Multiplex nucleic acid visual detection method and kit based on isothermal cascade amplification and cell-free transcription fluorescent RNA aptamer and application

Abstract

The application discloses a multiple nucleic acid visual detection method and kit based on isothermal cascade amplification and cell-free transcription fluorescent RNA aptamer and application, and provides a multifunctional probe which comprises a target complementary recognition sequence, a nicking enzyme recognition site sequence, a fluorescent RNA aptamer coding sequence and a promoter template sequence. When nucleic acid targets exist, the target triggers the extension of the probe to form a nick structure, and under the action of a nicking enzyme and a strand displacement polymerase, the probe is cyclically amplified to generate a large amount of transcription template containing a promoter and an aptamer coding region; then, the RNA polymerase transcribes the transcription template to generate fluorescent RNA aptamer, the aptamer is combined with corresponding fluorescent ligand to generate specific wavelength fluorescent signal, and nucleic acid target detection is realized. By changing the probe recognition sequence and the aptamer coding sequence, multiple detection and multicolor visual interpretation of different nucleic acid targets can be realized, and the application is suitable for on-site rapid detection and multiple target typing.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics and bioanalysis technology, specifically to a method for visualizing multiplex nucleic acid detection based on isothermal cascade amplification and cell-free transcription of fluorescent RNA aptamers, a kit for implementing the detection method, and its application in the detection of bacterial resistance genes and virulence genes. Background Technology

[0002] Nucleic acid testing plays a vital role in the early diagnosis, drug resistance monitoring, and virulence typing of infectious diseases. Current multiplex nucleic acid testing methods often rely on fluorescent probes / complex instruments or multiple rounds of operation, making it difficult to simultaneously meet the following requirements: 1) high sensitivity; 2) multiplex detection capability; 3) readability by the naked eye / simple devices; and 4) rapid on-site testing with low equipment dependence.

[0003] On the other hand, fluorescent RNA aptamers can bind to specific fluorescent ligands to generate signals and have programmability and multicolor output potential, but their detection sensitivity is often limited by template generation efficiency, background and crosstalk control, and transcription output scale. Current technologies still require a platform that can effectively couple efficient isothermal amplification with fluorescent RNA aptamer transcription output and support multi-channel visualization readout. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide a method for visual detection of multiplex nucleic acids based on isothermal cascade amplification and cell-free transcription of fluorescent RNA aptamers. The present invention provides a MAGIC detection platform that generates a large number of "T7 promoter and fluorescent RNA aptamer template strands" through target-triggered isothermal exponential amplification. After binding with the T7 promoter coding strand, cell-free transcription generates fluorescent RNA aptamers of different colors, achieving highly sensitive, multiplex, and visual nucleic acid detection. Furthermore, by replacing the target recognition region of the probe and the coding region of the fluorescent RNA aptamer, it can be extended to different nucleic acid targets. A second objective of the present invention is to provide a kit for implementing the aforementioned detection method. A third objective of the present invention is to provide the application of the aforementioned detection method in the detection, typing, and rapid on-site detection of pathogen nucleic acids, drug resistance genes, and / or virulence genes. A fourth objective of the present invention is to provide the application of the aforementioned kit in typing Klebsiella pneumoniae based on drug resistance genes and / or virulence genes.

[0005] To achieve the above objectives, the present invention provides the following technical solution: 1. A method for visualizing and detecting multiplex nucleic acids based on isothermal cascade amplification and cell-free transcription fluorescent RNA aptamers, comprising the following steps: (1) Provide at least two multifunctional probes for different targets. The multifunctional probe is a single-stranded DNA and includes, from the 5′ end to the 3′ end, a T7 promoter template sequence, a fluorescent RNA aptamer coding sequence, a nicking endonuclease recognition site, and a target nucleic acid complementary recognition sequence. Different probes can achieve multiplex nucleic acid detection by configuring complementary recognition sequences for different target nucleic acids and different fluorescent RNA aptamer coding sequences. (2) The multifunctional probe is brought into contact with the nucleic acid sample to be tested under isothermal conditions. In the presence of target nucleic acid, the target nucleic acid hybridizes with the complementary recognition sequence of the probe to form a double-stranded structure. Under the action of DNA polymerase, it extends along the probe to form a double-stranded DNA containing a nicking endonuclease recognition site. Subsequently, the nicking endonuclease produces a single-stranded nick on the new strand. The DNA polymerase continues to extend from the nick and strand replacement occurs. The above nick-extension-replacement process is repeated, thereby generating a large number of template strands of T7 promoter and fluorescent RNA aptamer exponentially without changing the reaction temperature. (3) The template strand generated in step (3) combines with the T7 promoter coding strand to form a promoter double-stranded structure that can be recognized by RNA polymerase. Cell-free in vitro transcription is performed under the action of RNA polymerase to generate the transcription product fluorescent RNA aptamer. (4) The transcription product is bound to the corresponding fluorescent ligand, and the fluorescence signal is observed by the naked eye or simple optical equipment under excitation light to realize the visual detection of the target nucleic acid.

[0006] In some embodiments of the present invention, in step (1), the sequence of the multifunctional probe is selected from any one or more of the sequences shown in SEQ ID NO.11 to SEQ ID NO.26.

[0007] In some embodiments of the present invention, in step (1), the fluorescent RNA aptamer is selected from at least one of Broccoli, Chili or Malachite Green Aptamer.

[0008] In some embodiments of the present invention, in step (2), the isothermal condition is 37°C.

[0009] In some embodiments of the present invention, in step (3), the sequence of the T7 promoter coding chain is shown in SEQ ID NO.1.

[0010] 2. A kit for implementing the detection method of the claim, comprising the following components: (1) Multifunctional probe: Its nucleotide sequence is selected from any one or more of the sequences shown in SEQ ID NO.10 to SEQ ID NO.24; (2) Reaction components for isothermal exponential amplification: including DNA polymerase Klenow Fragment, nicking endonuclease Nb.BbvCI, deoxyribonucleotide substrate and reaction buffer; (3) Cell-free in vitro transcription reaction components: including the T7 promoter coding strand, T7 RNA polymerase, and ribonucleotide substrate as shown in SEQ ID NO.1; (4) Fluorescent ligands: including at least one of DFHBI-1T, DMHBO+ or Malachite Green.

[0011] 3. The application of the detection method in the detection, typing and rapid on-site detection of pathogen nucleic acids, drug resistance genes and / or virulence genes.

[0012] 4. Application of the kit in typing Klebsiella pneumoniae based on drug resistance genes and / or virulence genes, wherein the drug resistance gene is KPC or NDM, and the virulence gene is iucA, rmpA, iroN, or peg 344.

[0013] The beneficial effects of this invention are as follows: Compared with the prior art, the nucleic acid detection system of this invention has the following significant advantages: 1) High sensitivity: Through isothermal exponential amplification and cascade transcription reaction, the signal can be effectively amplified, improving detection sensitivity; 2) Multiplex detection: Different fluorescent RNA aptamers correspond to different fluorescent ligands. By selecting different aptamers and probe sequences, multiple targets can be detected simultaneously. 3) Low equipment dependence and visual inspection: The system can be observed with the naked eye using a simple light source (such as blue light), making it suitable for rapid on-site inspection; 4) High scalability: By changing the recognition sequence and aptamer coding region of the probe, it can be quickly adjusted to adapt to different nucleic acid targets. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 This is a technical schematic diagram of the present invention (A: schematic diagram of exponential amplification reaction; B: schematic diagram of cascade transcription of fluorescent aptamers; C: generation of multiple fluorescent aptamers through isothermal cascade amplification).

[0015] Figure 2 For multiplex nucleic acid detection (A: fluorescent group structure; BD: fluorescence response of three fluorescence systems in different analytes; EG: fluorescence kinetic time curves of the MAGIC system targeting different genes; H: multiplex detection results of three genes simultaneously detected by orthogonal fluorescence system).

[0016] Figure 3 The performance evaluation of the MAGIC method was conducted as follows: (A: Real-time fluorescence curves obtained by MAGIC method for different target concentrations; B: Endpoint fluorescence intensity corresponding to different target concentrations; C: Fluorescence response of the MAGIC detection system targeting the iucA gene to different targets; D: Cross-reactivity evaluation of the MAGIC detection system; Six MAGIC probes designed for iucA, rmpA, iroN, peg344, KPC and NDM genes were cross-validated with their corresponding targets).

[0017] Figure 4 The results of this method for early detection of Klebsiella pneumoniae drug resistance genes are as follows: (A: Comparison of the speed of MAGIC technology and standard clinical methods for detecting carbapenem resistance in Klebsiella pneumoniae; B: Dynamic changes in fluorescence signal of KPC gene detected by MAGIC within 0-14 h of simulated infection; C: qPCR expression detection results of KPC gene in Klebsiella pneumoniae from 0-14 h post-infection; D: Agarose gel electrophoresis images of KPC gene amplification products from 24 Klebsiella pneumoniae isolates; E: qPCR detection results of KPC drug resistance genes in 24 clinical isolates; F: Performance comparison of MAGIC and qPCR for detecting KPC genes in 24 strains).

[0018] Figure 5 The results of this method for dual detection of virulence and drug resistance genes in clinical strains are shown below: (A: Schematic diagram of the principle of simultaneous detection of virulence and drug resistance genes in Klebsiella pneumoniae using orthogonal MAGIC; B: Fluorescence intensity and representative images of Broccoli and Chili reporter systems under four detection conditions; C: Fluorescence signal results of 12 carbapenem-resistant highly virulent Klebsiella pneumoniae strains after 2 hours of MAGIC reaction; D: Fluorescence signal results of 20 carbapenem-sensitive Klebsiella pneumoniae strains after 2 hours of MAGIC reaction; CR-hvKP: carbapenem-resistant highly virulent Klebsiella pneumoniae; hvKP: highly virulent Klebsiella pneumoniae; CRKP: carbapenem-resistant Klebsiella pneumoniae; NTC: non-drug-resistant, weakly virulent Klebsiella pneumoniae). Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention. Example 1. Composition and Multifunctional Probe Structure of the MAGIC Nucleic Acid Detection System

[0020] This embodiment provides a nucleic acid detection scheme that integrates target recognition, exponential amplification, and transcription template generation into a single probe. Its core lies in the structural design of a single multifunctional probe, which allows target recognition events to be directly converted into a fluorescent signal source that can be amplified through in vitro transcription. This structurally distinguishes it from traditional detection systems that separate the recognition, amplification, and reporter modules. The multifunctional probe is a single-stranded DNA, comprising, from the 5′ to the 3′ end, a T7 promoter template sequence, a fluorescent RNA aptamer coding sequence, an Nb.BbvCI nick endonuclease recognition site, and a target nucleic acid complementary recognition sequence. When the target is absent, the probe does not form a complete double-stranded structure and cannot trigger subsequent amplification and transcription processes. However, when the target is present, the target nucleic acid itself acts as a structural trigger factor, hybridizing with the probe to transform it into a functional nucleic acid that can be both exponentially amplified and used as an in vitro transcription template. This achieves direct coupling between target recognition and the signal amplification initiation point. Furthermore, the probe itself is the transcription template, eliminating the need for additional intermediate template construction and laying the structural foundation for subsequent exponential amplification and cascade transcription reactions. Figure 1 ). Example 2. A method for detecting the "synchronous generation" of target-triggered isothermal exponential amplification and transcription template.

[0021] This embodiment provides a detection method for target-triggered isothermal exponential amplification and simultaneous generation of transcription templates. It illustrates that MAGIC does not simply perform transcription after amplification, but rather generates a transcription template carrying the coding sequence of the fluorescent RNA aptamer exponentially under isothermal conditions through a cyclic mechanism combining nick endonucleation and strand displacement. This is how it achieves attomorlar-level (10^6) transcription. -18 The key to ultra-high sensitivity detection (mol / L) lies in the following: Specifically, at 37°C, a multifunctional probe, Nb.BbvCI nicking endonuclease, Klenow fragments with strand displacement capability, dNTPs, buffer, and the target nucleic acid are added to the reaction system. After hybridization between the target and the probe, the polymerase extends along the probe to form double-stranded DNA containing the Nb.BbvCI recognition site. Subsequently, Nb.BbvCI creates a single-strand nick on the nascent strand, and the polymerase continues to extend from the nick and undergo strand displacement. This nick-extension-displacement process is repeated cyclically, thereby exponentially generating a large amount of single-stranded DNA without changing the reaction temperature. This amplified product simultaneously encodes the T7 promoter and fluorescent RNA aptamer sequence, which can be directly used as a template for subsequent in vitro transcription, avoiding signal loss caused by intermediate conversion and forming a cascade amplification system for amplification and transcription. Figure 1 A). Example 3. An example of multiplex nucleic acid visualization detection based on multicolor fluorescent RNA aptamers.

[0022] This embodiment provides a multiplex nucleic acid visualization detection scheme based on multicolor fluorescent RNA aptamers, illustrating that the MAGIC system achieves true multiplex detection by "transing different RNA aptamers" rather than "distinguishing different amplification probes", thereby enabling intuitive differentiation of multiple targets without the need for complex optical systems. Taking the detection of the virulence genes iucA, rmpA, and iroN of Klebsiella pneumoniae (KP) as an example, three MAGIC probes targeting different genes—Broccoli, Chili, and MGA (SEQ ID NO.11, SEQ ID NO.18, SEQ ID NO.24)—were simultaneously added to the same reaction system. Each probe shared the same isothermal amplification and transcription system. After amplification, corresponding transcription templates were generated. Following the addition of the T7 promoter coding strand shown in SEQ ID NO.1, T7 RNA polymerase, rNTP mixture, and related buffer systems, in vitro transcription was performed at 37 ℃. Different transcription products formed RNA aptamers with specific fluorescent properties. Under single blue light excitation, these RNA aptamers, after binding to their respective ligands (DFHBI-1T, DMHBO+, MG), emitted green, orange, and red fluorescent signals, allowing for visual differentiation of the presence of different targets. Furthermore, there was no significant crosstalk between the different fluorescent channels, thus achieving multi-target detection in a single tube and color-coded identification. Figure 2 ). Example 4. Sensitivity and Specificity Validation of the MAGIC Method

[0023] In this embodiment, real-time fluorescence monitoring revealed a significant concentration-dependent change in fluorescence signal with increasing target nucleic acid concentration; high target concentrations significantly accelerated the amplification reaction kinetics. Analysis of the fluorescence signal at the reaction endpoint showed a good linear correlation between fluorescence intensity and target nucleic acid concentration across multiple orders of magnitude. Taking the artificially synthesized KPC target (SEQ ID NO.8) as an example, its detection limit reached 10 aM, demonstrating a sensitivity superior to most nucleic acid detection methods based on fluorescent RNA aptamer signal output. Figure 3 , AB). Meanwhile, validation of the detection specificity revealed that the probe designed for the iucA gene only produced a significant fluorescent signal in the presence of its perfectly complementary target sequence, while showing almost no response to non-complementary target sequences. Figure 3(C); further, cross-reactivity experiments were conducted using six probes (SEQ ID NO. 11-26) targeting iucA, rmpA, iroN, peg344, KPC, and NDM. Each probe produced a specific fluorescence signal only for its corresponding target, and no significant cross-interference was observed between different targets. This indicates that the method of the present invention achieves ultra-high sensitivity detection while possessing excellent specificity and reliability under multi-target detection conditions. Figure 3 D). Example 5. Application of the MAGIC method in the early combined detection of clinical drug resistance and virulence genes.

[0024] This embodiment demonstrates the specific application of the MAGIC method in the early combined detection of clinical drug resistance genes and virulence genes, aiming to illustrate that this method can achieve rapid and visual determination of key clinical phenotypes without the need for strain culture or complex DNA purification steps. In the specific implementation process, only a single colony of the clinically isolated strain needs to be taken, resuspended in nuclease-free water, and then lysed by boiling water and centrifugation. The supernatant can be directly added to the MAGIC detection template in the reaction system. In the same reaction tube, the Broccoli channel is set for the detection of virulence genes (such as iucA, rmpA, iroN, and peg344), and the Chili channel is set for the detection of carbapenem resistance genes (such as KPC or NDM). After the reaction, the results are interpreted based on the fluorescence color, where green fluorescence indicates a positive virulence gene, orange fluorescence indicates a positive drug resistance gene, and the simultaneous appearance of both colors indicates that the strain has a combined phenotype of drug resistance and high virulence. Based on oligonucleotide sequences (Table 1), this invention designs probes for different targets and aptamers (Table 2). Figure 4 This paper presents the results of early detection of the carbapenem resistance gene KPC in Klebsiella pneumoniae (KP) using this method. Figure 5 The results demonstrate the effectiveness of this method in achieving dual detection of virulence and drug resistance genes in clinical strains. Experimental results show that the detection conclusions of this method are completely consistent with qPCR results, and the overall detection time is shortened by about 46 hours compared with the traditional drug sensitivity and typing process. It has significant clinical application value and is suitable for point-of-care testing and rapid typing analysis.

[0025] Table 1. Oligonucleotide sequences used in this invention Table 2. Probes designed for different targets and aptamers The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for visualizing and detecting multiplex nucleic acids based on isothermal cascade amplification and cell-free transcription fluorescent RNA aptamers, characterized in that, Includes the following steps: (1) Provide at least two multifunctional probes for different targets. The multifunctional probe is a single-stranded DNA and includes, from the 5′ end to the 3′ end, a T7 promoter template sequence, a fluorescent RNA aptamer coding sequence, a nicking endonuclease recognition site, and a target nucleic acid complementary recognition sequence. Different probes can achieve multiplex nucleic acid detection by configuring complementary recognition sequences for different target nucleic acids and different fluorescent RNA aptamer coding sequences. (2) The multifunctional probe is brought into contact with the nucleic acid sample to be tested under isothermal conditions. In the presence of target nucleic acid, the target nucleic acid hybridizes with the complementary recognition sequence of the probe to form a double-stranded structure. Under the action of DNA polymerase, it extends along the probe to form a double-stranded DNA containing a nicking endonuclease recognition site. Subsequently, the nicking endonuclease produces a single-stranded nick on the new strand. The DNA polymerase continues to extend from the nick and strand replacement occurs. The above nick-extension-replacement process is repeated, thereby generating a large number of template strands of T7 promoter and fluorescent RNA aptamer exponentially without changing the reaction temperature. (3) The template strand generated in step (3) combines with the T7 promoter coding strand to form a promoter double-stranded structure that can be recognized by RNA polymerase. Cell-free in vitro transcription is performed under the action of RNA polymerase to generate the transcription product fluorescent RNA aptamer. (4) The transcription product is bound to the corresponding fluorescent ligand, and the fluorescence signal is observed by the naked eye or simple optical equipment under excitation light to realize the visual detection of the target nucleic acid.

2. The method according to claim 1, characterized in that, In step (1), the sequence of the multifunctional probe is selected from any one or more of the sequences shown in SEQ ID NO.11 to SEQ ID NO.

26.

3. The method according to claim 1, characterized in that, In step (1), the fluorescent RNA aptamer is selected from at least one of Broccoli, Chili or Malachite Green Aptamer.

4. The method according to claim 1, characterized in that, In step (2), the isothermal condition is 37°C.

5. The method according to claim 1, characterized in that, In step (3), the sequence of the T7 promoter coding chain is shown in SEQ ID NO.

1.

6. A kit for implementing the detection method according to any one of claims 1-5, characterized in that, Includes the following components: (1) Multifunctional probe: Its nucleotide sequence is selected from any one or more of the sequences shown in SEQ ID NO.11 to SEQ ID NO.26; (2) Reaction components for isothermal exponential amplification: including DNA polymerase Klenow Fragment, nicking endonuclease Nb.BbvCI, deoxyribonucleotide substrate and reaction buffer; (3) Cell-free in vitro transcription reaction components: including the T7 promoter coding strand, T7 RNA polymerase, and ribonucleotide substrate as shown in SEQ ID NO.1; (4) Fluorescent ligands: including at least one of DFHBI-1T, DMHBO+ or Malachite Green.

7. The application of the method according to any one of claims 1-6 in the detection, typing and rapid on-site detection of pathogen nucleic acids, drug resistance genes and / or virulence genes.

8. The application of the kit according to claim 6 in typing Klebsiella pneumoniae based on drug resistance genes and / or virulence genes, characterized in that: The resistance gene is KPC or NDM, and the virulence gene is iucA, rmpA, iroN, or peg344.

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