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

Abstract

The application discloses a kit for detecting pathogen nucleic acid based on catalytic hairpin assembly and rolling circle amplification technology and application thereof, 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, a ligase, a polymerase and a buffer. The method for detecting pathogen nucleic acid based on catalytic hairpin assembly and rolling circle amplification (CHAC-RCA) technology provided by the application utilizes the recyclable advantage of a catalytic hairpin assembly (CHA) target, integrates the CHA into a padlock probe (PLP) connection process, improves the loop forming efficiency of the PLP, improves the detection sensitivity of rolling circle amplification (RCA), provides theoretical support for new application of the RCA technology, and tests monkeypox as an example to provide an empirical method for pathogen detection including monkeypox virus.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and in particular to a kit for detecting pathogen nucleic acids based on catalytic hairpin assembly and rolling circle amplification technology and its application. Background Technology

[0002] Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), which belongs to the genus Orthopoxvirus in the family Poxviridae. MPXV is an enveloped double-stranded DNA virus with two genetic lineages: lineage I (from the Central African Republic / Congo Basin) and lineage II (from West Africa). Since May 2022, the World Health Organization (WHO) reported a sharp increase in monkeypox infections in non-endemic countries, with approximately 87,000 cases and 112 deaths reported in 110 countries, bringing monkeypox to the forefront of international public health attention. In 2024, the WHO reported the emergence of a new variant—branch Ib—which can be transmitted through droplets, posing a public health threat. In August 2024, the WHO declared monkeypox a second Public Health Emergency of International Concern.

[0003] Diagnosis of patients infected with pathogens such as monkeypox typically requires a combination of clinical symptoms, epidemiological information, and laboratory tests. Compared to other diagnostic methods, nucleic acid amplification testing is faster, more sensitive, and more specific, making it the primary choice for diagnosing this virus. According to WHO recommendations, nucleic acid amplification testing should be performed on suspected monkeypox patients. Real-time polymerase chain reaction (PCR) is the gold standard and most common strategy for monkeypox virus diagnosis. Conserved monkeypox genes commonly detected using this method include hemagglutinin (HA), extracellular envelope protein (B6R), and DNA polymerase gene (E9L). However, the lack of specialized nucleic acid testing laboratories and operators, the need for cold chain transportation of samples, and the requirement for rapid on-site testing significantly hinder the implementation of on-site diagnosis for suspected monkeypox patients in remote areas. Therefore, developing rapid, accurate, low-cost testing systems that do not rely on specialized facilities and personnel is particularly important.

[0004] Biomarkers such as proteins, nucleic acids, and ions are frequently used to monitor health and disease. Compared to protein biomarkers, nucleic acid biomarkers are small, programmable, and can respond to disease states at the transcriptional level; therefore, nucleic acid-based biosensors represent one of the most promising areas for early precision diagnosis and treatment. Nucleic acid amplification technology can dramatically amplify nucleic acid fragments associated with specific target nucleic acids, thus exhibiting high sensitivity and specificity even with low sample sizes. Nucleic acid amplification has been widely applied in forensic medicine, food safety, and clinical diagnostics, and many techniques have been used to develop commercially viable and scalable diagnostic technologies.

[0005] Polymerase chain reaction (PCR), as a mature and widely used gene amplification assay, has long been the gold standard in fields such as epidemiology, forensic medicine, and paleontology. However, PCR technology has some drawbacks, such as susceptibility to contamination, expensive equipment, and a high risk of false positives. Furthermore, the need for skilled personnel and precise thermal cycling controllers limits its application in resource-constrained environments.

[0006] To overcome the limitations of PCR in the molecular amplification stage, many isothermal nucleic acid amplification techniques have been widely proposed and applied in recent years. These methods mainly include loop-mediated isothermal amplification, strand displacement amplification, rolling circle amplification, and recombinase polymerase amplification. Compared with PCR, isothermal amplification is performed entirely at the same temperature, eliminating the need for a thermal cycler. Therefore, it simplifies instrument requirements, significantly shortens reaction time, and offers sensitivity similar to current real-time quantitative PCR, better meeting the rapid and convenient needs of modern molecular detection technologies. It has become a hot topic in academic research and industry. Isothermal amplification technology can detect a wide range of targets, and its combination with nanotechnology has attracted strong attention. Some techniques have already become mature commercial products, showing broad prospects in biosensing, bioimaging, and nanomedicine applications. Among these, rolling circle amplification (RCA), as an isothermal amplification method, has received considerable attention in biosensing applications.

[0007] The basic elements of the RCA reaction system are four: DNA polymerase (such as phi29 DNA polymerase), primers, circular templates, and dNTPs. Among them, cyclizable oligonucleotides are also known as "padlock probes" (PLPs). The target first hybridizes to the end of the PLP as a ligation template. The PLP is then circularized, forming a closed circular structure through enzymatic ligation. The primers then extend along the 5'→3' direction under the catalysis of the polymerase, generating a single-stranded product containing numerous tandem repeat sequences, which can then detect the presence of a specific DNA sequence. However, because traditional padlock probes use one target primer to ligate one PLP during the targeted circularization process, and due to limitations in molecular collision efficiency, ligase efficiency, and side reactions during the circularization process (non-specific circularization and intermolecular circularization), PLPs are often difficult to fully utilize, leading to reduced RCA sensitivity and limiting the application of RCA technology. Summary of the Invention

[0008] The purpose of this invention is to provide a kit for detecting pathogen nucleic acids based on catalytic hairpin assembly-and-circularization (CHAC) technology and its applications, thereby addressing the problems existing in the prior art. This invention provides a method for detecting pathogen nucleic acids based on catalytic hairpin assembly-and-circularization (CHAC) technology. Utilizing the recyclable nature of the CHA target, CHA is integrated into the PLP ligation process to improve the circulation efficiency of PLP, thus enhancing the detection sensitivity of RCA. This provides theoretical support for new applications of RCA technology and offers an empirical method for the detection of viral nucleic acids.

[0009] To achieve the above objectives, the present invention provides the following solution:

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

[0011] The hairpin H1 has a target recognition region and a CP binding region, and is formed into a hairpin structure through 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 hair clip H2 is provided with an H1 bonding area and an HP bonding area, and is formed into a hair clip structure through heat treatment; the H1 bonding area is complementary to the hair clip H1; the HP bonding area 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] Phosphorylation of the 5' ends of the hairpin H1 and the auxiliary probe HP.

[0016] Furthermore, 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] Furthermore, the heat treatment process involves reacting at 92℃-98℃ for 4-6 minutes, followed by a temperature reduction to 25℃ at a gradient of 1℃ / min.

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

[0020] Secondly, the present invention provides an application of the above-mentioned kit in the preparation of products for detecting pathogen nucleic acids, wherein the pathogen nucleic acids include monkeypox virus nucleic acid.

[0021] Thirdly, the present invention provides a method for using the above-mentioned reagent 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℃-38℃ for 30-120 min to obtain the catalytic hairpin assembly ligation (CHAC) reaction product.

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

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

[0026] The method of use described is not for diagnostic purposes.

[0027] This invention utilizes the combined detection of pathogen nucleic acids using CHA and RCA technologies. Leveraging the recyclability of CHA targets, CHA is integrated into the PLP ligation process. A padlock probe ligation reaction mediated by hairpin assembly is used to improve the cyclization efficiency of PLP. Specifically, hairpin H2 binds to hairpin H1 to displace the target strand, allowing the target to be reused without consumption. The CHA process enables a single target to generate multiple ligase recognition sites, improving the low efficiency of traditional RCA reactions where a single target can only be recognized and ligated by a single PLP, thus increasing the ligation efficiency of RCA. The CHA reaction can significantly accelerate the RCA reaction, synergistically generating amplified signals to achieve ultrasensitive nucleic acid detection.

[0028] The detection principle of the reagent kit of this invention is as follows:Figure 1 As shown, specifically, when the target is present, it first hybridizes with hairpin H1 using its anchor point and opens hairpin H1. Subsequently, the anchor point of hairpin H2 recognizes and binds to the exposed single-stranded portion of hairpin H1, hybridizing with H1 through anchor point-triggered strand displacement. Simultaneously, the target is released into the next cycle to generate more double-stranded products. CP and H2 act as ligation templates. Under the action of ligase, H1 and HP are linked end-to-end to form a circular template, which can then initiate the subsequent RCA reaction to generate long single-stranded DNA products. Qualitative analysis of the target can be achieved by detecting the final reaction products using agarose gel electrophoresis.

[0029] Optionally, in step S1, the working concentrations of 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 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; and the working concentration of the 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; and the working concentration of the 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 (RCA has good robustness) and can be used for detection in serum and skin exudate, etc.

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

[0036] (3) The detection method or kit provided by this invention is the first to combine catalytic hairpin assembly with padlock probe ligation for the detection of conserved region sequences of monkeypox virus. Leveraging the recyclability of the target in catalytic hairpin assembly, it overcomes the bottleneck of PLP circularization efficiency being less than 1 in traditional RCA, greatly improving the efficiency of RCA. The selected target gene sequences are widely distributed among orthopoxvirus species, expanding the applicability of poxvirus detection. Compared to RT-PCR and other existing detection methods, this method is low-cost, time-efficient, requires relatively low operator skill, and has high accuracy, making it suitable for on-site detection under limited environmental conditions.

[0037] (4) The molecular strategy underlying the detection method or kit provided by this invention can flexibly replace specific target sequences and their complementary pairs to construct a biosensing system adapted to different pathogens. Under limited conditions, this system can achieve efficient and real-time detection of a variety of pathogens. This capability is of great public health significance for monitoring and controlling pathogens with widespread transmission risks and high dangers, and provides strong technical support for the formulation and implementation of disease control strategies. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a schematic diagram illustrating the detection principle of the reagent kit used in this invention.

[0040] Figure 2 Electrophoretic verification results for the feasibility of the CHA and ligase reaction;

[0041] Figure 3 Figure showing the electrophoretic verification results of CHAC target cycling and its efficiency;

[0042] Figure 4 for Figure 3 Gray-scale analysis results of CHA reactants and products in the medium-sized CHA reaction;

[0043] Figure 5 Electrophoresis results are shown to verify the performance comparison between traditional rolling circle amplification and rolling circle amplification based on catalytic hairpin assembly. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] In this invention, the Rolling Circle Amplification (RCA) technique is an isothermal amplification method. The basic elements of the RCA reaction system are four: DNA polymerase (such as phi29 DNA polymerase), primers, a circular template, and dNTPs. Among these, cyclizable oligonucleotides are also known as "padlock probes" (PLPs). The target first hybridizes to the end of the PLP as a ligation template. The PLP is then circularized, forming a closed loop through enzymatic ligation. Then, the primers extend along the 5'→3' direction under the catalysis of the polymerase, generating a single-stranded product containing numerous tandem repeat sequences, which can then detect the presence of a specific DNA sequence.

[0050] In this invention, the catalytic hairpin assembly (CHA) is a chemical reaction using two partially complementary DNA hairpins (H1 and H2) and a single-stranded oligonucleotide (I). Both hairpins consist of three consecutive domains, each containing a foothold. First, I triggers an allosteric change in H1 by specifically binding to the foothold of H1, forming the H1-I intermediate. Subsequently, the exposed H1 foothold initiates an assembly reaction with H2, which is opened to generate the unstable complex H1-I-H2. H2 triggers branching migration, and I separates from H1 to enter the next cycle.

[0051] The formation of CHA products primarily relies on the free energy released from chain shifts, achieving signal amplification through hairpin self-assembly. Compared to PCR, this avoids false positive results caused by the accumulation of large amounts of target molecules. Furthermore, altering the base sequence of the initiating strand significantly affects reaction efficiency, giving it excellent ability to identify single-base mutations. More importantly, the initiating strand can cyclically trigger hairpin hybridization to obtain a large number of stable double-stranded complexes, achieving a 1:N target-to-signal ratio and amplifying the signal 50-100 times in a short time, breaking through the fundamental bottleneck of traditional DNA detection.

[0052] In general, the enzyme concentrations used in the following examples are in excess to ensure maximum reaction efficiency; lower concentrations will reduce reaction efficiency. The reaction times for each step are the shortest times to obtain the optimal signal, determined by the reaction efficiency. If the reaction efficiency is low, the same result can be achieved by extending the reaction time. The CHA ligase reaction temperature is the temperature with the optimal signal-to-noise ratio. The RCA reaction temperature depends on the enzyme's optimal operating temperature 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. Sequence screening of target genes

[0055] Monkeypox virus is a linear double-stranded DNA virus. Currently, several gene sequences have been developed for monkeypox virus detection, including the envelope protein gene (B6R), B7R, DNA polymerase gene E9L, complement-binding protein C3L, DNA-dependent RNA polymerase subunit 18 (RPO18), N3R, and F3L. This invention identifies the target gene by screening and comparing existing monkeypox virus gene sequences, performing conservation analysis, and ultimately selecting the conserved E9L fragment (gene name OPG071) from the monkeypox virus genome. This yielded a highly conserved target gene sequence containing 29 nt, as shown in Table 1.

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

[0057] The catalytic hairpin assembly ligation-mediated rolling circle amplification (RCA) technology proposed in this invention involves CHA, DNA ligation, and RCA reactions. The required components mainly include a single-stranded target of monkeypox virus gene, H1, H2, CP, HP, T4 DNA ligase, and phi29 DNA polymerase. Hairpins H1 and H2 have a special design: H1 recognizes the target and acts as a padlock probe after circularization; while H2 binds to H1 in the CHA reaction to form a double-stranded product, simultaneously serving as a ligation template and a primer for the amplification reaction to initiate RCA. In the absence of a target, H1 and H2 maintain a stable hairpin structure; however, with the introduction of a target, the target can catalyze the hybridization of H1 and H2 and be repeatedly recycled. Therefore, the stability of the hairpin structure must be ensured. H1 and HP participate in the ligation reaction as padlock probes, so their nucleotide sequence ends must be designed to perfectly match the ligation template (CP and H2), and the 5' ends of H1 and HP must be phosphorylated to meet the working conditions of the ligase. The binding affinity of H1 to the target gene (T) sequence was calculated using the online nucleic acid secondary structure prediction tool NUPACK software. Hairpin structures of H1 and H2 were predicted, and the free energy values ​​of H1 and H2 needed to be below -10 kcal / mol to ensure the stability of the hairpin structure. The absolute values ​​of the binding free energies of T and H1 were lower than those of H1 and H2 to improve amplification efficiency. Sequence information for each component is shown in Table 1.

[0058] Table 1 Sequence Information

[0059]

[0060]

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

[0062] Example 2: Validation of the CHAC reaction and performance of the CHAC-RCA detection method

[0063] The nucleic acid sequences involved in this embodiment are shown in Table 1 of Example 1. In agarose gel electrophoresis, circular DNA structures migrate much more slowly than linear DNA structures of the same length. This characteristic makes electrophoresis an effective qualitative verification method for detecting whether padlock probes have been successfully ligated into a circular shape by ligase. On the other hand, RCA products have complex structures and large molecular weights, resulting in extremely low migration rates in agarose gels. Therefore, by observing the migration of RCA products in agarose gels, the success of the amplification and hybridization reaction design can be qualitatively verified, and the expected nucleic acid fragments can be verified.

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

[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 SA, BY-R0100); 10×TBE buffer (Beijing Pulilai Gene Technology Co., Ltd., B1111); SYBR Gold (500 μL, Thermo Fisher, S11494); 6×DNA Loading buffer (Hunan Aikerui Biotechnology Co., Ltd., AG11902); GLDNA Marker 500 (Hunan Aikerui Biotechnology Co., Ltd., AG11906); GL DNA Marker 1000 (Shanghai Heyuan Liji Biotechnology Co., Ltd., AN33L226).

[0066] The agarose gel electrophoresis system utilizes four deoxyribonucleoside triphosphates (dNTPs) to provide raw materials for DNA amplification. SYBR Gold is a fluorescent dye with high affinity for DNA, which significantly enhances the fluorescence signal upon binding. The loading buffer increases sample density during agarose gel electrophoresis, ensuring uniform deposition of the DNA sample into the wells. Furthermore, the color indicator within the buffer displays the electrophoresis progress, contributing to improved accuracy and reliability. The DNA marker is added to the gel along with the sample for electrophoretic separation; by comparing the marker with the sample bands, the molecular weight and concentration of the sample DNA can be roughly estimated. Additionally, phi29 DNA polymerase is a polymerase with high continuous synthesis capacity (above 70 kb) and efficient isothermal DNA amplification. It possesses strong strand displacement activity and is a commonly used enzyme for RCA reactions. Phi29 DNA polymerase also exhibits 3'→5' exonuclease (correction reading) activity that preferentially acts on single-stranded DNA or RNA.

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

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

[0069] S1. Preparation of hairpins H1 and H2: Dissolve the dry powders of H1 and H2 to 10 μM using 1×TE buffer and store as a stock solution at -20℃. Before use, thaw thoroughly at 4℃, add pure H2O and phi29 DNA polymerase buffer (10×) to dilute the hairpins to 1 μM. After preparation, place in a metal bath at 95℃ for 5 min, then slowly cool to room temperature at a temperature gradient of 1℃ / min to form hairpin structures, obtaining hairpins H1 and H2, and then store at 4℃ for later use.

[0070] S2.CHAC-RCA ligase reaction: Mix 10 μL ligase buffer (10×), 5 μL target (20 nM), 2 μL each of H1, H2, CP and HP (1 μM), 10 μL phi29 DNA polymerase buffer (10×), 55 μL pure H2O, 2 μL T4 DNA ligase (5 U / μL) and 10 μL bovine serum albumin (2 mg / mL), and react at 37℃ for 60 min.

[0071] S3. RCA reaction: Mix 15 μL of diluted ligase reaction product (target concentration in 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 H2O, and 0.5 μL of phi29 DNA polymerase (10 U / μL), and react at 37℃ for 60 min to obtain the RCA reaction product.

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

[0073] 2. Electrophoresis to verify the CHA and CHAC reactions

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

[0075] Agarose gel electrophoresis (2.5%, 1×TAE buffer): 15 μL of the sample to be analyzed was mixed with 1 μL of SYBR Gold and 3 μL of loading buffer, and gel electrophoresis was performed at room temperature (100V, 60 min), and images were captured. The sample to be analyzed included:

[0076] (a) Lane M: Marker;

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

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

[0079] (d) Lane 3: The system contains hairpins H1 and H2, both with a final concentration of 200 nM. The specific system is as follows: 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 H2O are mixed and reacted at 37℃ for 60 min.

[0080] (e) Lane 4: The system contains a target with a final concentration of 20 nM and hairpins H1 and H2 with a final concentration of 200 nM each; the specific system is as follows: 3 μL of 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 H2O are mixed and reacted at 37℃ for 60 min;

[0081] (f) Lane 5: The system contains hairpin H1 and auxiliary probe CP with a final concentration of 200 nM. The specific system is as follows: 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 H2O are mixed and reacted at 37℃ for 60 min.

[0082] (g) Lane 6: The system contains hairpin H1, CP and HP, all with a final concentration of 200 nM; the specific system is as follows: 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 H2O are mixed and reacted at 37℃ for 60 min;

[0083] (h) Lane 7: The system contains a target at a final concentration of 20 nM and H1, CP and HP at a final concentration of 200 nM each; the specific system is as follows: mix 3 μL of target (initial concentration of 200 nM), 6 μL of hairpin H1 (initial concentration of 1 μM), 6 μL of CP (1 μM), 6 μL of HP (initial concentration of 1 μM), 3 μL of phi29 DNA polymerase buffer (10×) and 6 μL of pure H2O, and react at 37℃ for 60 min;

[0084] (i) Lane 8: The system contains H1, H2, CP and HP, all with a final concentration of 200 nM. The specific system is as follows: 6 μL of hairpin H1 (initial concentration of 1 μM), 6 μL of hairpin H2 (initial concentration of 1 μM), 6 μL of CP (initial concentration of 1 μM), 6 μL of HP (1 μM), 3 μL of phi29 DNA polymerase buffer (10×) and 3 μL of pure H2O are mixed and reacted at 37℃ for 60 min.

[0085] (j) Lane 9: The system contains a target at a final concentration of 20 nM and H1, H2, CP, and HP at a final concentration of 200 nM each; the specific system is as follows: 3 μL of 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), 6 μL of CP (initial concentration of 1 μM), 6 μL of HP (initial concentration of 1 μM), and 3 μL of phi29 DNA polymerase buffer (10×) are mixed and reacted at 37℃ for 60 min;

[0086] (k) Lane 10: The system contains a target at a final concentration of 10 nM, H1, H2, CP and HP at a final concentration of 100 nM, and ligase reaction mixture solution; the specific system is as follows: 3 μL ligase buffer (10×), 1.5 μL target gene (initial concentration of 200 nM), 3 μL each of H1, H2, CP and HP (initial concentration of 1 μM), 3 μL phi29 buffer (10×), 7 μL pure H2O, 0.5 μL T4 DNA ligase (initial concentration of 5 U / μL) and 3 μL bovine serum albumin (initial concentration of 2 mg / mL) are mixed and reacted at 37℃ for 60 min.

[0087] Electrophoresis verification results are shown below Figure 2 Analysis revealed that, compared to lane 4, the H1 and H2 bands in lane 3 were brighter, and there was no obvious band at the CHA product site, indicating that the hairpin H1 and H2 structures were stable and had almost no interaction in the absence of a target. Lanes 5-8 showed that H1, the capture probe CP, and the helper probe HP could bind stably in the CHA ligase system, and the probability of H2 binding to the H1-CP-HP complex was extremely low in the absence of a target, consistent with theoretical expectations. When a target was added to the system, a stable CHA product was generated, and with the help of HP, H2 served as a ligation template for PLP circularization, generating a circular template (lane 10).

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

[0089] Traditional rolling circle amplification (PLP-RCA) based on padlock probes requires the presence of a linker strand for PLP circularization to occur, ensuring high selectivity. Traditional RCA works by circularizing one PLP strand with one linker template. This invention utilizes the cyclic nature of the CHA target, integrating CHA into the padlock probe linker of RCA, theoretically allowing one target to circularize multiple PLP strands. This experiment verifies the CHA target cycling and its efficiency by constructing reaction systems with different target concentrations. The electrophoretic verification experimental scheme is as follows:

[0090] (1) Target gradient dilution: Add 5 μL of target (200 nM) to an EP tube containing 15 μL of pure H2O and mix thoroughly. Repeat this step, taking 5 μL of the diluent from the previous step each time to dilute and obtain target solutions of 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: Mix 6 μL, 3 μL or 7.5 μL of target solutions of 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×). Add pure H2O to the total volume to 30 μL and react at 37℃ for 120 min.

[0092] (3) Electrophoresis verification: Agarose gel electrophoresis (2.5%, 1×TAE buffer). Take 15 μL of the sample to be analyzed, mix it with 1 μL of SYBR Gold and 3 μL of loading buffer, and perform gel electrophoresis at room temperature (100V, 60 min) and take images. 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: 100nM target and 200nM DNA hairpins H1 and H2;

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

[0107] Electrophoresis verification results are shown below Figure 3Observing the electrophoresis results, it can be seen that the CHAC method exhibits significant reactivity at low target concentrations. For example, in lane 2, CHAC product bands begin to appear at low target concentrations. Simultaneously, we also verified that the tailing phenomenon observed when no target is present (as shown in lane 1) is not caused by non-specific reactions, thus ensuring the accuracy of the experimental results. As the target concentration gradually increases, the reaction efficiency of CHA is significantly improved. When the target concentration reaches 6.25 nM, the reaction is complete, meaning that the positions where H1 and H2 should be are blank. To more accurately evaluate the reaction efficiency of the CHAC method, further grayscale analysis of the electrophoresis bands was performed (e.g., ...). Figure 4 (As shown). The analysis results show that, under the condition of a target concentration of 6.25 nM, the CHA reaction reached complete reaction within a reaction time of 2 hours. If we take the efficiency of traditional RCA connecting the template (target) and padlock probe in 1 hour as a reference (1:1), then in this CHA system, the efficiency can be as high as 1:16.

[0108] In summary, the 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, providing strong support for its further application in the fields of biosensing and molecular diagnostics.

[0109] 4. Performance verification of the CHAC-RCA detection method

[0110] Based on the aforementioned experimental results, concentration gradient experiments were conducted using PLP-RCA technology and the CHAC-RCA detection method of this invention to further verify the superior 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) PLP-RCA ligase reaction: Mix 2 μL padlock probe (1 μM), 6 μL target gene (1 μM), 70 μL pure H2O, 10 μL ligase buffer (10×), 2 μL T4 DNA ligase (5 U / μL) with 10 μL bovine serum albumin (2 mg / mL) and react at 37℃ for 60 min.

[0115] (2) CHAC-RCA ligase reaction: 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 H2O, 10 μL of ligase 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℃ for 60 min.

[0116] (3) Mix 15 μL of diluted ligase reaction product (the target concentrations in the diluted ligase 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, and 49 fM), 3 μL of polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 1 μL of deoxyribonucleotide dNTP (10 mM), 7.5 μL of pure H2O, 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). Mix 15 μL of the sample to be analyzed with 1 μL of SYBR Gold and 3 μL of loading buffer, and perform gel electrophoresis at room temperature (100V, 60 min) and capture images. The sample to be analyzed includes:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0142] Electrophoresis verification results are shown below Figure 5Observing the electrophoresis results, in the traditional PLP-RCA system, RCA products begin to appear in the lanes when the concentration is 12.5 pM (lane 9). In contrast, the CHA-RCA system shows RCA product bands at a concentration as low as 391 fM (lane 16), and the band brightness gradually increases with increasing concentration. Compared with traditional PLP-RCA, the CHAC-RCA technology proposed in this 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 earlier. The reason for this may be:

[0143] (i) The conclusions drawn above may be based on the time and target concentration at which the hairpin reaction is complete. However, in the CHA reaction, the concentration of unreacted hairpins gradually decreases as the reaction proceeds, leading to a gradual decrease in the reaction rate. In the CHA-RCA reaction, we calculate the signal amplification efficiency based on the time at which the product is observed and the concentration of the cyclic template. At this point, the concentration of reactants in the system is higher, and therefore it is not limited by the reaction rate, thus obtaining a higher signal amplification factor.

[0144] (ii) The padlock probe used to generate the ring template in the traditional PLP-RCA reaction is not from the same batch as the padlock probe used in CHAC-RCA, which may have a certain impact on the reaction efficiency.

[0145] (iii) Compared with the simple CHA reaction described above, this performance comparison experiment also included the linkage reaction and RCA. With the formation of products, the forward reaction may proceed faster, thereby further improving the signal amplification efficiency.

[0146] In the above embodiments, the qualitative verification of the amplification products was performed by electrophoresis. To ensure the reliability of the data, some electrophoresis results were analyzed for grayscale using ImageJ software.

[0147] Example 3: Detection of monkeypox virus

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

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

[0150] S2. Dissolve the dry powders of H1 and H2 to 10 μM using 1×TE buffer and store as a stock solution at -20℃. Before use, thaw thoroughly at 4℃, add pure H2O and phi29 DNA polymerase buffer (10×) to dilute the hairpins to 1 μM. After preparation, place in a metal bath and react at 95℃ for 5 min, then slowly cool to room temperature at a temperature gradient of 1℃ / min to form hairpin structures, obtaining hairpins H1 and H2.

[0151] S3. Mix 10 μL ligase buffer (10×), 5 μL monkeypox virus genome, 2 μL each of H1, H2, CP and HP (1 μM), 10 μL phi29 DNA polymerase buffer (10×), 55 μL pure H2O, 2 μL T4 DNA ligase (5 U / μL) and 10 μL bovine serum albumin (2 mg / mL), and react at 37°C for 60 min.

[0152] S4. Mix 15 μL of 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 H2O, and 0.5 μL of phi29 DNA polymerase (10 U / μL). React at 37°C for 60 min to obtain the RCA reaction product.

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

[0154] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those skilled in the art to the technical solutions of the present invention (including technical solutions for detecting other pathogens based on the present invention) should fall within the protection scope defined by the claims of the present invention.

Claims

1. A kit for detecting monkeypox virus nucleic acid, characterized in that, It contains hairpin H1, hairpin H2, capture probe CP, helper probe HP, dNTP, bovine serum albumin, T4 DNA ligase, phi29 DNA polymerase and buffer; The hairpin H1 has a target recognition region and a CP binding region, and is formed into a hairpin structure through heat treatment; the target recognition region is complementary to the monkeypox virus nucleic acid; the CP binding region is complementary to the capture probe CP; The hair clip H2 is provided with an H1 bonding area and an HP bonding area, and is formed into a hair clip structure through heat treatment; the H1 bonding area is complementary to the hair clip H1; the HP bonding area 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; Phosphorylation of the 5' ends of the hairpin H1 and the auxiliary probe HP; The nucleotide sequence of hairpin H1 is shown in SEQ ID NO.2; the nucleotide sequence of hairpin H2 is shown in SEQ ID NO.3; the nucleotide sequence of the capture probe CP is shown in SEQ ID NO.4; and the nucleotide sequence of the auxiliary probe HP is shown in SEQ ID NO.

5.

2. The reagent kit according to claim 1, characterized in that, The heat treatment process involves reacting at 92℃-98℃ for 4-6 minutes, followed by a temperature reduction to 25℃ at a gradient of 1℃ / min.

3. The use of the kit according to claim 1 or 2 in the preparation of products for detecting pathogen nucleic acids, characterized in that, The pathogen nucleic acid includes monkeypox virus nucleic acid.

4. A method of using the kit according to claim 1 or 2, characterized in that, Includes the following steps: S1. Extract the genome from the sample to be tested; S2. Mix the genome, hairpin H1, hairpin H2, capture probe CP, helper probe HP, T4 DNA ligase, bovine serum albumin and buffer, and incubate at 36℃-38℃ for 30-120 min to obtain the catalytic hairpin assembly ligation reaction product; S3. The catalytic hairpin assembly and ligation reaction product is mixed with phi29 DNA polymerase, bovine serum albumin, dNTPs and buffer, and incubated at 36℃-38℃ for 60-120 min to obtain the RCA product; S4. Perform agarose gel electrophoresis on the RCA product to observe whether long single-chain products are generated; The method of use described is not for diagnostic purposes.

5. The method of use according to claim 4, characterized in that, In step S1, the working concentrations of hairpin H1, hairpin H2, capture probe CP, and auxiliary probe HP are all 100-200 nM.

6. The method of use according to claim 4, characterized in that, In step S1, the molar ratio of hairpin H1, hairpin H2, capture probe CP, and auxiliary probe HP is 1:1:1:

1.

7. The method of use according to claim 4, characterized in that, In step S1, the working concentration of the T4 DNA ligase is 0.05-0.15 U / μL; the working concentration of the bovine serum albumin is 0.1-0.3 mg / mL.

8. The method of use according to claim 4, characterized in that, In step S2, the working concentration of the phi29 DNA 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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