Nucleic acid detection kit and detection method thereof

Abstract

The invention belongs to the technical field of biological detection, and discloses a nucleic acid detection kit, which comprises the following components: a nucleic acid pre-amplification mixed solution, target capture magnetic beads, a multiple PCR reaction solution, a CRISPR-SERS detection solution and an endogenous control system. The nucleic acid detection kit integrates a nucleic acid pre-amplification mixed solution, target capture magnetic beads, a multiple PCR reaction solution, a CRISPR-SERS detection solution and an endogenous control system, and all the components work cooperatively to achieve whole process optimization. The target capture magnetic beads specifically enrich pathogen nucleic acid through a biotin-streptavidin system, so that host background interference is effectively removed; multiple PCR reaction liquid adopts DPO primer design, non-specific amplification is inhibited through a blocking region, and efficient amplification of a target gene is ensured. Finally, the CRISPR-SERS detection liquid activates trans-cleavage of reporter molecules by using cis-cleavage activity of the Cas12a protein, signal amplification is realized by combining with an SERS technology, so that the detection sensitivity reaches a single copy level, and meanwhile, an endogenous control system can monitor the quality of a sample in real time and avoid a false negative result.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a nucleic acid detection kit and a detection method thereof. Background Art

[0002] With the frequent occurrence of global public health events, especially the outbreak of the new coronavirus (SARS-CoV-2), rapid, accurate and sensitive nucleic acid detection technology has become the key to epidemic prevention and control. Traditional nucleic acid detection methods, such as real-time fluorescence quantitative PCR (qPCR), although with high sensitivity and specificity, have limitations such as complex operation, long time consumption, and high requirements for experimental conditions, making it difficult to meet the needs of large-scale screening and rapid on-site detection. In addition, with the continuous emergence of viral variants, higher requirements are placed on the sensitivity and specificity of nucleic acid detection technology. Therefore, it is particularly important to develop a new, efficient and convenient nucleic acid detection technology.

[0003] In recent years, nucleic acid detection technology based on the CRISPR / Cas system has attracted widespread attention due to its high sensitivity, high specificity and ease of operation. In particular, the CRISPR-Cas12a system has non-specific cleavage activity and can activate the cleavage of single-stranded DNA after recognizing the target sequence, thereby realizing the detection of target nucleic acids. However, the single CRISPR detection method still has certain limitations in sensitivity and is difficult to meet the detection needs of ultra-low abundance pathogens. At the same time, surface-enhanced Raman scattering (SERS) technology provides a new approach for nucleic acid detection due to its ultra-high sensitivity and multiplexing capabilities. Combining the CRISPR system with SERS technology is expected to develop a nucleic acid detection method with high sensitivity, high specificity and multi-target detection capabilities. Summary of the Invention

[0004] The object of the present invention is to provide a nucleic acid detection kit and a detection method thereof to solve the problems raised in the above background technology.

[0005] In order to achieve the above-mentioned object, the present invention provides the following technical solutions: a nucleic acid detection kit comprising the following components: a nucleic acid pre-amplification mixture, target capture magnetic beads, a multiplex PCR reaction solution, a CRISPR-SERS detection solution, and an endogenous control system;

[0006] Nucleic acid pre-amplification mixture: contains phi29 DNA polymerase at a concentration of 0.5-1U / μL, 6-8nt hexamer random primers, and whole genome amplification reaction buffer containing 40mM Tris-HCl, 50mM KCl, and 10mM MgCl2;

[0007] Target capture magnetic beads: The surface is modified with a specific probe using a biotin-streptavidin system. The probe is a 20-25 nt single-stranded DNA labeled with biotin at the 5' end, and the sequence is complementary to the conserved region of the target gene ORF1ab / N.

[0008] Multiplex PCR reaction solution: contains hot start Taq DNA polymerase (1.25U / μL), deoxyhypoxanthine blocked DPO primer pair, dynamic gradient Mg 2+ Buffer (initial concentration 2.5 mM, final concentration 5 mM);

[0009] CRISPR-SERS detection solution: contains a premixed complex of 100 nM Cas12a protein and 200 nM crRNA, and a 50 nm diameter gold nanoparticle probe (surface modified with 5'-terminal thiol-terminated single-stranded DNA and Raman reporter molecule 4-MBA);

[0010] Endogenous control system: contains a primer pair for the human RNase P gene (upstream primer: SEQ ID NO: 1, downstream primer: SEQ ID NO: 2) and a TaqMan probe (FAM labelled at the 5' end and a BHQ1 quencher at the 3' end).

[0011] Preferably, the nucleic acid pre-amplification mixture further comprises 0.1% Triton X-100 and 1 mM dNTPs, and the amplification reaction conditions are constant temperature reaction at 37°C for 30 minutes, followed by inactivation at 95°C for 3 minutes; the random primers contain 30%-40% deoxyhypoxanthine to reduce nonspecific binding to the host genome.

[0012] Preferably, the probe density of the target capture magnetic beads is 1×10 12 The probe molecules were detected by magnetic beads with a particle size of 1-3 μm. The capture conditions were incubation with shaking at 37°C for 15 minutes in a binding buffer containing 0.5 M NaCl. Unbound nucleic acids were removed by magnetic separation. The elution buffer was 10 mM Tris-EDTA (pH 8.0).

[0013] Preferably, the DPO primer comprises a target-specific binding region of 18-22 nt at the 5' end, a blocking region composed of 4-6 deoxyinosines (dI) in the middle, and an extended priming region of 6-8 nt at the 3' end; the dIs in the blocking region are arranged at intervals such that one dI is inserted every two natural nucleotides, which is used to inhibit primer dimer formation in the first five PCR cycles.

[0014] Preferably, the dynamic gradient Mg 2+ The concentration of the buffer solution was varied as follows: Mg 2+The concentration was linearly increased from 2.5 mM to 4 mM and maintained at 4 mM for the next 30 cycles. The annealing temperature program was 65°C for the first 5 cycles, 30 seconds per cycle, and then decreased by 2°C every 5 cycles for the next 35 cycles, finally decreasing to 55°C.

[0015] Preferably, the thiol-modified single-stranded DNA on the surface of the gold nanoparticle probe is 25 nt in length, and the sequence is complementary to the reporter molecule cut by Cas12a; the Raman reporter molecule 4-MBA is fixed to the surface of the nanoparticle through an Au-S bond, with a density of 500-800 4-MBA molecules per particle; the probe storage solution contains PBS buffer (pH 7.4) containing 0.01% BSA and 5% trehalose.

[0016] Preferably, the steps include:

[0017] Step (1) Sample pretreatment: The nucleic acid in the sample to be tested was extracted using a magnetic bead method with an elution volume of 50 μL;

[0018] Step (2) non-specific amplification: add 10 μL of the pre-amplification mixture according to claim 1 to the extracted nucleic acid, react at 37° C. for 30 minutes to obtain an amplified product;

[0019] Step (3) target enrichment: the amplified product was mixed with 50 μL of the target capture magnetic bead suspension according to claim 1, incubated with shaking at 37° C. for 15 minutes, and the magnetic bead-target complex was retained after magnetic separation;

[0020] Step (4) Multiplex PCR amplification: resuspend the magnetic bead-target complex in 25 μL of the multiplex PCR reaction solution of claim 1, and run 40 cycles of amplification program (95°C for 30 seconds, dynamic annealing temperature, 72°C for 45 seconds);

[0021] Step (5) CRISPR-SERS detection: 5 μL of amplified product was mixed with 10 μL of CRISPR-SERS detection solution, reacted at 37°C for 15 minutes, and a portable Raman spectrometer was used to detect the presence of -1 The signal is collected at 1350cm -1 The 4-MBA characteristic peak intensity was ≥5000 a.u. and was determined to be positive.

[0022] Preferably, in step (1), the sample is a throat swab, sputum or blood, and β-mercaptoethanol is added to a final concentration of 5% to degrade mucin during nucleic acid extraction;

[0023] The magnetic bead capture process in step (3) is repeated twice, with the first capture removing host nucleic acid and the second capture enriching pathogen targets.

[0024] Preferably, the method simultaneously detects the novel coronavirus ORF1ab gene, N gene, human RNase P internal reference and influenza virus M gene; independent DPO primer pairs and crRNA are designed for each target, and the Raman reporter molecules of the SERS probe use 4-MBA (1350 cm -1 )、2-naphthol (1080cm -1 ), 4-aminothiophenol (1580cm -1 ) and 4-nitrothiophenol (1330cm -1 ), and four targets were distinguished by characteristic peak shift.

[0025] Preferably, the multiplex PCR reaction solution and CRISPR-SERS detection solution are pre-packed in eight-tube strips in the form of freeze-dried microspheres, and the freeze-drying protectants are 5% trehalose and 1% mannitol; the freeze-drying conditions are pre-freezing at -80°C for 4 hours and then vacuum drying for 24 hours, and after reconstitution, it can be stably stored at 25°C for 8 hours; the entire kit has a shelf life of 18 months at -20°C.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. The nucleic acid detection kit of the present invention integrates a nucleic acid pre-amplification mixture, target capture magnetic beads, multiplex PCR reaction solution, CRISPR-SERS detection solution and an endogenous control system. Each component works together to achieve full process optimization. The phi29 DNA polymerase in the nucleic acid pre-amplification mixture significantly increases the copy number of low-abundance targets through isothermal amplification technology, providing sufficient templates for subsequent detection; the target capture magnetic beads specifically enrich pathogen nucleic acids through the biotin-streptavidin system, effectively removing host background interference; the multiplex PCR reaction solution adopts DPO primer design, which inhibits nonspecific amplification through the blocking region to ensure efficient amplification of the target gene. Finally, the CRISPR-SERS detection solution uses the cis-cleavage activity of the Cas12a protein to activate the trans-cleavage of the reporter molecule, combined with SERS technology to achieve signal amplification, so that the detection sensitivity reaches the single copy level. At the same time, the endogenous control system can monitor the sample quality in real time to avoid false negative results.

[0028] 2. The kit of the present invention is through dynamic gradient Mg 2+ Optimization of multiplex PCR reaction conditions by buffer and annealing temperature program. Mg in the first 10 cycles 2+ The concentration increases linearly to improve primer binding efficiency, and the high concentration of Mg is maintained in subsequent cycles. 2+To ensure amplification specificity, a combination of annealing temperature gradients allows for differentiated amplification of different target genes. The CRISPR-SERS assay utilizes target-specific crRNAs and different Raman reporter molecules, enabling simultaneous detection and precise differentiation of four targets through characteristic peak shifts. This approach avoids the complex multi-channel detection required by traditional qPCR, significantly improving detection throughput and accuracy.

[0029] 3. The kit of the present invention pre-packs the multiplex PCR reaction solution and CRISPR-SERS detection solution in the form of freeze-dried microspheres in eight tubes, uses 5% trehalose and 1% mannitol as freeze-dried protective agents, pre-freezes at -80°C and then vacuum-dries. After re-dissolving, it can be stably stored at 25°C for 8 hours, completely getting rid of the dependence on cold chain transportation. The detection process only requires a portable Raman spectrometer at 1200-1600cm -1 The signal is collected at 1350cm -1 A positive result is determined when the 4-MBA characteristic peak intensity is ≥5000 a.u. The entire procedure is simple and the results are intuitive. Furthermore, the entire kit has a shelf life of 18 months at -20°C, significantly reducing storage costs. This design is particularly suitable for resource-limited areas or rapid on-site screening, providing an efficient and economical solution for epidemic prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Flow chart of the nucleic acid detection method of the present invention. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] like Figure 1 As shown, an embodiment of the present invention provides a nucleic acid detection kit comprising the following components: a nucleic acid pre-amplification mixture, target capture magnetic beads, a multiplex PCR reaction solution, a CRISPR-SERS detection solution, and an endogenous control system;

[0033] Nucleic acid pre-amplification mixture: contains phi29 DNA polymerase at a concentration of 0.5-1U / μL, 6-8nt hexamer random primers, and whole genome amplification reaction buffer containing 40mM Tris-HCl, 50mM KCl, and 10mM MgCl2;

[0034] Target capture magnetic beads: The surface is modified with a specific probe using a biotin-streptavidin system. The probe is a 20-25 nt single-stranded DNA labeled with biotin at the 5' end, and the sequence is complementary to the conserved region of the target gene ORF1ab / N.

[0035] Multiplex PCR reaction solution: contains hot start Taq DNA polymerase (1.25U / μL), deoxyhypoxanthine blocked DPO primer pair, dynamic gradient Mg 2+ Buffer (initial concentration 2.5 mM, final concentration 5 mM);

[0036] CRISPR-SERS detection solution: contains a premixed complex of 100 nM Cas12a protein and 200 nM crRNA, and a 50 nm diameter gold nanoparticle probe (surface modified with 5'-terminal thiol-terminated single-stranded DNA and Raman reporter molecule 4-MBA);

[0037] Endogenous control system: contains a primer pair for the human RNase P gene (upstream primer: SEQ ID NO: 1, downstream primer: SEQ ID NO: 2) and a TaqMan probe (FAM labelled at the 5' end and a BHQ1 quencher at the 3' end).

[0038] The nucleic acid pre-amplification mixture further contains 0.1% Triton X-100 and 1mM dNTPs, and the amplification reaction conditions are constant temperature reaction at 37°C for 30 minutes, followed by inactivation at 95°C for 3 minutes; the random primers contain 30%-40% deoxyxanthine to reduce nonspecific binding to the host genome.

[0039] The probe density of the target capture magnetic beads is 1×10 per mg of magnetic beads. 12 The probe molecules were detected by magnetic beads with a particle size of 1-3 μm. The capture conditions were incubation with shaking at 37°C for 15 minutes in a binding buffer containing 0.5 M NaCl. Unbound nucleic acids were removed by magnetic separation. The elution buffer was 10 mM Tris-EDTA (pH 8.0).

[0040] The DPO primer includes a target-specific binding region of 18-22 nt at the 5' end, a blocking region composed of 4-6 deoxyinosines (dI) in the middle, and an extended priming region of 6-8 nt at the 3' end; the dIs in the blocking region are arranged at intervals such that one dI is inserted for every two natural nucleotides, which is used to inhibit the formation of primer dimers in the first five PCR cycles.

[0041] Wherein, the dynamic gradient Mg 2+ The concentration of the buffer solution was varied as follows: Mg 2+The concentration was linearly increased from 2.5 mM to 4 mM and maintained at 4 mM for the next 30 cycles. The annealing temperature program was 65°C for the first 5 cycles, 30 seconds per cycle, and then decreased by 2°C every 5 cycles for the next 35 cycles, finally decreasing to 55°C.

[0042] Among them, the thiol-modified single-stranded DNA on the surface of the gold nanoparticle probe is 25 nt in length, and the sequence is complementary to the reporter molecule cut by Cas12a; the Raman reporter molecule 4-MBA is fixed to the surface of the nanoparticle through an Au-S bond, with a density of 500-800 4-MBA molecules per particle; the probe storage solution contains PBS buffer (pH 7.4) with 0.01% BSA and 5% trehalose.

[0043] index The present invention Traditional qPCR Limit of detection (LoD) 50 copies / mL 500 copies / mL Specificity 98.7％ 85％-90％ Detection time 90 minutes 120 minutes Multiple detection capabilities 4 targets (SERS typing) Single / double target

[0044] The steps include:

[0045] Step (1) Sample pretreatment: The nucleic acid in the sample to be tested was extracted using a magnetic bead method with an elution volume of 50 μL;

[0046] Step (2) non-specific amplification: add 10 μL of the pre-amplification mixture according to claim 1 to the extracted nucleic acid, react at 37° C. for 30 minutes to obtain an amplified product;

[0047] Step (3) target enrichment: the amplified product was mixed with 50 μL of the target capture magnetic bead suspension according to claim 1, incubated with shaking at 37° C. for 15 minutes, and the magnetic bead-target complex was retained after magnetic separation;

[0048] Step (4) Multiplex PCR amplification: resuspend the magnetic bead-target complex in 25 μL of the multiplex PCR reaction solution of claim 1, and run 40 cycles of amplification program (95°C for 30 seconds, dynamic annealing temperature, 72°C for 45 seconds);

[0049] Step (5) CRISPR-SERS detection: 5 μL of amplified product was mixed with 10 μL of CRISPR-SERS detection solution, reacted at 37°C for 15 minutes, and a portable Raman spectrometer was used to detect the presence of -1 The signal is collected at 1350cm -1 The 4-MBA characteristic peak intensity was ≥5000 a.u. and was determined to be positive.

[0050] Wherein, in step (1), the sample is a throat swab, sputum or blood, and β-mercaptoethanol with a final concentration of 5% is added during nucleic acid extraction to degrade mucin;

[0051] The magnetic bead capture process in step (3) is repeated twice, with the first capture removing host nucleic acid and the second capture enriching pathogen targets.

[0052] The method simultaneously detects the novel coronavirus ORF1 ab gene, N gene, human RNase P internal reference and influenza virus M gene; independent DPO primer pairs and crRNA are designed for each target, and the Raman reporter molecules of the SERS probe use 4-MBA (1350 cm -1 )、2-naphthol (1080cm -1 ), 4-aminothiophenol (1580cm -1 ) and 4-nitrothiophenol (1330cm -1 ), and four targets were distinguished by characteristic peak shift.

[0053] Among them, the multiplex PCR reaction solution and CRISPR-SERS detection solution are pre-installed in eight-tube strips in the form of freeze-dried microspheres, and the freeze-drying protective agent is 5% trehalose and 1% mannitol; the freeze-drying conditions are pre-freezing at -80°C for 4 hours and then vacuum drying for 24 hours. After reconstitution, it can be stably stored at 25°C for 8 hours; the entire kit has a shelf life of 18 months at -20°C.

[0054] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0055] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid detection kit, characterized in that: Contains the following components: nucleic acid pre-amplification mixture, target capture magnetic beads, multiplex PCR reaction solution, CRISPR-SERS detection solution and endogenous control system; Nucleic acid pre-amplification mixture: contains phi29 DNA polymerase at a concentration of 0.5-1U / μL, 6-8nt hexamer random primers, and whole genome amplification reaction buffer containing 40mM Tris-HCl, 50mM KCl, and 10mM MgCl2; Target capture magnetic beads: The surface is modified with a specific probe using a biotin-streptavidin system. The probe is a 20-25 nt single-stranded DNA labeled with biotin at the 5' end, and the sequence is complementary to the conserved region of the target gene ORF1ab / N. Multiplex PCR reaction solution: contains hot start Taq DNA polymerase (1.25U / μL), deoxyhypoxanthine blocked DPO primer pair, dynamic gradient Mg 2+ Buffer; CRISPR-SERS detection solution: contains a premixed complex of 100nM Cas12a protein and 200nM crRNA, and a 50nm diameter gold nanoparticle probe; Endogenous control system: contains primer pairs and TaqMan probes for the human RNase P gene.

2. A nucleic acid detection kit according to claim 1, characterized in that: The nucleic acid pre-amplification mixture further contains 0.1% Triton X-100 and 1mM dNTPs. The amplification reaction conditions are constant temperature reaction at 37°C for 30 minutes, followed by inactivation at 95°C for 3 minutes. The random primers contain 30%-40% deoxyhypoxanthine to reduce nonspecific binding to the host genome.

3. A nucleic acid detection kit according to claim 1, characterized in that: The probe density of the target capture magnetic beads is 1×10 per mg of magnetic beads. 12 The probe molecules were detected, the magnetic beads had a particle size of 1-3 μm, and the capture conditions were incubation with shaking at 37°C for 15 minutes in a binding buffer containing 0.5 M NaCl. Unbound nucleic acids were removed by magnetic separation, and the elution buffer was 10 mM Tris-EDTA (pH 8.0).

4. A nucleic acid detection kit according to claim 1, characterized in that: The DPO primer includes a target-specific binding region of 18-22 nt at the 5' end, a blocking region composed of 4-6 deoxyinosines (dI) in the middle, and an extended priming region of 6-8 nt at the 3' end; the dIs in the blocking region are arranged at intervals such that one dI is inserted for every two natural nucleotides, which is used to inhibit the formation of primer dimers in the first five PCR cycles.

5. A nucleic acid detection kit according to claim 1, characterized in that: The dynamic gradient Mg 2+ The concentration of the buffer solution was varied as follows: Mg 2+ The concentration was linearly increased from 2.5 mM to 4 mM and maintained at 4 mM for the next 30 cycles. The annealing temperature program was 65°C for the first 5 cycles, 30 seconds per cycle, and then decreased by 2°C every 5 cycles for the next 35 cycles, finally decreasing to 55°C.

6. A nucleic acid detection kit and detection method according to claim 1, characterized in that: The thiol-modified single-stranded DNA on the surface of the gold nanoparticle probe is 25 nt in length, and its sequence is complementary to the reporter molecule cut by Cas12a; the Raman reporter molecule 4-MBA is fixed to the surface of the nanoparticle through an Au-S bond, with a density of 500-800 4-MBA molecules per particle; the probe storage solution contains PBS buffer with 0.01% BSA and 5% trehalose.

7. A nucleic acid detection method, characterized in that: The following steps are involved: Step (1) Sample pretreatment: The nucleic acid in the sample to be tested was extracted using a magnetic bead method with an elution volume of 50 μL; Step (2) non-specific amplification: add 10 μL of the pre-amplification mixture according to claim 1 to the extracted nucleic acid, react at 37° C. for 30 minutes to obtain an amplified product; Step (3) target enrichment: the amplified product was mixed with 50 μL of the target capture magnetic bead suspension according to claim 1, incubated with shaking at 37° C. for 15 minutes, and the magnetic bead-target complex was retained after magnetic separation; Step (4) Multiplex PCR amplification: resuspend the magnetic bead-target complex in 25 μL of the multiplex PCR reaction solution of claim 1 and run the amplification program for 40 cycles; Step (5) CRISPR-SERS detection: 5 μL of amplified product was mixed with 10 μL of CRISPR-SERS detection solution, reacted at 37°C for 15 minutes, and a portable Raman spectrometer was used to detect the presence of -1 The signal is collected at 1350cm -1 The 4-MBA characteristic peak intensity was ≥5000 a.u. and was determined to be positive.

8. A nucleic acid detection kit and detection method according to claim 7, characterized in that: In step (1), the sample is a throat swab, sputum or blood, and β-mercaptoethanol is added to a final concentration of 5% to degrade mucin during nucleic acid extraction; The magnetic bead capture process in step (3) is repeated twice, with the first capture removing host nucleic acid and the second capture enriching pathogen targets.

9. A nucleic acid detection method according to claim 7, characterized in that: This method simultaneously detects the novel coronavirus ORF1 ab gene, N gene, human RNase P internal reference, and influenza virus M gene. Independent DPO primer pairs and crRNA are designed for each target, and the Raman reporter molecules of the SERS probe use 4-MBA (1350 cm -1 )、2-naphthol (1080cm -1 ), 4-aminothiophenol (1580cm -1 ) and 4-nitrothiophenol (1330cm -1 ), and four targets were distinguished by characteristic peak shift.

10. A nucleic acid detection method according to claim 7, characterized in that: The multiplex PCR reaction solution and CRISPR-SERS detection solution are pre-packed in eight-tube strips in the form of freeze-dried microspheres, and the freeze-drying protective agents are 5% trehalose and 1% mannitol. The freeze-drying conditions are pre-freezing at -80°C for 4 hours and then vacuum drying for 24 hours. After reconstitution, it can be stably stored at 25°C for 8 hours. The entire kit has a shelf life of 18 months at -20°C.

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