Nucleotide detection method based on FEN1 mediated linear amplification reaction and universal microarray and application

Abstract

The invention discloses a nucleotide detection method based on FEN1 mediated linear amplification reaction and a universal microarray and application. The method comprises the following steps: 1) triggering template activation by an invasive reaction based on FEN1 enzyme structure specific recognition, and further triggering linear isothermal amplification; 2) realizing colorimetric, optical, Raman spectrum and electrochemical results by utilizing multi-type signals formed by combining probes with various signal labels and spatial positioning output; and (3) constructing a standardized array, and enabling the microarray platform to have extremely strong adaptive compatibility to different targets through the universal design of a primer region and a recognition region. According to the detection method, the whole process from amplification to reading can be completed within 48 minutes, the sensitivity can reach 101 copies / mu L, and the detection method is suitable for rapid screening of methylation, gene mutation, SNP and pathogens, is particularly suitable for a microfluidic or paper-based chip integrated platform, and has the advantages of low cost, high throughput, simplicity and convenience in operation and the like.

Description

Technical Field

[0001] This invention relates to a nucleotide detection method and its application based on FEN1-mediated linear amplification reaction and universal microarray, belonging to the field of ship energy conservation and emission reduction. Background Technology

[0002] In recent years, with the increase in global population density, frequent international travel, and the emergence of drug-resistant pathogens, the pressure on the prevention and control of infectious diseases has been increasing, especially in the areas of emerging viral outbreaks, the spread of multidrug-resistant bacteria, and environmental nucleic acid contamination monitoring. Effective, rapid, sensitive, and multi-target nucleic acid detection technologies are key technological supports for ensuring public health security, hospital infection control, and environmental monitoring.

[0003] Currently, polymerase chain reaction (PCR) and its variants are widely used as the gold standard for nucleic acid detection due to their high sensitivity and specificity. However, they rely on complex thermal cycling instruments, have long reaction times, high equipment costs, and complex primer design for multiplex amplification, which limits on-site real-time detection and high-throughput multi-target applications.

[0004] To overcome the limitations of thermal cycling, isothermal amplification techniques such as FEN1-mediated invasion reaction, hybridization chain reaction (HCR), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) have emerged, offering the advantage of rapid, isothermal amplification; some techniques can complete detection within tens of minutes. Nevertheless, isothermal amplification often faces challenges in multi-target detection, including uneven amplification efficiency, primer cross-reactivity, and poor system compatibility, affecting the accuracy and throughput of results.

[0005] Nucleic acid chips and microarray technologies rely on spatial coding to achieve simultaneous detection of multiple targets, demonstrating high-throughput potential. However, traditional microarrays largely depend on direct hybridization signals between targets and capture probes, resulting in long hybridization times, high temperature control requirements, and limited sensitivity. Furthermore, signal detection generally relies on fluorescent or chemiluminescent labeling, limiting amplification efficiency. In particular, traditional microarrays are highly dependent on probe sequences, making it difficult to achieve universal design. Modular signal conversion leads to poor multi-target scalability, restricting the flexible detection of diverse targets.

[0006] To overcome the problems of low signal amplification efficiency and poor versatility of traditional microarrays, hybridization detection using probes with various signal tags (such as fluorescein, HRP, electrochemical groups, SERS nanostructures, gold nanoparticles, or quantum dots), or linear or nonlinear nucleic acid chains formed by hybridization chain reaction (HCR), or various types of signal probes formed by rolling circle amplification (RCA) has become a new trend. These signal probes generate strong and diverse detection signals by hybridizing with amplification products or target sequences, making them suitable for diversified and modular designs, and easily achieving high sensitivity and multiplex recognition.

[0007] However, there is currently no technology that can convert the nucleic acid signal from FEN1-aided structure-specific cleavage to combine with linear amplification products of a uniform sequence, and use labeled signal probes to form a universal, standardized, multi-target compatible microarray signal detection system to achieve high-sensitivity, low-background, rapid and portable nucleic acid detection. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to provide a nucleic acid detection method based on FEN1-mediated structure-specific cleavage. By designing a probe with a 5′flap structure and combining it with a transformation template and a signal output module, sensitive, specific, and high-throughput detection of nucleic acids of various pathogens can be achieved.

[0009] Technical Solution: The present invention provides a nucleotide detection method based on FEN1-mediated linear amplification reaction and a universal microarray, the method comprising the following steps:

[0010] (1) Using the conserved sequence of the analyte as the target nucleic acid region, Up probe, Dp probe, transformation template and capture probe are designed according to the target nucleic acid region; among them, the 3′ recognition region of the Dp probe is completely complementary to the downstream region of the target nucleic acid region, and the 5′ protrusion region is designed as a flap structure that does not pair with the target nucleic acid region for FEN1 enzyme recognition and cleavage; the Up probe is completely complementary to the upstream sequence of the target nucleic acid region and forms a three-base overlap region with the 3′ end of the flap structure of the Dp probe to enhance the recognition stability of FEN1 enzyme; the transformation template includes a flap binding region, a nicking enzyme recognition region and an amplification region, the 3′ initiation region is complementary to the free end of the flap fragment after FEN1 enzyme cleavage of the Dp probe, the nicking enzyme recognition region is the complementary sequence recognized by the nicking enzyme, the amplification region is the sequence that is completely complementary to the capture probe and the initiation sequence for activating hybridization; the capture probe is the sequence that is completely complementary to the amplification region of the transformation template and needs to be modified at the 5′ end with functional groups for immobilization on the surface of the microarray chip;

[0011] (2) Mix the single-stranded DNA template of the test sample, Up / Dp probe, FEN1 enzyme, transformation template, polymerase, nicking enzyme and dNTPs to form a reaction system and incubate;

[0012] (3) Fix the capture probe on the surface of the microarray chip, then add the reaction system in step (2) to the surface of the microarray chip, incubate, and elute;

[0013] (4) Read the probe signal.

[0014] Specifically, the design of Dp probes should avoid self-complementarity and hairpin structures, as these can prevent the protrusions from forming stable secondary structures. The design of Up probes should avoid significant complementarity with the Dp sequence to prevent probe pairing rather than target recognition.

[0015] Furthermore, the functional groups mentioned in step (1) include amino or thiol groups.

[0016] Furthermore, step (2) also includes the steps of extracting DNA from the analyte, designing primers to amplify the target nucleic acid region, and making it single-stranded.

[0017] Furthermore, the reaction system described in step (2) also includes 10×ThermoPol Reaction Buffer, 10×NEBuffer 3.1 and H2O.

[0018] Furthermore, the reaction system described in step (2) also includes SSC and SDS.

[0019] Furthermore, the incubation conditions described in step (2) are to first incubate at 25-75°C for 5-120 minutes, and then incubate at 95°C for 5 minutes.

[0020] Furthermore, the incubation conditions described in step (3) are 25-60℃ and the incubation time is 5-120 minutes.

[0021] Furthermore, the types of microarray chips mentioned in step (3) include those made from one or more of the following materials: paper-based chips, glass slide microarrays, gels, plastics, and metals.

[0022] Furthermore, the detection method of the probe signal in step (4) includes colorimetric, optical, Raman spectroscopy or electrochemical methods.

[0023] The present invention relates to the application of the nucleotide detection method based on FEN1-mediated linear amplification reaction and universal microarray in the detection of pathogenic bacteria.

[0024] Furthermore, the pathogens include Legionella pneumophila (Lp), Staphylococcus aureus (Sa), and Pseudomonas aeruginosa (Pa).

[0025] Furthermore, the Up probe sequence for detecting Staphylococcus aureus (Sa) is shown in SEQ ID NO.1, the Dp probe sequence is shown in SEQ ID NO.2, the transformation template sequence is shown in SEQ ID NO.3, and the capture probe sequence is shown in SEQ ID NO.4; the Up probe sequence for detecting Legionella pneumophila (Lp) is shown in SEQ ID NO.5, the Dp probe sequence is shown in SEQ ID NO.6, the transformation template sequence is shown in SEQ ID NO.7, and the capture probe sequence is shown in SEQ ID NO.8; the Up probe sequence for detecting Pseudomonas aeruginosa (Pa) is shown in SEQ ID NO.9, the Dp probe sequence is shown in SEQ ID NO.10, the transformation template sequence is shown in SEQ ID NO.11, and the capture probe sequence is shown in SEQ ID NO.12.

[0026] A schematic diagram of the principle of the method of the present invention is shown below. Figure 1 As shown, the process begins by linking the single-stranded DNA (target) of the analyte's target nucleic acid region with upstream and downstream probes to form a 5' flap structure. The FEN1 enzyme recognizes the Dp probe structure and specifically cleaves it, releasing a flap fragment unrelated to the target. This process occurs at a temperature near the Tm of the Dp probe, allowing for brief hybridization between the Dp probe and its complementary sequence on the target nucleic acid region. The cleaved Dp probe rapidly dissociates from the template, making it easier for the remaining intact probes to rebind, forming a cleavage-dissociation-annealing cycle that achieves the first stage of signal amplification.

[0027] To further realize the functional transformation of the flap fragment, an editable template (i.e., the transformation template (Tp template), containing a flap binding region, an Nt.BstNBI recognition region, and an amplification region) is introduced. The flap fragment can be extended to form a double-stranded structure under the action of polymerase and dNTPs, generating an Nt.BstNBI recognition site. Subsequently, Nt.BstNBI mediates cleavage and releases the downstream strand (amplification region), and the second amplification cycle is completed by strand displacement activity, generating a large amount of Trigger DNA (amplification region, i.e., 1′+a, n′+a), thus realizing the second signal amplification.

[0028] The trigger DNA is designed with a modular structure, with one end containing a complementary sequence to the capture probe (Cp probe) on the recognition microarray chip, and the other end containing a unified activation hybridization start region. When the trigger is present, it hybridizes with multiple types of signal probes to achieve a third level of signal amplification, supporting multiple readout methods including visual, optical, Raman spectroscopy, and electrochemical methods, thus achieving signal amplification and localization readout.

[0029] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: The detection method of this application, through the standardized design of the FEN1-aided LIAR module, can convert the amplification products of all targets into a unified trigger sequence (Trigger DNA), significantly improving system compatibility in multiplex detection; it employs diverse labeled signal probes to achieve multimodal signal amplification and detection; the spatially encoded microarray structure enables parallel detection of multiple targets, adapting to microfluidic chips or paper-based platforms, simplifying the operation process and reducing costs; the platform's detection limit can reach 10^1 copies / μL, and the entire process of nucleic acid amplification, signal conversion, and readout can be completed within 48 minutes, significantly superior to traditional PCR or hybridization array schemes; the detection results can be read visually or by devices through multiple methods such as colorimetry, fluorescence, electrochemistry, or Raman signals, suitable for rapid on-site screening and deployment in low-resource areas; no thermal cycling equipment is required, the entire process is carried out at a constant temperature, and the operation is simple; it is compatible with various sample types and vectors, suitable for rapid screening of methylation, gene mutations, SNPs, and pathogens. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0031] Figure 2 The results are shown in the gel electrophoresis diagram. M: DNA ladder marker; 1: Single-stranded DNA template; 2: Upstream probe; 3: Downstream probe; 4: Single-stranded DNA template + upstream probe; 5: Single-stranded DNA template + downstream probe; 6: Single-stranded DNA template + upstream probe + downstream probe; 7: Flap; 8: Single-stranded DNA template + upstream probe + downstream probe after Flap fragment release + FEN1 (FEN1-mediated invasion reaction); 9: Transformation template; 10: FEN1-mediated invasion reaction product + transformation template; 11: Trigger DNA; 12: Linear amplification product.

[0032] Figure 3 This is the sensitivity result of this method. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0034] Example 1: Establishment of the detection method

[0035] 1. Sequence design:

[0036] Using the conserved sequence of the analyte as the target nucleic acid region, an upstream probe (Up probe), a downstream probe (Dp), a transformation template (Tp template), and a capture probe (Cp probe) are designed according to the target nucleic acid region. The 3′ recognition region of the Dp probe is completely complementary to the downstream region of the target nucleic acid region, and the 5′ protrusion region is designed as a flap structure that does not pair with the target nucleic acid region for recognition and cleavage by the FEN1 enzyme. The Up probe is completely complementary to the upstream sequence of the target nucleic acid region and forms a three-base overlap region with the 3′ end of the flap structure of the Dp probe to enhance the stability of FEN1 enzyme recognition. The transformation template includes a flap binding region, a nicking enzyme recognition region, and an amplification region. The 3′ initiation region is complementary to the free end of the flap fragment after FEN1 enzyme cleavage of the Dp probe. The nicking enzyme recognition region is a complementary sequence recognized by the nicking enzyme, and the amplification region is a sequence completely complementary to the capture probe and an activation hybridization initiation sequence. The capture probe is a sequence completely complementary to the amplification region of the transformation template and requires modification at the 5′ end with functional groups for immobilization on the surface of the microarray chip.

[0037] 2. Reaction steps:

[0038] (1) Construction of the reaction system (25 μL): containing template DNA, Up / Dp probe, FEN1 enzyme, polymerase (Vent(exo-)DNA polymerase), nicking enzyme (Nt.BstNBI), and dNTPs; specifically, 5.0 μL of single-stranded template DNA, 2.5 μL of 10×ThermoPol Reaction Buffer, 1.25 μL of 10×NEBuffer 3.1, and 2.5 μL of 2.5×10 -6 MUp probe, 2.5 μL of 2.5 × 10⁻⁶ -6 M Dp probe, 2.5 μL of 2.5 mM dNTPs, 2.5 μL of 2.5 × 10⁻⁶ mM dNTPs -6M transformation template, 0.5 μL FEN1, 0.625 μL Vent(exo-) DNA polymerase, 0.5 μL Nt.BstNBI, 4.625 μL H2O. (2) Incubate at 55℃ for 25 minutes to start the FEN1-aided LIAR reaction, and then react at 95℃ for 5 minutes; (3) Add the incubated reaction product to the SSC and SDS hybridization reaction solution, and the total reaction volume after mixing is 250 μL (final concentration 2×SSC+0.1%SDS). Place it on the surface of the microarray chip with the capture probe fixed on the surface, and incubate at 37℃ for 10 minutes to form a spatial localization signal; (4) Wash 3 times with 2×SSC+0.1%SDS, 2 min each time, and then wash once with 0.1M citrate buffer (pH=5.0) for 2 min. Finally, read the probe signal to obtain the result.

[0039] Example 2: Detection of highly pathogenic bacteria

[0040] 1. Legionella pneumophila (Lp), Staphylococcus aureus (Sa), and Pseudomonas aeruginosa (Pa) are common highly pathogenic bacteria, and infections caused by them often lead to many serious health problems. Therefore, this experiment uses the detection of these three pathogenic bacteria as an example. The specific primers used in the detection are shown in Table 1.

[0041] Table 1 Primer sequence information Oligos Sequence (5′-3′)

[0042] Sa-Up GTT GTT TCA GCA GCG ACA G

[0043] Sa-Dp GAC GGA CGC TCG CGG CAT GTATTC CAT TCT TA

[0044] Sa-Tp CCT GGACGTACGACACTT CCT CCC CGC TGAAACAGACTC CGC GAG CGT CCGTC

[0045] Sa-Cp NH2-C6-TTT TTT TTT TAATGAGCC TGGACG TAC GAC A

[0046] Lp-Up CAA TGG CTA AAG GCA TGC AA

[0047] L.p-Dp CGT CGC ACG CTG GCA GAC GCT ATG AGT GG

[0048] L.p-Tp AAC GCT TGA TAC AGG CTT CCT CCC CGC TGA AACAGACTC GCCAGC GTGCGACG

[0049] L.p-Cp NH2-C6-TTT TTT TTT TTT GCC GAAAAC GCT TGATACAGG

[0050] P.a-Up AAG CCG CTC TGC GTG CG

[0051] P.a-Dp CGT CGG CAG GCA GGG ATC ACC ACC TTC TAC

[0052] P.a-Tp GCG TAT CAA TAC TAC CTT CCT CCC CGC TGA AAC AGA CTC CCT GCCTGC CGA CG

[0053] P.a-Cp NH2-C6-TTT TTT TTT TTG AAG ACA TGG ACAAAG CGT ATC AATACT AC

[0054] Conserved sequences of Legionella pneumophila (L.p, NCBI accession number HQ-645041.1), Staphylococcus aureus (S.a, NCBI accession number CP 157769.1), and Pseudomonas aeruginosa (P.a, NCBI accession number CP-148035.1).

[0055] Because the three bacteria mentioned above are highly pathogenic, the DNA template used in this embodiment was synthesized by Shanghai Sangon Biotech. The capture probe was pre-fixed onto the surface of the microarray chip. Immobilization of the capture probe: The Biodyne C membrane was cut to the desired size and then immersed in a 5% EDC solution prepared with MES buffer, activated at room temperature for 30 minutes. After activation, the membrane was rinsed three times with deionized water and dried at 65°C for 35 minutes. The membrane was incubated with 10 μM capture probe at room temperature for 40 minutes. Subsequently, it was treated with 100 mM NaOH at room temperature for 8 minutes to block unreacted active sites. The membrane was then rinsed three more times with deionized water, dried at 65°C for 30 minutes, and stored at 4°C for later use.

[0056] The template DNA of the three bacteria was mixed at equal concentrations (total concentration of 10^2–10^7 copies / μL), and then mixed with Up / Dp probe, FEN1 enzyme, polymerase (Vent(exo-)DNA polymerase), nicking enzyme (Nt.BstNBI), dNTPs and other reaction system components as described in Example 1. The mixture was incubated at 55°C for 25 minutes, followed by reaction at 95°C for 5 minutes. The hybridization reaction solution was then added to the surface of a microarray chip with the capture probe immobilized on its surface and incubated at 37°C for 10 minutes. After elution for 8 minutes, the colorimetric signal was read.

[0057] The results are as follows Figure 3 As shown, the sensitivity of this method reaches 10^1 copies / μL, and the total detection time is approximately 48 minutes.

[0058] 2. Verification of method feasibility

[0059] Figure 2 This study presents the step-by-step validation results of the core module of the Universal Signal Conversion System (USCS), confirming the correctness of each key reaction step and the product formation through gel electrophoresis. The products detected are from the process involving Staphylococcus aureus (Sa).

[0060] Specifically, in the figure, M represents the DNA marker, lane 8 is the reaction after adding FEN1 enzyme digestion, showing the release of the flap fragment, and lane 12 is the linear amplification reaction (LIAR) product after adding amplification template and polymerase, producing a trigger DNA sequence, indicating that the universal signal conversion system is feasible.

[0061] Experimental results show that FEN1 achieves structure-dependent cleavage in lane 8; the LIAR module completes triggered sequence amplification in lane 12; verifying the effectiveness of the system and the compatibility between modules. This figure visually demonstrates the sequential triggering and efficient coupling capabilities between USCS system modules, which is the fundamental support for the entire detection platform to achieve sensitive, visible, and high-throughput signal output.

[0062] Example 3: Paper-based platform operation

[0063] A microarray is applied to a paper substrate, and reagents and samples are loaded manually using pipetting. After the reaction is complete, the colorimetric spot signals are visible to the naked eye, enabling field detection without the need for specialized equipment. This method differs from existing technologies in the following ways:

[0064]

Claims

1. A nucleotide detection method based on FEN1-mediated linear amplification reaction and a universal microarray, characterized in that, The method includes the following steps: (1) Using the conserved sequence of the analyte as the target nucleic acid region, Up probe, Dp probe, transformation template and capture probe are designed according to the target nucleic acid region; among them, the 3′ recognition region of the Dp probe is completely complementary to the downstream region of the target nucleic acid region, and the 5′ protrusion region is designed as a flap structure that does not pair with the target nucleic acid region for FEN1 enzyme recognition and cleavage; the Up probe is completely complementary to the upstream sequence of the target nucleic acid region and forms a three-base overlap region with the 3′ end of the flap structure of the Dp probe to enhance the recognition stability of FEN1 enzyme; the transformation template includes a flap binding region, a nicking enzyme recognition region and an amplification region, the 3′ initiation region is complementary to the free end of the flap fragment after FEN1 enzyme cleavage of the Dp probe, the nicking enzyme recognition region is the complementary sequence recognized by the nicking enzyme, the amplification region is the sequence that is completely complementary to the capture probe and the initiation sequence for activating hybridization; the capture probe is the sequence that is completely complementary to the amplification region of the transformation template and needs to be modified at the 5′ end with functional groups for immobilization on the surface of the microarray chip; (2) Mix the single-stranded DNA template of the test sample, Up / Dp probe, FEN1 enzyme, transformation template, polymerase, nicking enzyme and dNTPs to form a reaction system and incubate; (3) Fix the capture probe on the surface of the microarray chip, then add the reaction system in step (2) to the surface of the microarray chip, incubate, and elute; (4) Read the probe signal.

2. The nucleotide detection method according to claim 1, characterized in that, The functional groups mentioned in step (1) include amino or thiol groups.

3. The nucleotide detection method according to claim 1, characterized in that, The reaction system described in step (2) also includes 10×ThermoPol Reaction Buffer, 10×NEBuffer 3.1 and H2O.

4. The nucleotide detection method according to claim 1, characterized in that, The incubation conditions described in step (2) are to first incubate at 25-75℃ for 5-120 minutes, and then incubate at 80-95℃ for 3-30 minutes.

5. The nucleotide detection method according to claim 1, characterized in that, The incubation conditions described in step (3) are 25-60℃ and the incubation time is 5-120 minutes.

6. The nucleotide detection method according to claim 1, characterized in that, The types of microarray chips mentioned in step (3) include those made from one or more of the following materials: paper-based chips, glass slide microarrays, gels, plastics, and metals.

7. The nucleotide detection method according to claim 1, characterized in that, The detection methods for the probe signal in step (4) include colorimetric, optical, Raman spectroscopy or electrochemical methods.

8. The application of the nucleotide detection method based on FEN1-mediated linear amplification reaction and universal microarray as described in any one of claims 1 to 7 in the detection of pathogenic bacteria.

9. The application according to claim 8, characterized in that, The pathogens include Legionella pneumophila (Lp), Staphylococcus aureus (Sa), and Pseudomonas aeruginosa (Pa).

10. The application according to claim 9, characterized in that, The Up probe sequence for detecting Staphylococcus aureus (Sa) is shown in SEQ ID NO.1, the Dp probe sequence is shown in SEQ ID NO.2, the transformation template sequence is shown in SEQ ID NO.3, and the capture probe sequence is shown in SEQ ID NO.

4. The Up probe sequence for detecting Legionella pneumophila (Lp) is shown in SEQ ID NO.5, the Dp probe sequence is shown in SEQ ID NO.6, the transformation template sequence is shown in SEQ ID NO.7, and the capture probe sequence is shown in SEQ ID NO.8; the Up probe sequence for detecting Pseudomonas aeruginosa (Pa) is shown in SEQ ID NO.9, the Dp probe sequence is shown in SEQ ID NO.10, the transformation template sequence is shown in SEQ ID NO.11, and the capture probe sequence is shown in SEQ ID NO.12.

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