection system and method based on exponential amplification reaction and Argonaute nuclease. Background technique [0002] Mature microRNA (miRNA) is a kind of single-stranded non-coding RNA molecule with a length of 18-28 bases, which plays an important role in various life activities such as the immune system, cell growth and development, proliferation and differentiation, and apoptosis. Regulatory effect. miRNA has been widely concerned by researchers in various fields such as development, biology, aging and metabolism, disease pathology research, and disease treatment. miRNA is transcribed from some DNA into RNA and processed. It can bind to mRNA, thereby inhibiting gene expression after transcription. The abnormal level of miRNA is closely related to many diseases. For example, the upregulation of miRNA-21, miRNA-92a, miRNA-31 and miRNA-141 levels ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6883
CPCC12Q1/6851C12Q1/6886C12Q2600/178C12Q2521/301C12Q2531/113C12Q2563/107C12Q2525/301C12Q2525/207C12Q2527/127C12Q2537/143
Inventor 陈惠林秋媛孔继烈
Owner FUDAN UNIV
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