Kit for detecting pathogen nucleic acid based on catalytic hairpin assembly and rolling circle amplification technology and application of kit

Abstract

The invention discloses a kit for detecting pathogen nucleic acid based on a catalytic hairpin assembly and rolling circle amplification technology and application of the kit, and belongs to the technical field of pathogen detection. The kit comprises a hairpin H1, a hairpin H2, a capture probe CP, an auxiliary probe HP, dNTP, bovine serum albumin, ligase, polymerase and a buffer solution. According to the method for detecting the pathogen nucleic acid based on the catalytic hairpin assembly connection mediated rolling circle amplification (CHAC-RCA) technology, the advantage that a catalytic hairpin assembly (CHA) target can be recycled is utilized, CHA is fused into a padlock probe (PLP) connection process, so that the ring forming efficiency of the PLP is improved, the detection sensitivity of rolling circle amplification (RCA) is improved, and the detection sensitivity of the pathogen nucleic acid is improved. Theoretical support is provided for new application of the RCA technology, testing is carried out by taking monkey pox as an example, and an empirical method is provided for detection of pathogens including monkey pox viruses.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and particularly to a kit for detecting pathogen nucleic acids based on catalytic hairpin assembly and rolling circle amplification technology and its application. Background Art

[0002] Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV). MPXV belongs to the Orthopoxvirus genus of the Poxviridae family, along with the highly pathogenic smallpox virus and vaccinia virus. MPXV is an enveloped double-stranded DNA virus, and its genetic lineages are divided into lineage I (from the Central African-Congo Basin) and lineage II (from West Africa). The World Health Organization reported that since May 2022, cases of monkeypox infection that were previously prevalent in Africa have increased sharply in non-endemic countries. Approximately 87,000 cases and 112 deaths have been reported in 110 countries, and monkeypox has received extensive attention in international public health events. In 2024, the World Health Organization reported a new variant of monkeypox - clade Ib, which can be transmitted via droplets, making it a public health threat. In August 2024, the World Health Organization declared that monkeypox constitutes the second public health emergency of international concern.

[0003] Diagnosis of patients infected with pathogens represented by monkeypox usually requires a combination of clinical symptoms, epidemiological information, and laboratory tests. Compared with other diagnostic methods, nucleic acid amplification testing is faster, more sensitive, and more specific, and is the main choice for diagnosing this virus. According to the recommendations of the World Health Organization, nucleic acid amplification testing should be performed on suspected monkeypox patients. Real-time polymerase chain reaction is the gold standard and the most common strategy for monkeypox virus diagnosis. Conserved genes of monkeypox commonly used for this method of detection include hemagglutinin (HA), extracellular envelope protein (B6R), and DNA polymerase gene (E9L), etc. However, the lack of specialized nucleic acid testing laboratories and operators, the cold chain transportation for sample preservation, and the need for on-site rapid detection have greatly hindered the on-site diagnosis of suspected monkeypox patients in remote areas. Therefore, it is particularly important to develop a detection system that is fast, accurate, low-cost, and does not rely on professional sites and personnel.

[0004] Biomarkers such as proteins, nucleic acids, and ions are often used to monitor health and diseases. Compared with protein biomarkers, nucleic acid biomarkers are small in size, programmable, and can respond to disease states at the transcriptional level. Therefore, nucleic acid-based biosensors are the most promising fields for early precise diagnosis and treatment. Nucleic acid amplification technology can greatly amplify nucleic acid fragments related to specific target nucleic acids, and thus has high sensitivity and specificity even at low sample volumes. Nucleic acid amplification has been widely used in fields such as forensic medicine, food safety, and clinical diagnosis, and many have been used to develop commercial and scalable diagnostic technologies.

[0005] Polymerase Chain Reaction (PCR), as a mature and widely used gene amplification assay technology, has long been the gold standard in fields such as epidemiology, forensic medicine, and paleontology. However, PCR technology has some drawbacks, such as being prone to contamination, having expensive equipment, and being prone to false positives. Coupled with the need for skilled staff and precise thermal cyclers, it limits its application in resource-limited environments.

[0006] To circumvent the limitations of PCR in the molecular amplification stage, many isothermal nucleic acid amplification technologies have been widely proposed and applied in recent years. These methods mainly include loop-mediated isothermal amplification, strand displacement amplification, rolling circle amplification, recombinase polymerase amplification, etc. Compared with PCR, isothermal amplification is carried out at the same temperature throughout the process and does not require a thermal cycler. Therefore, the requirements for instruments are simplified, the reaction time is significantly shortened, and it has similar sensitivity to current real-time fluorescence quantitative PCR tests, better meeting the requirements of modern molecular detection technology for being fast and simple. It has become a hot topic of concern in academic research and industry. Isothermal amplification technology can detect a wide range of target types, and its combination with nanotechnology has also attracted strong attention. Some have formed mature commercial products and have broad prospects in the applications of biosensing, bioimaging, and nanomedicine. Among them, Rolling Circle Amplification (RCA), as an isothermal amplification method, has received great attention in biosensing applications.

[0007] The basic elements of the RCA reaction system are four: DNA polymerase (such as phi29 DNA polymerase), primer, circular template, and dNTP. Among them, the cyclizable oligonucleotide is also called the "padlock" probe (Padlock Probe, PLP). The target first hybridizes to the end of the PLP as a ligation template, and the PLP is then cyclized, forming a closed loop through enzymatic ligation. Then, the primer extends along the 5'→3' direction under the catalysis of the polymerase to generate a single-stranded product containing a large number of tandem repeats, and the presence of a specific DNA sequence can be detected. However, since one target primer is used to ligate one PLP during the process of traditional padlock probe targeting cyclization, limited by the molecular collision efficiency, ligase working efficiency, and side reactions (non-specific cyclization and intermolecular cyclization) during the cyclization process, PLP is usually difficult to be fully utilized, resulting in a reduction in the sensitivity of RCA and limiting the application of RCA technology. Summary of the Invention

[0008] The object of the present invention is to provide a kit for detecting pathogen nucleic acid based on catalytic hairpin assembly and rolling circle amplification techniques and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a method for detecting pathogen nucleic acid based on catalytic hairpin assembly-and-circularization (CHAC) technology. By taking advantage of the recyclable target of catalytic hairpin assembly (CHA), CHA is incorporated into the PLP ligation process to improve the ligation efficiency of PLP, thereby enhancing the detection sensitivity of RCA, providing theoretical support for new applications of RCA technology and providing an empirical method for the detection of viral nucleic acid.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] In the first aspect, the present invention provides a kit for detecting pathogen nucleic acid, comprising hairpin H1, hairpin H2, capture probe CP, auxiliary probe HP, dNTP, bovine serum albumin, ligase, polymerase and buffer;

[0011] The hairpin H1 is provided with a target recognition region and a CP binding region, and forms a hairpin structure after heat treatment; the target recognition region is complementary to the pathogen nucleic acid; the CP binding region is complementary to the capture probe CP;

[0012] The hairpin H2 is provided with an H1 binding region and an HP binding region, and forms a hairpin structure after heat treatment; the H1 binding region is complementary to the hairpin H1; the HP binding region is complementary to the auxiliary probe HP;

[0013] The capture probe CP is provided with an HP binding region and an H1 binding region; the HP binding region is complementary to the auxiliary probe HP; the H1 binding region is complementary to the hairpin H1;

[0014] The auxiliary probe HP is provided with a CP binding region and an H2 binding region; the CP binding region is complementary to the capture probe CP; the H2 binding region is complementary to the hairpin H2;

[0015] The 5'-ends of the hairpin H1 and the auxiliary probe HP are phosphorylated.

[0016] Further, the ligase is a DNA ligase; the polymerase is a DNA polymerase.

[0017] Optionally, the ligase is T4 DNA ligase; the polymerase is phi29 DNA polymerase.

[0018] Further, the heat treatment process is to react at 92°C - 98°C for 4 - 6 min and then cool down to 25°C at a gradient of 1°C / min.

[0019] Further, when the pathogen is monkeypox virus, the nucleotide sequence of the hairpin H1 is as shown in SEQ ID NO.2; the nucleotide sequence of the hairpin H2 is as shown in SEQ ID NO.3; the nucleotide sequence of the capture probe CP is as shown in SEQ ID NO.4; the nucleotide sequence of the helper probe HP is as shown in SEQ ID NO.5.

[0020] In a second aspect, the present invention provides an application of the above kit in the preparation of a product for detecting pathogen nucleic acid, wherein the pathogen nucleic acid includes monkeypox virus nucleic acid.

[0021] In a third aspect, the present invention provides a method for using the above kit, comprising the following steps:

[0022] S1. Extract the genome from the sample to be tested;

[0023] S2. Mix the genome, hairpin H1, hairpin H2, capture probe CP, helper probe HP, ligase, bovine serum albumin and buffer, and incubate at 36°C - 38°C for 30 - 120 min to obtain a catalytic hairpin assembly ligation (CHAC) reaction product;

[0024] S3. Mix the CHAC reaction product with polymerase, bovine serum albumin, dNTP and buffer, and incubate at 36°C - 38°C for 60 - 120 min to obtain an RCA product;

[0025] S4. Perform agarose gel electrophoresis on the RCA product and observe whether long single-stranded products are generated;

[0026] The above method of use is for non-diagnostic purposes.

[0027] The present invention uses the combination of CHA and RCA technologies to detect pathogen nucleic acid. Utilizing the advantage of the recyclability of the CHA target, CHA is incorporated into the PLP ligation process, and through the catalytic hairpin assembly-mediated padlock probe ligation reaction, the ligation efficiency of PLP is improved. Specifically, the hairpin H2 is used to bind to the hairpin H1 to displace the target strand, so that the target can be reused without being consumed. The CHA process enables a single target to generate multiple ligase recognition sites, improving the low efficiency problem in the traditional RCA reaction where a single target can only be recognized and ligated into a loop by a single PLP, and enhancing the ligation efficiency of RCA. The CHA reaction can greatly promote the RCA reaction and synergistically generate amplified signals to achieve ultrasensitive nucleic acid detection.

[0028] The detection principle of the method of using the kit of the present invention is asFigure 1 As shown, specifically, when the target is present, the target first hybridizes with the hairpin H1 by means of a foothold and opens the hairpin H1. Subsequently, the foothold of the hairpin H2 recognizes and binds to the single-stranded part exposed by the hairpin H1, hybridizes with H1 through strand displacement triggered by the foothold, and at the same time the target is released into the next cycle to produce more double-stranded products. CP and H2 act as connecting templates, and under the action of ligase, H1 and HP are connected end to end to form a circular template, thereby initiating the subsequent RCA reaction to generate long single-stranded DNA products. Qualitative analysis of the target can be achieved by detecting the final reaction product using agarose gel electrophoresis.

[0029] Optionally, in step S1, the working concentrations of the hairpin H1, hairpin H2, capture probe CP, and auxiliary probe HP are all 100-200 nM.

[0030] Optionally, in step S1, the molar ratio of the hairpin H1, hairpin H2, capture probe CP, and auxiliary probe HP is 1:1:1:1.

[0031] Optionally, in step S1, the working concentration of the ligase is 0.05-0.15 U / μL; the working concentration of bovine serum albumin is 0.1-0.3 mg / mL.

[0032] Optionally, in step S2, the working concentration of the polymerase is 0.15-0.18 U / μL; the working concentration of bovine serum albumin is 0.1-0.3 mg / mL.

[0033] The present invention discloses the following technical effects:

[0034] (1) The detection method provided by the present invention has low requirements for sample pretreatment during the detection process (the robustness of RCA is relatively good), and can be used for detection in serum, skin exudate, etc.

[0035] (2) The kit provided by the present invention can greatly reduce the detection cost during the outbreak of disease epidemics.

[0036] (3) The detection method or kit provided by the present invention combines catalytic hairpin assembly and padlock probe ligation for the first time to detect the conserved region sequence of monkeypox virus. By taking advantage of the recyclable target of catalytic hairpin assembly, the bottleneck that the cyclization efficiency of PLP in traditional RCA is less than 1 is broken through, and the efficiency of RCA is greatly improved. The selected target gene sequence is widely present in the orthopoxvirus genus, improving the applicable range of poxvirus detection. Compared with RT-PCR and other existing detection methods, this method has low cost, short time consumption, relatively low technical requirements for operators, high accuracy, and is applicable to on-site detection under limited environmental conditions.

[0037] (4) The molecular strategy on which the detection method or kit provided by the present invention is based can construct a biosensing system adapted to different pathogens by flexibly replacing specific target sequences and their complementary pairing sequences. Under limited conditions, this system can achieve efficient and instant detection of multiple pathogens. This ability has extremely significant public health significance for monitoring and preventing pathogens with a wide spread risk and high danger, and provides strong technical support for the formulation and implementation of disease prevention and control strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0039] Figure 1 It is the detection principle diagram of the usage method of the kit of the present invention;

[0040] Figure 2 It is the electrophoretic verification result diagram of the feasibility of the CHA and ligase reactions;

[0041] Figure 3 It is the electrophoretic verification result diagram of the CHAC target cycle and its efficiency;

[0042] Figure 4 For Figure 3 the gray-scale analysis result diagram of the CHA reactants and products bands in

[0043] Figure 5 It is the electrophoretic result diagram of the performance comparison verification between traditional rolling circle amplification and rolling circle amplification based on catalytic hairpin assembly ligation. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0048] As used herein, terms such as "comprising", "including", "having", "containing", etc. are open-ended terms, meaning including but not limited to.

[0049] In the present invention, the rolling circle amplification (RCA) technology is an isothermal amplification method. The basic elements of the RCA reaction system are four: DNA polymerase (such as phi29 DNA polymerase), primer, circular template, and dNTP. Among them, the circularizable oligonucleotide is also called the "padlock" probe (PLP). The target first hybridizes to the end of the PLP as a ligation template, and the PLP is immediately circularized, forming a closed loop through enzymatic ligation. Then, the primer extends along the 5'→3' direction under the catalysis of the polymerase, generating a single-stranded product containing a large number of tandem repeats, and thus the presence of a specific DNA sequence can be detected.

[0050] In the present invention, the catalytic hairpin assembly (CHA) is a chemical reaction that uses two partially complementary DNA hairpins (H1 and H2) and a single-stranded oligonucleotide (I). Both hairpins consist of three consecutive domains, and each domain contains a toehold. First, I triggers the allosteric change of H1 by specifically binding to the toehold of H1, forming an H1-I intermediate product; subsequently, the exposed toehold of H1 initiates the assembly reaction with H2, H2 is opened, generating an unstable complex H1-I-H2; H2 triggers branch migration, and I separates from H1 and enters the next cycle.

[0051] The formation process of the CHA product mainly relies on the free energy released by strand displacement, realizes signal amplification through hairpin self-assembly, and can avoid false positive results caused by the accumulation of a large number of targets compared with PCR. In addition, changing the base sequence of the primer strand will significantly affect the reaction efficiency, endowing it with excellent ability to recognize single-base mutations. More importantly, the primer strand can cyclically trigger the hybridization of hairpins to obtain a large number of stable double-stranded complexes, achieving a target signal ratio of 1:N, amplifying the signal by 50-100 times in a short time, and breaking through the fundamental bottleneck of traditional DNA detection.

[0052] General description: In the following examples, the enzyme concentration involved is used in excess to ensure the maximum reaction efficiency. If the concentration is low, the reaction efficiency will be reduced. The reaction time for each step is the shortest time to obtain the best signal, which is determined by the reaction efficiency. If the reaction efficiency is low, the same reaction result can be achieved by extending the reaction time. The reaction temperature of the CHA ligase is the temperature with the best signal-to-noise ratio, and the reaction temperature of RCA depends on the optimal working temperature of the enzyme and can be adjusted according to the enzyme used.

[0053] Sequence design in the method for detecting pathogen nucleic acid in Example 1

[0054] 1. Screening of the target gene sequence

[0055] Monkeypox virus is a linear double-stranded DNA virus. Currently, envelope protein gene (B6R), B7R, DNA polymerase gene E9L, complement-binding protein C3L, DNA-dependent RNA polymerase subunit 18 (RPO18), N3R, F3L, etc. have been developed for the detection of monkeypox virus. The target for detection in this invention is obtained by screening and aligning the existing monkeypox virus gene sequences, conducting conservation analysis, and finally selecting the conserved fragment of E9L in the monkeypox virus genome (gene name: OPG071), obtaining a highly conserved target gene sequence containing 29 nt, and the sequence information is shown in Table 1.

[0056] 2. Design of the nucleic acid sequences of other components

[0057] The technology of catalytic hairpin assembly ligation-mediated rolling circle amplification proposed by the present invention involves CHA, DNA ligation reaction and RCA reaction. The required components mainly include single-stranded target of monkeypox virus gene, H1, H2, CP, HP, T4 DNA ligase and phi29 DNA polymerase, etc. Hairpins H1 and H2 have special designs: H1 can recognize the target and act as a padlock probe after cyclization; while H2 binds to H1 in the CHA reaction to form a double-stranded product, and at the same time serves as a ligation template and a primer for the amplification reaction to initiate RCA. In the absence of the target, H1 and H2 maintain a stable hairpin structure; after the introduction of the target, the target can catalyze the hybridization of H1 and H2 and be recycled repeatedly. Therefore, it is necessary to ensure the stability of the hairpin structure; H1 and HP participate in the ligation reaction as padlock probes, so the nucleotide sequence ends need to be designed to perfectly match the ligation template (CP and H2), and the 5'-ends of H1 and HP need to be phosphorylated to meet the working conditions of the ligase. The binding force between H1 and the target gene (T) sequence and the hairpin structures of H1 and H2 are calculated by the online nucleic acid secondary structure prediction tool NUPACK software. The free energy values of H1 and H2 need to be lower than -10 kcal / mol to ensure the stability of the hairpin structure. The absolute value of the binding free energy of T and H1 should be lower than the absolute value of the free energy of H1 and H2 to improve the amplification efficiency. The sequence information of each component is shown in Table 1.

[0058] Table 1 Sequence Information

[0059]

[0060]

[0061] Note: The stem regions in the hairpin structures of H1 and H2 are underlined; the sequences in the table are all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0062] Example 2 Verification of CHAC Reaction and Verification of the Performance of CHAC-RCA Detection Method

[0063] The nucleic acid sequences involved in this example are shown in Table 1 of Example 1. In agarose gel electrophoresis, the migration rate of the circular DNA structure is slower than that of the linear DNA structure of the same length. This characteristic makes electrophoresis an effective qualitative verification method for detecting whether the padlock probe has been successfully ligated into a circle by the ligase. On the other hand, the products of RCA have complex structures and large molecular weights, which result in extremely low migration rates in agarose gels. Therefore, by observing the migration of RCA products in agarose gels, the success of the reaction amplification and hybridization reaction design can be qualitatively verified, and whether the expected nucleic acid fragments are generated can be verified.

[0064] The following are the main reagents and equipment involved in this example:

[0065] (1) Reagents: T4 DNA ligase (5 U / μL, Thermo Fisher, EL0014), 10×T4 DNA ligase buffer (Thermo Fisher, EL0014), phi29 DNA polymerase (10 U / μL, Thermo Fisher, EP0094), 10×phi29 DNA polymerase buffer (Thermo Fisher, EP0094), bovine serum albumin (BSA) (Beijing Solarbio Science & Technology Co., Ltd., A8010); dNTP mixture (10 mM / each, Beijing Solarbio Science & Technology Co., Ltd., PC2200), TE buffer (pH 8.0, Beijing Solarbio Science & Technology Co., Ltd., PC2200); agarose gel (100 g, Hispanagar S.A., BY-R0100); 10×TBE buffer (Beijing Primegene Biotechnology Co., Ltd., B1111) SYBR Gold (500 μL, ThermoFisher, S11494); 6×DNA Loading buffer (Hunan Akribiotech Co., Ltd., AG11902); GLDNA Marker 500 (Hunan Akribiotech Co., Ltd., AG11906); GL DNA Marker 1000 (Shanghai Heyuan Liji Biotechnology Co., Ltd., AN33L226).

[0066] Among them, dNTP is four kinds of deoxynucleoside triphosphates, which provide raw materials for DNA amplification; SYBR Gold is a fluorescent dye with a relatively high affinity for DNA, and can greatly enhance the fluorescence signal after binding to DNA; Loading buffer (loading buffer) can increase the sample density in agarose gel electrophoresis, ensuring that the DNA sample evenly sinks into the loading hole. In addition, the color indicator in it plays a role in showing the electrophoresis process, which helps to improve the accuracy and reliability of the experimental results; DNA marker is added to the gel together with the sample for electrophoresis separation. By comparing it with the sample band, the molecular weight size of the sample DNA and its concentration in the solution can be roughly estimated. In addition, phi29 DNA polymerase is a polymerase with high processive synthesis ability (higher than 70 kb) and can achieve efficient isothermal DNA amplification, and has strong strand displacement activity. It is a commonly used tool enzyme for RCA reaction. phi29 DNA polymerase also has 3'→5' exonuclease (proofreading reading) activity that preferentially acts on single-stranded DNA or RNA.

[0067] (2) Equipment: Gel imaging system (BIO-OI Biotechnology Co., Ltd., Guangzhou, OI100); Metal bath thermostat (Naisi Biotechnology Co., Ltd., Wuxi, Jiangsu, HS11000); Electrophoresis apparatus (Junyi Oriental Electrophoresis Equipment Co., Ltd., Beijing, JY600C).

[0068] 1. Basic steps of the CHAC-RCA detection method

[0069] S1. Preparation of hairpins H1 and H2: Dissolve the dry powders of H1 and H2 in 1×TE buffer to 10 μM and store as the mother liquor at -20 °C. Thaw thoroughly at 4 °C before use, and add pure H 2 O and phi29 DNA polymerase buffer (10×) to dilute the hairpins to 1 μM. After the configuration is completed, place it in a metal bath at 95 °C for 5 min, and then slowly cool it to room temperature at a temperature gradient of 1 °C / min to form the hairpin structure, obtaining hairpins H1 and H2, and then store them at 4 °C for later use.

[0070] S2. Ligation reaction of CHAC-RCA: Mix 10 μL of ligation buffer (10×), 5 μL of target (20 nM), 2 μL each of H1, H2, CP, and HP (1 μM), 10 μL of phi29 DNA polymerase buffer (10×), 55 μL of pure H 2 O, 2 μL of T4 DNA ligase (5 U / μL), and 10 μL of bovine serum albumin (2 mg / mL), and react at 37 °C for 60 min.

[0071] S3. RCA reaction: Mix 15 μL of the diluted ligation reaction product (the target concentration in the diluted reaction product is 200 pM), 3 μL of phi29 DNA polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 1 μL of deoxynucleotide dNTP (10 mM), 7.5 μL of pure H 2 O, and 0.5 μL of phi29 DNA polymerase (10 U / μL), and react at 37 °C for 60 min to obtain the RCA reaction product.

[0072] S4. Electrophoresis verification: Agarose gel electrophoresis (2.5%, 1×TAE buffer). After mixing 15 μL of the RCA reaction product with 1 μL of SYBR Gold and 3 μL of loading buffer, perform gel electrophoresis at room temperature (100 V, 60 min) and take images.

[0073] 2. Electrophoresis verification of the CHA reaction and the CHAC reaction

[0074] The principle of the detection method of the present invention is that the hairpin structures of H1 and H2 are stable. Only in the presence of the target, CHA will be initiated. CP and H2 assist the ligation process as ligation templates. DNA ligase is added to ligate the gap between CP and the H1 strand to form a circular template. In the subsequent RCA reaction, phi29 DNA polymerase has exonuclease (proofreading) activity that preferentially acts on single-stranded DNA or RNA in the 3'→5' direction. Phi29 DNA polymerase preferentially recognizes the 3' end of the free single strand, and after proofreading, it extends to obtain the RCA reaction product. Therefore, in this experiment, different reaction systems were constructed to detect the CHA products and verify whether the CHA circular template was ligated successfully. The electrophoresis verification scheme is as follows:

[0075] For agarose gel electrophoresis (2.5%, 1×TAE buffer), after mixing 15 μL of the sample to be analyzed with 1 μL of SYBR Gold and 3 μL of loading buffer, gel electrophoresis was carried out at room temperature (100 V, 60 min) and an image was taken. The samples to be analyzed include:

[0076] (a) Lane M: Marker;

[0077] (b) Lane 1: 200 nM hairpin H1;

[0078] (c) Lane 2: 200 nM hairpin H2;

[0079] (d) Lane 3: The system contains hairpin H1 and H2 with a final concentration of 200 nM each; the specific system is: Mix 6 μL of hairpin H1 (initial concentration of 1 μM), 6 μL of hairpin H2 (initial concentration of 1 μM), 3 μL of phi29 DNA polymerase buffer (10×), and 15 μL of pure H 2 O, and react at 37 °C for 60 min.

[0080] (e) Lane 4: The system contains a target with a final concentration of 20 nM and hairpin H1 and H2 with a final concentration of 200 nM each; the specific system is: Mix 3 μL of the target (initial concentration of 200 nM), 6 μL of hairpin H1 (initial concentration of 1 μM), 6 μL of hairpin H2 (initial concentration of 1 μM), 3 μL of phi29 DNA polymerase buffer (10×), and 12 μL of pure H 2 O, and react at 37 °C for 60 min;

[0081] (f) Lane 5: The system contains hairpin H1 and the auxiliary probe CP with a final concentration of 200 nM each; the specific system is: Mix 6 μL of hairpin H1 (initial concentration of 1 μM), 6 μL of CP (initial concentration of 1 μM), 3 μL of phi29 DNA polymerase buffer (10×), and 15 μL of pure H 2Mix, react at 37 °C for 60 min;

[0082] (g) Lane 6: The system contains hairpin H1, CP, and HP with final concentrations of 200 nM each; specific system: Mix 6 μL of hairpin H1 (initial concentration 1 μM), 6 μL of CP (initial concentration 1 μM), 6 μL of HP (initial concentration 1 μM), 3 μL of phi29 DNA polymerase buffer (10×), and 9 μL of pure H 2 O, react at 37 °C for 60 min;

[0083] (h) Lane 7: The system contains a target with a final concentration of 20 nM and H1, CP, and HP with final concentrations of 200 nM each; specific system: Mix 3 μL of the target (initial concentration 200 nM), 6 μL of hairpin H1 (initial concentration 1 μM), 6 μL of CP (1 μM), 6 μL of HP (initial concentration 1 μM), 3 μL of phi29 DNA polymerase buffer (10×), and 6 μL of pure H 2 O, react at 37 °C for 60 min;

[0084] (i) Lane 8: The system contains H1, H2, CP, and HP with final concentrations of 200 nM each; specific system: Mix 6 μL of hairpin H1 (initial concentration 1 μM), 6 μL of hairpin H2 (initial concentration 1 μM), 6 μL of CP (initial concentration 1 μM), 6 μL of HP (1 μM), 3 μL of phi29 DNA polymerase buffer (10×), and 3 μL of pure H 2 O, react at 37 °C for 60 min;

[0085] (j) Lane 9: The system contains a target with a final concentration of 20 nM and H1, H2, CP, and HP with final concentrations of 200 nM each; specific system: Mix 3 μL of the target (initial concentration 200 nM), 6 μL of hairpin H1 (initial concentration 1 μM), 6 μL of hairpin H2 (initial concentration 1 μM), 6 μL of CP (initial concentration 1 μM), 6 μL of HP (initial concentration 1 μM), 3 μL of phi29 DNA polymerase buffer (10×), and react at 37 °C for 60 min;

[0086] (k) Lane 10: The system contains a target with a final concentration of 10 nM, H1, H2, CP, and HP with final concentrations of 100 nM each, and add ligase reaction mixture solution; specific system: Mix 3 μL of ligase buffer (10×), 1.5 μL of target gene (initial concentration 200 nM), 3 μL each of H1, H2, CP, and HP (initial concentration 1 μM), 3 μL of phi29 buffer (10×), 7 μL of pure H 20.5 μL of T4 DNA ligase (initial concentration: 5 U / μL) was mixed with 3 μL of bovine serum albumin (initial concentration: 2 mg / mL), and the reaction was carried out at 37 °C for 60 min.

[0087] The results of electrophoresis verification are shown in Figure 2 , and it can be analyzed that compared with lane 4, the bands of H1 and H2 in lane 3 are bright, and there is no obvious band at the CHA product, indicating that in the absence of the target, the hairpin H1 and H2 structures are stable and there is almost no interaction. Lanes 5-8 show that H1, capture probe CP, and helper probe HP in the CHA ligase system can stably bind, and the probability of H2 binding to the H1-CP-HP complex in the absence of the target is extremely low, which is in line with the theoretical expectation. When the target is added to the system, stable CHA products are generated, and with the help of HP, H2 serves as the ligation template for PLP cyclization and generates a circular template (lane 10).

[0088] 3. Electrophoresis verification of CHAC target cycling and its efficiency

[0089] In traditional padlock probe-based rolling circle amplification (PLP-RCA), the ligation reaction for PLP cyclization can only occur in the presence of the ligation strand, and the fragments of the ligation strand are complementary to the 3' and 5' ends of the linear PLP, thus ensuring high selectivity. The working mode of traditional RCA is that one ligation template cyclizes one PLP. The detection method of the present invention utilizes the characteristic that the CHA target can cycle, incorporates CHA into the padlock probe ligation link of RCA, and theoretically one target can cyclize multiple PLPs. In this experiment, reaction systems with different target concentrations were constructed to verify CHAC target cycling and its efficiency. The electrophoresis verification experimental protocol is as follows:

[0090] (1) Target gradient dilution: 5 μL of the target (200 nM) was added to an eppendorf tube containing 15 μL of pure H 2 O, and mixed well. This step was repeated, and each time 5 μL of the dilution from the previous step was taken for dilution to obtain target solutions with different concentrations (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78 nM, 0.39 nM).

[0091] (2) CHAC: 6 μL, 3 μL, or 7.5 μL of target solutions with different concentrations (1 μM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78 nM, 0.39 nM, 0), 6 μL of H1 and H2 (1 μM), and 3 μL of phi29 DNA polymerase buffer (10×) were mixed, and pure H 2Make up to a total volume of 30 μL with O and react at 37 °C for 120 min.

[0092] (3) Electrophoresis verification: Agarose gel electrophoresis (2.5%, 1×TAE buffer). After mixing 15 μL of the sample to be analyzed with 1 μL of SYBR Gold and 3 μL of loading buffer, perform gel electrophoresis at room temperature (100 V, 60 min) and take an image. The sample to be analyzed includes:

[0093] (a) Lane M: Marker;

[0094] (b) Lane 1: 200 nM DNA hairpins H1 and H2;

[0095] (c) Lane 2: 0.1 nM target and 200 nM DNA hairpins H1 and H2;

[0096] (d) Lane 3: 0.195 nM target and 200 nM DNA hairpins H1 and H2;

[0097] (e) Lane 4: 0.39 nM target and 200 nM DNA hairpins H1 and H2;

[0098] (f) Lane 5: 0.78 nM target and 200 nM DNA hairpins H1 and H2;

[0099] (g) Lane 6: 1.5625 nM target and 200 nM DNA hairpins H1 and H2;

[0100] (h) Lane 7: 3.125 nM target and 200 nM DNA hairpins H1 and H2;

[0101] (i) Lane 8: 6.25 nM target and 200 nM DNA hairpins H1 and H2;

[0102] (j) Lane 9: 12.5 nM target and 200 nM DNA hairpins H1 and H2;

[0103] (k) Lane 10: 25 nM target and 200 nM DNA hairpins H1 and H2;

[0104] (L) Lane 11: 50 nM target and 200 nM DNA hairpins H1 and H2;

[0105] (M) Lane 12: 100 nM target and 200 nM DNA hairpins H1 and H2;

[0106] (N) Lane 13: 200 nM target and 200 nM DNA hairpins H1 and H2.

[0107] The results of electrophoresis verification are shown inFigure 3 , by observing the electrophoresis pattern, it can be observed that the CHAC method exhibits significant reactivity at low target concentrations. As shown in lane 2, at low target concentrations, CHAC product bands begin to appear. At the same time, we also verified that when there is no target (as shown in lane 1), the observed trailing phenomenon is not caused by non-specific reactions, thus ensuring the accuracy of the experimental results. As the target concentration gradually increases, it can be found that the reaction efficiency of CHA is significantly improved. When the target concentration reaches 6.25 nM, the reaction has been completely carried out, that is, there is a blank at the positions where H1 and H2 should be. To more accurately evaluate the reaction efficiency of the CHAC method, further gray-scale analysis of the electrophoresis bands was performed (as Figure 4 shown). The analysis results show that under the condition of a target concentration of 6.25 nM, the CHA reaction reaches complete reaction within 2 hours of reaction time. If the efficiency of the traditional RCA in connecting the template (target) and the padlock probe within 1 hour is taken as 1:1 as a reference, then in this CHA system, its efficiency can be as high as 1:16.

[0108] In summary, the above experimental results not only verify the high efficiency and sensitivity of the CHAC method at low target concentrations, but also verify the recycling of the target in the CHAC reaction, and also provide strong support for its further application in the fields of biosensing and molecular diagnosis.

[0109] 4. Performance Verification of the CHAC-RCA Detection Method

[0110] According to the aforementioned experimental results, concentration gradient experiments were respectively carried out using the PLP-RCA technique and the CHAC-RCA detection method of the present invention to further verify the excellent performance of the CHAC-RCA detection method. The sequence information involved in the PLP-RCA reaction is shown in Table 2.

[0111] Table 2 Sequence Information

[0112]

[0113] The specific experimental steps are as follows:

[0114] (1) Ligation reaction of PLP-RCA: Mix 2 μL of padlock probe (1 μM), 6 μL of target gene (1 μM), 70 μL of pure H 2 O, 10 μL of ligation buffer (10×), 2 μL of T4 DNA ligase (5 U / μL) and 10 μL of bovine serum albumin (2 mg / mL), and react at 37 °C for 60 min.

[0115] (2) Ligation reaction of CHAC-RCA: Mix 5 μL of target gene (20 nM), 2 μL each of H1, H2, CP, and HP (1 μM), 10 μL of phi29 DNA polymerase buffer (10×), 55 μL of pure H 2 O, 10 μL of ligation buffer (10×), 2 μL of T4 DNA ligase (5 U / μL), and 10 μL of bovine serum albumin (2 mg / mL), and react at 37 °C for 60 min.

[0116] (3) Mix 15 μL of the diluted ligation reaction product (the target concentrations in the diluted ligation reaction product are: 100 pM, 50 pM, 25 pM, 12.5 pM, 6.25 pM, 3.125 pM, 1.5625 pM, 781 fM, 319 fM, 195 fM, 98 fM, 49 fM), 3 μL of polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 1 μL of deoxynucleotide dNTP (10 mM), 7.5 μL of pure H 2 O, and 0.5 μL of phi29 DNA polymerase (10 U / μL), and react at 37 °C for 60 min.

[0117] (4) Electrophoresis verification: Agarose gel electrophoresis (2.5%, 1× TAE buffer). After mixing 15 μL of the sample to be analyzed with 1 μL of SYBR Gold and 3 μL of loading buffer, perform gel electrophoresis at room temperature (100 V, 60 min) and take images. The samples to be analyzed include:

[0118] (a) Lane 1: PLP-RCA product with a target concentration of 49 fM;

[0119] (b) Lane 2: PLP-RCA product with a target concentration of 98 fM;

[0120] (c) Lane 3: PLP-RCA product with a target concentration of 195 fM;

[0121] (d) Lane 4: PLP-RCA product with a target concentration of 391 fM;

[0122] (e) Lane 5: PLP-RCA product with a target concentration of 781 fM;

[0123] (f) Lane 6: PLP-RCA product with a target concentration of 1.56 pM;

[0124] (g) Lane 7: PLP-RCA product with a target concentration of 3.125 pM;

[0125] (h) Lane 8: PLP-RCA product with a target concentration of 6.25 pM;

[0126] (i) Lane 9: PLP-RCA product with a target concentration of 12.5 pM;

[0127] (j) Lane 10: PLP-RCA product with a target concentration of 25 pM;

[0128] (k) Lane 11: PLP-RCA product with a target concentration of 50 pM;

[0129] (l) Lane 12: PLP-RCA product with a target concentration of 100 pM;

[0130] (m) Lane 13: CHA-RCA product with a target concentration of 49 fM;

[0131] (n) Lane 14: CHA-RCA product with a target concentration of 98 fM;

[0132] (o) Lane 15: CHA-RCA product with a target concentration of 195 fM;

[0133] (p) Lane 16: CHA-RCA product with a target concentration of 391 fM;

[0134] (q) Lane 17: CHA-RCA product with a target concentration of 781 fM;

[0135] (r) Lane 18: CHA-RCA product with a target concentration of 1.56 pM;

[0136] (s) Lane 19: CHA-RCA product with a target concentration of 3.125 pM;

[0137] (t) Lane 20: CHA-RCA product with a target concentration of 6.25 pM;

[0138] (u) Lane 21: CHA-RCA product with a target concentration of 12.5 pM;

[0139] (v) Lane 22: CHA-RCA product with a target concentration of 25 pM;

[0140] (w) Lane 23: CHA-RCA product with a target concentration of 50 pM;

[0141] (x) Lane 24: CHA-RCA product with a target concentration of 100 pM.

[0142] The results of electrophoresis verification are shown in Figure 5, Observe the electrophoresis pattern. In the traditional PLP-RCA system, when the concentration is 12.5 pM (lane 9), RCA products that can be observed with the naked eye start to appear in the lane. In contrast, in the CHA-RCA system, RCA product bands can be observed when the concentration is as low as 391 fM (lane 16), and as the concentration increases, the brightness of the bands gradually increases. Compared with traditional PLP-RCA, the CHAC-RCA technology proposed in the present invention exhibits a higher nucleic acid signal amplification efficiency, approximately 32 times. This value is higher than the 16-fold signal amplification efficiency of CHA mentioned above. The reasons may be as follows:

[0143] (i) The conclusions obtained previously may be based on the time and target concentration when the hairpin reaction is complete. However, during the CHA reaction, as the reaction progresses, the concentration of unreacted hairpins gradually decreases, resulting in a gradual decrease in the reaction rate. In the CHA-RCA reaction, we calculate the signal amplification efficiency based on the time when the product is observed and the concentration of circular templates. At this time, the concentration of reactants in the system is relatively high, so it is not limited by the reaction rate, thus obtaining a higher signal amplification multiple;

[0144] (ii) The padlock probes used to generate circular templates in the traditional PLP-RCA reaction and those used in CHAC-RCA are not from the same batch, which may have a certain impact on the reaction efficiency;

[0145] (iii) Compared with the simple CHA reaction mentioned above, this performance comparison experiment also includes a ligation reaction and RCA. As the product is generated, the forward reaction rate may be faster, thereby further improving the signal amplification efficiency.

[0146] In the above-mentioned embodiment, the qualitative verification of the amplification product is verified by electrophoresis. To ensure the reliability of the data, the gray-scale analysis of the electrophoresis bands is performed on some electrophoresis result images using the software Image J.

[0147] Example 3 Detection of Monkeypox Virus

[0148] A method for detecting monkeypox virus nucleic acid, comprising the following steps:

[0149] S1. Pretreat the sample to be tested. The pretreatment method is: use a nucleic acid extraction kit to extract the monkeypox virus genome from the sample to be tested (from Changsha Center for Disease Control and Prevention).

[0150] S2. Dissolve the dry powders of H1 and H2 in 1×TE buffer to 10 μM and store them as mother liquors at -20°C. Thaw them thoroughly at 4°C before use, and add pure H 2The O and phi29 DNA polymerase buffer (10×) was used to dilute the hairpin to 1 μM. After the configuration was completed, it was placed in a metal bath and reacted at 95 °C for 5 min, and then slowly cooled to room temperature at a temperature gradient of 1 °C / min to form a hairpin structure, obtaining hairpins H1 and H2.

[0151] S3. Mix 10 μL of ligase buffer (10×), 5 μL of monkeypox virus genome, 2 μL each of H1, H2, CP, and HP (1 μM), 10 μL of phi29 DNA polymerase buffer (10×), 55 μL of pure H 2 O, 2 μL of T4 DNA ligase (5 U / μL), and 10 μL of bovine serum albumin (2 mg / mL), and react at 37 °C for 60 min.

[0152] S4. Mix 15 μL of the ligase reaction product, 3 μL of phi29 DNA polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 1 μL of deoxynucleotide dNTP (10 mM), 7.5 μL of pure H 2 O, and 0.5 μL of phi29 DNA polymerase (10 U / μL), and react at 37 °C for 60 min to obtain the RCA reaction product.

[0153] S5. Mix 15 μL of the RCA reaction product with 1 μL of SYBR Gold and 3 μL of loading buffer, and then perform agarose gel electrophoresis at room temperature (100 V, 60 min) to observe whether long single-stranded products are generated. The results showed that there were RCA product bands, indicating that the CHAC-RCA method has practical application value in the nucleic acid detection of monkeypox virus.

[0154] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention (including technical solutions for detecting other pathogens based on this solution) shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A kit for detecting pathogen nucleic acid, characterized in that: It contains hairpin H1, hairpin H2, capture probe CP, auxiliary probe HP, dNTP, bovine serum albumin, ligase, polymerase and buffer; The hairpin H1 is provided with a target recognition region and a CP binding region, and forms a hairpin structure after heat treatment; the target recognition region is complementary to the pathogen nucleic acid; the CP binding region is complementary to the capture probe CP; The hairpin H2 is provided with an H1 binding region and an HP binding region, and forms a hairpin structure after heat treatment; the H1 binding region is complementary to the hairpin H1; the HP binding region is complementary to the auxiliary probe HP; The capture probe CP is provided with an HP binding region and an H1 binding region; the HP binding region is complementary to the auxiliary probe HP; the H1 binding region is complementary to the hairpin H1; The auxiliary probe HP is provided with a CP binding region and an H2 binding region; the CP binding region is complementary to the capture probe CP; the H2 binding region is complementary to the hairpin H2; The 5' ends of the hairpin H1 and the auxiliary probe HP are phosphorylated.

2. The kit according to claim 1, characterized in that The ligase is DNA ligase; the polymerase is DNA polymerase.

3. The kit according to claim 1, characterized in that The heat treatment process is as follows: react at 92° C.-98° C. for 4-6 minutes, and then cool to 25° C. at a gradient of 1° C. / min.

4. The kit according to claim 1, characterized in that When the pathogen is monkeypox virus, the nucleotide sequence of the hairpin H1 is shown as SEQ ID NO.2; the nucleotide sequence of the hairpin H2 is shown as SEQ ID NO.3; the nucleotide sequence of the capture probe CP is shown as SEQ ID NO.4; and the nucleotide sequence of the auxiliary probe HP is shown as SEQ ID NO.

5.

5. Use of the kit according to any one of claims 1 to 4 in preparing a product for detecting pathogen nucleic acid, characterized in that: The pathogen nucleic acid includes monkeypox virus nucleic acid.

6. A method for using the kit according to any one of claims 1 to 4, characterized in that: The steps include: S1. Extract the genome from the sample to be tested; S2. The genome, hairpin H1, hairpin H2, capture probe CP, auxiliary probe HP, ligase, bovine serum albumin and buffer are mixed, and incubated at 36° C.-38° C. for 30-120 min to obtain a catalytic hairpin assembly ligation reaction product; S3. The CHAC reaction product is mixed with a polymerase, bovine serum albumin, dNTPs and a buffer, and incubated at 36° C. to 38° C. for 60 to 120 min to obtain an RCA product; S4. performing agarose gel electrophoresis on the RCA product to observe whether a long single-stranded product is generated; The described method of use is for non-diagnostic purposes.

7. The method of use according to claim 6, characterized in that: In step S1, the working concentrations of the hairpin H1, hairpin H2, capture probe CP and auxiliary probe HP are all 100-200 nM.

8. The method of use according to claim 6, characterized in that: In step S1, the molar ratio of the hairpin H1, the hairpin H2, the capture probe CP and the auxiliary probe HP is 1:1:1:

1.

9. The method of use according to claim 6, characterized in that: In step S1, the working concentration of the ligase is 0.05-0.15 U / μL; the working concentration of the bovine serum albumin is 0.1-0.3 mg / mL.

10. The method of use according to claim 6, characterized in that: In step S2, the working concentration of the polymerase is 0.15-0.18 U / μL; the working concentration of the bovine serum albumin is 0.1-0.3 mg / mL.

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