Ultrashort helicase-dependent isothermal amplification detection reagent based on CRISPR / Cas12a

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an ultrashort helicase dependent isothermal amplification detection reagent based on CRISPR / Cas12a. According to the invention, a large amount of ultrashort amplicons are generated through the usHDA reaction and are combined with the CRISPR / Cas12a detection technology, the reaction is rapid, the whole experiment time does not exceed one hour, the experiment time is greatly shortened, the defect of insufficient specificity of the traditional HDA technology is overcome, the uncovering operation is avoided, and the pollution risk is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an ultrashort helicase-dependent isothermal amplification detection reagent based on CRISPR / Cas12a. Background Technology

[0002] Nucleic acid amplification detection technology is based on enzyme-catalyzed reactions and achieves highly sensitive nucleic acid detection by amplifying specific target sequences. Commonly used nucleic acid amplification techniques are mainly divided into two categories: polymerase chain reaction (PCR) and isothermal amplification techniques. PCR technology uses thermal cycling to denature, anneal, and extend the DNA double helix, allowing primers to bind to the target DNA. Under the action of DNA polymerase, the DNA daughter strands are extended. After repeated cycles, the target DNA undergoes exponential amplification. Isothermal amplification, on the other hand, achieves exponential amplification of nucleic acids under a single temperature condition. [1] Common isothermal amplification techniques mainly include recombinase polymerase amplification (RPA). [2] Loop-mediated isothermal amplification (AMP) [3] Rolling cycle amplification (RCA) [4] Strand-displacement amplification (SDA) technology [5] Nucleic acid sequence-based amplification (NASBA) [6] And other technologies. Isothermal amplification (LAMP) is highly valued because it does not require a thermal cycler, has low equipment requirements, is simple to operate, and is relatively quick, making it easy to promote and apply. However, each technology has its own drawbacks. For example, LAMP technology requires multiple primer pairs, and its primer design is complex, leading to false positives. [7] RPA requires primer optimization; otherwise, partial non-specific amplification is likely to occur. [8] During the RCA reaction, non-circularized locking probes and unbound template DNA may generate some background signals. [9] SDA requires the addition of non-standard nucleotides to the reaction mixture, which affects amplification efficiency.

[10] NASBA has a complex reaction composition and high reagent costs. [6]In addition, there is a particularly PCR-like isothermal amplification technique—helicase-dependent amplification (HDA). This technique uses helicase instead of heat to open the DNA double helix. After the primer binds to the nucleic acid template, a double helix is ​​formed under the action of polymerase. The newly synthesized DNA double helix serves as a template for a new round of amplification, and the amplification is repeated cyclically.

[11] The advantages of HDA (High-Intensity Depression Amplification) are its simplicity, requiring only one pair of primers in the reaction system and simple design, making it considered a simplified version of PCR. However, it also has certain disadvantages, including low sensitivity and a tendency to form primer dimers. Precisely because of its low sensitivity, HDA has received relatively few research reports compared to other isothermal amplification techniques, leading to its immaturity. HDA is simple to operate, and if its low sensitivity problem can be solved, it has promising application prospects. The low sensitivity of HDA is mainly due to the limitation of helicase speed in the amplification reaction. Improving the efficiency of helicase in the amplification reaction is key to solving this problem. Since the amplification efficiency of HDA depends on the unwinding speed of the helicase, amplifying longer target gene fragments requires longer time and more helicase, which to some extent reduces amplification efficiency and increases detection costs.

[12] Traditional HDA amplification has relatively low efficiency, resulting in a generally long reaction time (usually about 2 hours).

[13] The limitation of reaction kinetics mainly stems from the unwinding rate of helicase. Amplifying longer fragments of the target gene requires more helicase than amplifying shorter fragments, thus affecting the efficiency of the amplification reaction. Therefore, the same amount of helicase can achieve higher amplification efficiency for shorter fragments of the target gene than for longer fragments. Thus, this invention found that shortening the target gene fragment significantly improves the amplification efficiency and sensitivity of HDA. HDA is classified into mesophilic types.

[13] and thermophilic

[14] There are two types. The mesophilic HDA can react under mild conditions at 37°C, while the thermophilic HDA typically reacts at 65°C. The inventors' research group previously reported a detection technique combining thermophilic HDA with CRISPR-Cas12a.

[15] However, thermophilic HDA reacts at a relatively high temperature of 65°C, while CRISPR-Cas12a typically reacts at 37°C. These temperatures are incompatible, and when they are reacted in the same tube, the enzyme activity of Cas12a is easily affected.

[15] However, the amplification efficiency of mesophilic HDA is lower than that of thermophilic HDA, resulting in very few research reports on mesophilic HDA to date. If the amplification efficiency of mesophilic HDA can be improved and a highly sensitive mesophilic HDA can be established, it will help promote the widespread application of mesophilic HDA.

[0003] In addition, traditional HDA technology faces another challenge in practical applications: HDA is prone to primer dimer formation, which affects its specificity and may lead to false positives.

[14] Therefore, the widespread application of HDA urgently requires addressing the potential for non-specific interference. In recent years, several highly specific emerging molecular diagnostic technologies have developed rapidly, among which CRISPR detection technology is a highly specific and noteworthy emerging molecular diagnostic technology. Regularly spaced clustered short palindromic repeats (CRISPR) and their associated protein (Cas) systems, especially CRISPR-Cas12a, have attracted widespread attention and application due to their high specificity in molecular diagnostics.

[16] The CRISPR / Cas system is an immune defense system used by bacteria to resist the invasion of foreign genetic material; it is a self-protection mechanism of prokaryotes.

[16] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) consists of clustered, regularly spaced short palindromic repeats. Together with CRISPR-associated (Cas) proteins, they form the CRISPR / Cas system. Cas is a nuclease that utilizes the RNA corresponding to the spacer sequences in the CRISPR sequence to recognize and cleave specific DNA strands complementary to its sequence.

[17] Guided by guide RNA (crRNA), Cas12a specifically recognizes the target DNA sequence and is then activated to perform trans-cleavage, non-specifically cleaving single-stranded DNA reporter molecules in the surrounding environment (such as fluorescently and quenched reporter probes). This transforms the invisible nucleic acid recognition event into a strong, readable fluorescent signal.

[18] This "instantaneous" signal amplification mechanism makes it an ideal detection system for building highly sensitive and specific molecular diagnostic platforms. However, the CRISPR system itself does not have nucleic acid amplification capabilities; its detection sensitivity usually relies on effective combination with pre-amplification techniques.

[19] .

[0004] [1]Jiang H, Li Y, Lv X, et al. Recent advances in cascade isothermalamplification techniques for ultra-sensitive nucleic acid detection[J]. Talanta, 2023, 260: 124645. [2]Feng X, Liu Y, Zhao Y, et al. Recombinase PolymeraseAmplification-Based Biosensors for Rapid Zoonoses Screening[J]. Int JNanomedicine, 2023, 18: 6311-6331. [3]Aglietti C, Benigno A, Cacciola S O, et al. LAMP Reaction in PlantDisease Surveillance: Applications, Challenges, and Future Perspectives[J].Life, 2024, 14(12): 1549. [4]Zhu L, Hu Q, Wang Z, et al. CRISPR / Cas12a-Mediated Rolling CircleAmplification for the Development of Liquid Crystal-Based Sensors[J]. AnalChem, 2025, 97(32): 17825-17832. [5]Gong S, Zhang S, Wang X, et al. Strand Displacement AmplificationAssisted CRISPR-Cas12a Strategy for Colorimetric Analysis of Viral NucleicAcid[J]. Anal Chem, 2021, 93(45): 15216-15223. [6]Mao Z, Lei H, Chen R, et al. CRISPR / Cas13a analysis based on NASBAamplification for norovirus detection[J]. Talanta, 2024, 280: 126725. [7]Gieroń M, arnowiec P, Zegad o K, et al. Loop-Mediated IsothermalAmplification of DNA (LAMP) as an Alternative Method for Determining Bacteriain Wound Infections[J]. International Journal of Molecular Sciences, 2023, 25(1): 411. [8]Zhao Y, Zhang Y, Wu W, et al. Rapid and sensitive detection ofMycoplasma synoviae using RPA combined with Pyrococcus furiosus Argonaute[J].Poultry Science, 2024, 103(3): 103244. [9]Chen Z, Wu C, Yuan Y, et al. CRISPR-Cas13a-powered electrochemicalbiosensor for the detection of the L452R mutation in clinical samples ofSARS-CoV-2 variants[J]. J Nanobiotechnology, 2023, 21(1): 141.

[10] Du Y, Qi Y, Kang Q, et al. A fluorescent sensor based on stranddisplacement amplification and primer exchange reaction coupling for label-free detection of miRNA[J]. Anal Chim Acta, 2023, 1279: 341780.

[11] Barreda-Garcia S, Miranda-Castro R, de-Los-Santos-Alvarez N, etal. Helicase-dependent isothermal amplification: a novel tool in thedevelopment of molecular-based analytical systems for rapid pathogendetection[J]. Anal Bioanal Chem, 2018, 410(3): 679-693.

[12] Kim U, Lee S Y, Oh S W. Thermophilic helicase-dependentamplification-based CRISPR / Cas12a system: Detection of stx2 in Escherichiacoli O157:H7 by controlling primer dimers[J]. Anal Chim Acta, 2023, 1239:340679.

[13] Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNAamplification[J]. EMBO Rep, 2004, 5(8): 795-800.

[14] Yu L, Tang Y, Sun Y, et al. DMSO enhanced one-pot HDA-CRISPR / Cas12a biosensor for ultrasensitive detection of Monkeypox virus[J]. Talanta,2025, 287: 127660.

[15] Weng Z, You Z, Li H, et al. CRISPR-Cas12a Biosensor Array forUltrasensitive Detection of Unamplified DNA with Single-NucleotidePolymorphic Discrimination[J]. ACS Sens, 2023, 8(4): 1489-1499.

[16] Mao Z, Chen R, Wang X, et al. CRISPR / Cas12a-based technology: Apowerful tool for biosensing in food safety[J]. Trends in Food Science&Technology, 2022, 122: 211-222.

[17] Li Y, Liu L, Liu G. CRISPR / Cas Multiplexed Biosensing: AChallenge or an Insurmountable Obstacle?[J]. Trends Biotechnol, 2019, 37(8):792-795.

[18] Santiago-Frangos A, Nemudryi A, Nemudraia A, et al. CRISPR-Cas,Argonaute proteins and the emerging landscape of amplification-freediagnostics[J]. Methods, 2022, 205: 1-10.

[19] Rahimi S, Balusamy S R, Perumalsamy H, et al. CRISPR-Cas targetrecognition for sensing viral and cancer biomarkers[J]. Nucleic Acids Res,2024, 52(17): 10040-10067. Summary of the Invention To address the aforementioned issues, this invention proposes an innovative cascaded amplification detection strategy: by establishing a fast-response HDA system as an amplifier and combining it with CRISPR-Cas12a detection technology as a highly specific signal output, a novel HDA-Cas12a biosensor is constructed. This invention constructs an HDA technology platform for amplifying ultrashort gene fragments, specifically ultrashort gene fragments of approximately 40 bp in size, which is named ultrashort HDA (usHDA). The established usHDA reaction is fast, yielding a large number of amplicones in just 30 minutes. The usHDA amplified fragment contains only the primer-binding region and the crRNA complementary binding region, thus meeting the requirements for CRISPR-Cas12a detection. The crRNA complementary binding region (CBR) sequence is 20 nt. Primers are designed on both sides of the CBR. Each primer binding region for mesophilic HDA is approximately 15-16 nt. Therefore, the size of the mesophilic HDA amplification fragment for CRISPR-Cas12a detection, designed conventionally, is approximately 51-53 bp. To improve the efficiency of the helicase, this invention further shortens the amplification fragment. Taking the detection of common influenza A virus as an example, the usHDA-Cas12a detection of influenza A virus produces an amplification product fragment of only 42 bp. Combining this with CRISPR / Cas12a technology to detect ultrashort amplicon significantly improves the sensitivity and specificity of detection. The usHDA-Cas12a detection system constructed in this invention exhibits higher speed, sensitivity, and specificity, thereby improving its practicality. It can also be extended to the detection of other target nucleic acids, showing good application prospects.

[0005] To address the aforementioned technical problems, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides an ultrashort helicase-dependent isothermal amplification detection reagent based on CRISPR / Cas12a.

[0006] In some embodiments of the present invention, the detection reagent includes crRNA and upstream and downstream primers.

[0007] In some embodiments of the present invention, the amplification fragment length of the upstream and downstream primers is 40~45bp.

[0008] In some embodiments of the present invention, the upstream and downstream primers are partially complementary to the crRNA.

[0009] In some embodiments of the present invention, the amplification fragments of the upstream and downstream primers include primer-binding regions and complementary binding regions of crRNA.

[0010] In some embodiments of the present invention, the nucleic acid target includes DNA or RNA.

[0011] In some embodiments of the present invention, the RNA includes RNA viruses.

[0012] In some embodiments of the present invention, the RNA virus is an influenza A virus.

[0013] This invention provides a reagent for detecting influenza A virus based on CRISPR / Cas12a ultrashort helicase-dependent isothermal amplification, using influenza A virus as an example.

[0014] In some embodiments of the present invention, the sequence of the crRNA is shown in SEQ ID NO: 7.

[0015] In some embodiments of the present invention, the sequences of the upstream and downstream primers are shown as SEQ ID NO: 2 and 5, respectively.

[0016] A second aspect of the present invention provides the use of the reagents of the first aspect of the present invention in the preparation of products for detecting nucleic acids.

[0017] In some embodiments of the present invention, the nucleic acid includes influenza A virus.

[0018] A third aspect of the present invention provides a detection kit comprising a fluorescent reporter probe, an enzyme, and a reaction buffer.

[0019] In some embodiments of the present invention, the enzyme includes Cas protein, polymerase, helicase, single-strand binding protein, and repair protein.

[0020] In some embodiments of the present invention, the Cas protein includes Cas12a.

[0021] In some embodiments of the present invention, the helicase includes T4gp41 helicase, PcrA helicase, and T7gp4A helicase.

[0022] A fourth aspect of the present invention provides a method for detecting nucleic acids for non-disease treatment or diagnostic purposes, comprising the following steps: The reagents, fluorescent reporter probes, enzymes, and reaction buffers in the detection kit described in the third aspect of this invention are used to react with the test sample to detect the target gene.

[0023] In some embodiments of the present invention, the reaction temperature is 35~40°C; In some embodiments of the present invention, the reaction time is 15 to 90 minutes.

[0024] Specifically, the present invention provides a one-step method for detecting nucleic acids: The reactions of the usHDA and Cas12a systems are integrated into one EP tube, placed at the bottom of the EP tube or on the reaction cap (in any order).

[0025] The sample is first incubated with the usHDA reaction system at 37°C for 15-90 min, preferably 30 min; then the amplification product is mixed with the Cas12a system and incubated at 37°C for 1 hour, and the fluorescence is detected.

[0026] The usHDA reaction system includes: upstream and downstream primers, dNTPs, helicase, Klenow Fragment (Exo-), T4gene protein 32, repair enzyme, and detection sample.

[0027] The Cas12a system includes Cas protein, crRNA, and fluorescent reporter probe.

[0028] This invention provides a one-tube nucleic acid detection technology based on usHDA-CRISPR / Cas12a, taking the detection of influenza A virus as an example. Its technical principle is as follows: To conduct a single-tube detection using the usHDA-CRISPR / Cas12a method, usHDA and Cas12a reagents are first added to the bottom and cap of the reaction tube, respectively. The usHDA-Cas12a primer pair is designed specifically for influenza A virus and can amplify ultrashort amplicon under isothermal conditions of 37°C for 30 min. The experiment begins with a 30-min isothermal amplification reaction at the bottom of the reaction tube. Helicase opens the DNA double strand, forming a single DNA strand, and the primer binds to the complementary region of the single-stranded DNA. DNA polymerase then amplifies and extends the DNA to form a new double strand. This new double strand is then amplified again, and the cycle repeats to generate a large amount of double-stranded DNA product containing the PAM sequence. The usHDA amplification product binds to Cas12a, activating its trans-cleavage activity, which cleaves the fluorescent reporter probe and releases a fluorescent signal. A schematic diagram is shown below. Figure 1 As shown.

[0029] Based on this principle, those skilled in the art can also apply it to the preparation of detection reagents for other viruses.

[0030] The beneficial effects of this invention are: (1) Solving the problem of low detection sensitivity of HDA. The HDA reaction system is similar to PCR, requiring only two primers for amplification. It is considered a simplified version of PCR, and is easy to operate, but its sensitivity is relatively low. Precisely because of its low sensitivity, compared with other isothermal amplification techniques, there are fewer application studies of HDA, resulting in its immature development. HDA is simple to operate, and if its sensitivity problem can be solved, it has good application prospects. The low sensitivity of HDA is mainly due to the limitation of helicase speed in the amplification reaction. For longer target sequences, a longer amplification time and more helicase are required. Since the amplification efficiency of HDA depends on the unwinding speed of helicase, improving the working efficiency of helicase in the amplification reaction is the solution to this problem. This invention innovatively proposes the concept of ultra-short HDA (usHDA), which significantly improves the detection sensitivity, with a detection limit (LOD) of 5 aM.

[0031] (2) Solving the problem of non-specific interference in HDA. HDA reactions are prone to producing primer dimers, affecting their specificity and potentially leading to false positives, thus hindering their widespread application. Therefore, improving the specificity of HDA is a key technical issue, and the problem of non-specific interference in HDA reactions urgently needs to be solved. This invention, based on USHDA, combines CRISPR / Cas12a detection technology to perfectly solve the problem of non-specific interference in HDA.

[0032] (3) This invention innovatively proposes the concept of ultra-short HDA (usHDA) and establishes a usHDA technology that completes the amplification reaction within 30 minutes. Traditional HDA reactions, whether mesophilic or thermophilic, typically take 1.5 to 2 hours or even longer, making it difficult to complete the amplification reaction within 1 hour. Therefore, the long reaction time of traditional HDA amplification is a key problem that urgently needs to be solved. This invention innovatively proposes the concept of ultra-short HDA (usHDA) and establishes a usHDA technology that completes the amplification reaction within 30 minutes, significantly shortening the experimental time.

[0033] (4) A CRISPR-Cas12a-usHDA detection technology based on the same tube reaction was established, which solved the technical problem that isothermal amplification and CRISPR-Cas12a detection require opening the lid and performing step-by-step experiments, and reduced the risk of contamination. HDA is divided into two types: mesothermal

[13] and thermophilic. Mesothermal HDA can react under mild conditions of 37℃, while thermophilic HDA usually reacts under conditions of 65℃. The inventors' research group has reported a detection technology of thermophilic HDA combined with CRISPR-Cas12a, but thermophilic HDA reacts under higher temperature conditions, which makes the enzyme activity of Cas12a easily affected during the same tube reaction. Mesothermal HDA can react under mild conditions of 37℃, but the amplification efficiency of mesothermal HDA is lower than that of thermophilic HAD. This invention innovatively proposes the concept of ultra-short HDA (usHDA), which improves the detection sensitivity and is also compatible with the reaction temperature conditions of CRISPR-Cas12a. However, isothermal amplification and CRISPR-Cas12a detection usually adopt a two-step method, which is prone to aerosol contamination. This invention establishes a CRISPR-Cas12a-usHDA detection technology based on co-tube reaction, which solves the technical problem of requiring open-cap stepwise experiments for isothermal amplification and CRISPR-Cas12a detection. It also solves the problems of low sensitivity and non-specific interference detection of HDA, and shortens the amplification reaction time to 30 min, with the entire detection experiment taking no more than one hour. Attached Figure Description

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The experimental principle of the usHDA-CRISPR / Cas12a one-tube method for nucleic acid detection.

[0035] Figure 2 The results of usHDA component deficiency verification include: M) DNA marker, 1) no target, 2) no helicase, 3) no polymerase, and 4) complete reaction components.

[0036] Figure 3 The results show the time optimization for amplifying ultrashort gene fragments of usHDA, where M) DNA marker, 1.) NC, 2, 3, 4, and 5 represent amplification times of 15 min, 30 min, 45 min, and 60 min, respectively.

[0037] Figure 4The amplification effect of different gene fragments of usHDA at 30 min was compared, where: M) DNA marker; 1) 42 bp NC group (no target); 2) 42 bp experimental group (with target); 3) 61 bp NC group (no target); 4) 61 bp experimental group (with target); 5) 90 bp NC group (no target); 6) 90 bp experimental group (with target).

[0038] Figure 5 The results of the feasibility verification of usHDA-CRISPR / Cas12a detection of influenza A virus include: A) Exposure image of the feasibility verification of influenza A virus detection; B) Fluorescence curve of the feasibility verification of influenza A virus detection.

[0039] Figure 6 Sensitivity analysis of usHDA-CRISPR / Cas12a for detecting influenza A virus, including: A) fluorescence curve for sensitivity analysis of influenza A virus detection; B) linearity analysis graph for sensitivity analysis of influenza A virus detection.

[0040] Figure 7 Fluorescence curve results for specificity analysis of usHDA-CRISPR / Cas12a for detecting influenza A virus. Detailed Implementation

[0041] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0042] The materials involved in this invention are as follows: LbCas12a protein (purchased from New England Biotechnology Co., Ltd.), Klenow Fragment (Exo-), T4gene protein 32, DNase-free water (purchased from Shanghai Beyotime Biotechnology Co., Ltd.), T4 gp41 DNA helicase (purchased from Shanghai Yubo Biotechnology Co., Ltd.), MutL protein (Shanghai Taoshu Biotechnology Co., Ltd.), dNTPs, ATP (purchased from Sangon Biotech Co., Ltd.). The synthesis of all DNA and RNA was outsourced to Sangon Biotech.

[0043] Table 1 Synthetic Sequences

[0044] Example 1: Feasibility Verification of usHDA Amplification of Ultrashort Gene Fragments To verify the feasibility of amplifying ultrashort gene fragments with usHDA, a verification experiment was first conducted in this embodiment.

[0045] Using 100 pM synthesized DNA as a template and primer concentration of 10 μM, usHDA amplification experiments were performed. The experiments were divided into an experimental group with complete components, a template deletion group, a helicase deletion group, and a polymerase deletion group.

[0046] usHDA amplification system. Primers FP1 and RP1, 3 mM ATP, 0.4 mM dNTP, 12 ng / uL LT4 gp 41, 0.16 U / uL Klenow Fragment (Exo-), 60 ng / uL T4 gene protein 32, 8 ng / uL MutL, and the influenza A virus target nucleotide template were prepared in 1× mixing buffer to a final concentration of 400 nM. The volume was adjusted to 20 μL with RNase-free water and added to the bottom of an EP tube. The mixture was incubated at 37 °C for 30 min. Agarose gel electrophoresis results showed that the experimental group had a clear specific amplification band below 50 bp, and the band size matched the expected fragment. No electrophoretic bands were observed in the template-deficient group, helicase-deficient group, and polymerase-deficient group. (See attached diagram). Figure 2 .

[0047] Example 2: Time optimization for amplifying ultrashort gene fragments with usHDA Based on the usHDA component deletion verification experiment, the amplification time for amplifying ultrashort gene fragments was optimized.

[0048] usHDA amplification experiments were performed using 100 pM synthesized DNA as a template and primers at a concentration of 10 μM. The experiment was divided into an experimental group containing the target template and a control group (NC group) without a template, with primer concentrations of 10 μM in each group. To determine the amplification reaction time, amplification was performed at different times, namely 15 min, 30 min, 45 min, and 60 min, and named the 15 min group, 30 min group, 45 min group, and 60 min group, respectively.

[0049] The usHDA amplification system was prepared in 1× mixing buffer with a final concentration of 400 nM primers FP1 and RP1, 3 mM ATP, 0.4 mM dNTP, 12 ng / uL L4 gp 41, 0.16 U / uL Klenow Fragment (Exo-), 60 ng / uL T4 gene protein 32, 8 ng / uL MutL, and influenza A virus target nucleotide template. The volume was adjusted to 20 μL with RNase-free water and added to the bottom of an EP tube. The mixture was incubated at 37 °C for different times. Agarose gel electrophoresis results showed that a clear specific amplification band was visible after 15 min of amplification, and the specific amplification product band was more obvious after 30 min. Further amplification at 45 min and 60 min did not show a significant increase in the amplification product band. Therefore, this experiment determined 30 min as the optimal amplification time for usHDA amplification of ultrashort gene fragments. Results are shown below. Figure 3 .

[0050] Example 3: Comparison of amplification effects of usHDA on gene fragments of different sizes after 30 min. To compare the amplification effect of usHDA on gene fragments of different sizes after 30 min, primers for amplifying fragments of different sizes, such as 42 bp, 61 bp, and 90 bp, were synthesized and tested.

[0051] Using 100 pM DNA as a template, the primer concentration for each group was 10 μM. The experiment was divided into experimental groups of 42 bp, 61 bp, and 90 bp and a control group without template.

[0052] usHDA amplification system. Forward and reverse primers with a final concentration of 400 nM were prepared in 1× mixing buffer (FP1 and RP1 were for amplifying a 42 bp target). StandardPrimer pairs for the fragment (FP2 and RP1 for amplifying the 61 bp target fragment, FP3 and RP2 for amplifying the 90 bp target fragment), 3 mM ATP, 0.4 mM dNTP, 12 ng / uL L4 gp 41, 0.16 U / uL Klenow Fragment (Exo-), 60 ng / uL T4 gene protein 32, 8 ng / uL MutL, and target DNA were added to the bottom of an EP tube, and the volume was adjusted to 20 μL with RNase-free water. The mixture was incubated at 37 ℃ for 30 minutes. Agarose gel electrophoresis results showed that when amplified for 30 minutes, only the 42 bp experimental group showed an electrophoretic band of 42 bp size slightly below 50 bp, while the control group did not show any corresponding amplification product electrophoretic band. No electrophoretic bands were observed in other experimental groups or the control group, indicating that 30 minutes of amplification of usHDA is suitable for amplifying ultrashort gene fragments. See results Figure 4 .

[0053] Example 4: Feasibility Experiment of usHDA-CRISPR / Cas12a One-Tube Method for Detecting Influenza A Virus (H1N1) After establishing the usHDA technology platform, the usHDA-CRISPR / Cas12a one-tube detection method was further validated.

[0054] Using 100 pM synthetic DNA as a template and primer concentration of 10 μM, the experiment included an experimental group with complete reaction components, a helicase-deficient group, a polymerase-deficient group, a Cas12a-deficient group, a crRNA-deficient group, a fluorescent reporter probe-deficient group, and a control group without template DNA.

[0055] The reactions of the usHDA amplification system and the Cas12a system were integrated into a single EP tube. Primers FP1 and RP1, 3 mM ATP, 0.4 mM dNTP, 12 ng / uL L4 gp41, 0.16 U / uL Klenow Fragment (Exo-), 60 ng / uL T4 gene protein 32, 8 ng / uL MutL, and the influenza A virus target nucleotide template were prepared in 1× mixing buffer to a final concentration of 400 nM and added to the bottom of the EP tube with RNase-free water. 50 nM LbCas12a, 100 nM crRNA, and 500 nM probe were prepared in 1× mixing buffer and adjusted to a final volume of 5 μL with RNase-free water, then placed on the EP tube cap. The tube was incubated at 37 °C for 30 minutes, followed by simple centrifugation to mix the Cas12a system from the cap with the amplification products at the bottom of the tube, and incubated at 37 °C for 1 hour. The fluorescence signal of the reaction was detected using a real-time quantitative PCR instrument, and the corresponding fluorescence curves were obtained. The results showed that the experimental group had obvious amplification curves, while no fluorescence amplification curves were observed in the helicase-deficient group, polymerase-deficient group, Cas12a-deficient group, crRNA-deficient group, fluorescent reporter probe-deficient group, and the control group without template DNA. (See attached results). Figure 5 .

[0056] Example 5: Sensitivity test of usHDA-CRISPR / Cas12a single-tube method for detecting influenza A virus. To evaluate the methodology of the established usHDA-CRISPR / Cas12a one-tube method, the detection sensitivity was first evaluated.

[0057] The synthesized influenza A DNA template was serially diluted, with a 10-fold standard dilution, from 1 nM to 1 aM, resulting in a total of 9 template concentrations for the experimental groups. Primers were at a concentration of 10 μM, and a blank control was also included.

[0058] The reactions of the usHDA amplification system and the Cas12a system were integrated into a single EP tube. Primers FP1 and RP1, 3 mM ATP, 0.4 mM dNTP, 12 ng / uL L4 gp 41, 0.16 U / uL Klenow Fragment (Exo-), 60 ng / uL T4 gene protein 32, 8 ng / uL MutL, and 2 μL of influenza A virus target nucleotide template were prepared in 1× mixing buffer and adjusted to a volume of 20 μL with RNase-free water before being added to the bottom of the EP tube. 50 nM LbCas12a, 100 nM crRNA, and 500 nM probe were prepared in 1× mixing buffer and adjusted to a volume of 5 μL with RNase-free water before being placed on the EP tube cap. Initially, the mixture was incubated at 37 °C for 30 minutes, followed by centrifugation to mix the Cas12a system with the amplification products, and then incubated at 37 °C for 1 hour. The fluorescence signal of the reaction was detected using a real-time quantitative PCR instrument, and the corresponding fluorescence curves were obtained. The fluorescence curves of the experimental group showed that the fluorescence intensity increased with increasing template concentration, while no obvious fluorescence signal was observed in the control group. The results are shown in the figure below. Figure 6 In section A, a sensitivity linearity plot for H1N1 virus detection was further plotted. The results showed a good linear relationship between reaction time and fluorescence intensity in the range of 10 aM to 1 pM. The lowest detection limit (LOD) was calculated to be 5 aM, with the mean fluorescence intensity of the lowest concentration group plus three times the standard deviation as the lowest detection fluorescence value. See section A. Figure 6 B.

[0059] Example 6: Specificity assay for detecting H1N1 influenza virus using the usHDA-CRISPR / Cas12a one-tube method. After evaluating the sensitivity of the usHDA-CRISPR / Cas12a one-tube method, its detection specificity was further evaluated.

[0060] Using 100 pM synthetic target DNA as a template and 10 μM primers, usHDA-CRISPR / Cas12a one-tube method experiments were conducted. Different experimental groups were set up for influenza A virus (Inf-A), influenza B virus (Inf-B), respiratory syncytial virus A (RSV-A), respiratory syncytial virus B (RSV-B), human parainfluenza virus 1 (HPIV1), human parainfluenza virus 3 (HPIV3), and metapneumovirus (HMPV). A template-free control group was also set up.

[0061] The reactions of the usHDA amplification system and the Cas12a system were integrated into a single EP tube. Primers FP1 and RP1, 3 mM ATP, 0.4 mM dNTP, 12 ng / uL L4 gp41, 0.16 U / uL Klenow Fragment (Exo-), 60 ng / uL T4 gene protein 32, 8 ng / uL MutL, and 2 μL of influenza A virus target nucleotide template were prepared in 1× mixing buffer and adjusted to a volume of 20 μL with RNase-free water before being added to the bottom of the EP tube. 50 nM LbCas12a, 100 nM crRNA, and 500 nM probe were prepared in 1× mixing buffer and adjusted to a volume of 5 μL with RNase-free water before being placed on the EP tube cap. The mixture was first incubated at 37 °C for 30 minutes, followed by brief centrifugation to mix the Cas12a system with the amplification products, and then incubated at 37 °C for 1 hour. The fluorescence signal of the reaction was detected using a real-time quantitative PCR instrument, and the corresponding fluorescence curves were obtained. The results showed that only the influenza A virus experimental group showed a strong fluorescence signal, while no obvious fluorescence signal was detected in the other non-influenza virus groups. (See attached results.) Figure 7 .

[0062] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A reagent for detecting nucleic acid targets, characterized in that: The detection reagent includes crRNA and upstream and downstream primers; The amplified fragment length of the upstream and downstream primers is 40~45bp; The upstream and downstream primers are partially complementary to the crRNA; The amplification fragments of the upstream and downstream primers include primer-binding regions and complementary binding regions of crRNA.

2. The reagent according to claim 1, characterized in that: The nucleic acid targets include DNA or RNA.

3. The reagent according to claim 2, characterized in that: The RNA includes RNA viruses; Preferably, the RNA virus is an influenza A virus.

4. The reagent according to claim 3, characterized in that: The sequence of the crRNA is shown in SEQ ID NO: 7; The sequences of the upstream and downstream primers are shown in SEQ ID NO: 2 and 5, respectively.

5. The use of the reagents described in claims 1-4 in the preparation of products for detecting nucleic acids; Preferably, the nucleic acid includes influenza A virus.

6. A test kit, characterized in that: The detection kit also includes a fluorescent reporter probe, an enzyme, and a reaction buffer.

7. The detection kit according to claim 5, characterized in that: The enzymes include Cas proteins, polymerases, helicases, single-chain binding proteins, and repair proteins.

8. The detection kit according to claim 6, characterized in that: The Cas protein includes Cas12a.

9. A method for detecting nucleic acid for non-disease treatment or diagnostic purposes, comprising the following steps: The reagents, fluorescent reporter probes, enzymes, and reaction buffers in the detection kit according to any one of claims 6 to 7 are used to react with the test sample for detection.

10. The method according to claim 9, characterized in that: The reaction temperature is 35~40℃; The reaction time is 15-90 min.

Images