A time-resolved fluorescence immunochromatographic test strip-based chikungunya virus detection system

Abstract

The application discloses a CHIKV detection system based on a time-resolved fluorescent immunochromatographic test strip, and belongs to the technical field of CHIKV nucleic acid detection. The detection system comprises a nucleic acid probe and a time-resolved fluorescent immunochromatographic test strip. The nucleic acid probe comprises a hairpin probe H1 and a hairpin probe H2. The nucleotide sequence of the hairpin probe H1 is shown as SEQ ID NO: 2. The nucleotide sequence of the hairpin probe H2 is shown as SEQ ID NO: 3. The time-resolved fluorescent immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad which are sequentially connected. The application realizes target nucleic acid signal amplification by using a catalytic hairpin self-assembly reaction, and combines the time-resolved fluorescent immunochromatographic test strip for detection, so that the application does not need to participate in the enzyme, is simple to operate, short in detection time and low in cost. The detection system has high sensitivity and specificity for CHIKV nucleic acid detection, and is suitable for rapid detection of CHIKV under limited resources.

Description

Technical Field

[0001] This invention belongs to the field of chikungunya virus nucleic acid detection technology, specifically relating to a chikungunya virus detection system based on a time-resolved fluorescence immunochromatographic test strip. The detection system amplifies the target nucleic acid signal through a catalytic hairpin self-assembly reaction and, combined with the time-resolved fluorescence immunochromatographic test strip, achieves rapid detection of chikungunya virus-specific nucleic acid sequences. Background Technology

[0002] Chikungunya virus (CHIKV) is a single-stranded positive-sense RNA virus transmitted by Aedes mosquitoes, belonging to the genus *Alphavirus* of the family Oculaviridae. Since its discovery, this virus has been continuously circulating in many tropical and subtropical regions and is gradually expanding to temperate areas, posing a persistent threat to global public health. Chikungunya virus infection can cause symptoms such as fever, joint pain, rash, and myalgia; some patients may also experience long-term joint pain, severely impacting their quality of life. Because there are currently no specific antiviral drugs, early, rapid, sensitive, and specific detection is of great importance.

[0003] Existing methods for detecting Chikungunya virus mainly include virus isolation, serological detection, and molecular biological detection. While virus isolation can serve as a confirmatory method, it has a long detection cycle and requires stringent experimental conditions, making it difficult to meet the demand for rapid detection. Serological methods, such as enzyme-linked immunosorbent assay (ELISA), can detect specific antibodies, but antibody titers are low in the early stages of infection and are susceptible to cross-reactivity, limiting sensitivity and specificity. Molecular detection methods, such as RT-PCR, offer high sensitivity and specificity, but typically rely on expensive equipment, skilled personnel, and standardized experimental conditions, limiting their application in grassroots or resource-constrained areas. Although some isothermal amplification methods avoid thermal cycling, they often still require multiple enzymes and have high requirements for reaction temperature and operating conditions, resulting in significant detection costs and implementation complexity.

[0004] Catalytic hairpin assembly (CHA) is an enzyme-free isothermal nucleic acid signal amplification strategy that triggers a cyclic self-assembly reaction between two complementary hairpin probes based on a target sequence, thereby amplifying the signal. This method is characterized by being enzyme-free, having low background leakage, relatively stable reaction processes, and ease of coupling with various detection methods. Time-resolved fluorescence immunochromatographic assays (TRFIA) combine the advantages of simple chromatographic detection and high sensitivity of time-resolved fluorescence signals, making them suitable for rapid, portable, and on-site detection. Combining CHA and TRFIA could potentially achieve a balance between the advantages of nucleic acid signal amplification and the convenience of chromatographic detection, thereby improving detection sensitivity and operability.

[0005] Therefore, there is an urgent need to provide a chikungunya virus detection system that is structurally sound, easy to operate, has a short detection time, high sensitivity, and good specificity, in order to overcome the problems of strong equipment dependence, complex reaction system, and unfavorable conditions for rapid on-site detection in existing technologies. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a chikungunya virus detection system based on time-resolved fluorescence immunochromatographic test strips. This detection system combines a catalytic hairpin self-assembly reaction with time-resolved fluorescence immunochromatographic test strips to achieve rapid, sensitive, and specific detection of chikungunya virus-specific nucleic acid sequences.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention relates to a chikungunya virus detection system based on a time-resolved fluorescence immunochromatographic test strip, comprising a nucleic acid probe and a time-resolved fluorescence immunochromatographic test strip; The nucleic acid probes include hairpin probe H1 and hairpin probe H2; the nucleotide sequence of hairpin probe H1 is shown in SEQ ID NO:2, and the 3' end of hairpin probe H1 is modified with digoxigenin; the nucleotide sequence of hairpin probe H2 is shown in SEQ ID NO:3, and the 3' end of hairpin probe H2 is modified with biotin. The time-resolved fluorescence immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad connected in sequence; the conjugate pad is immobilized with streptavidin-labeled time-resolved fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with anti-digoxigenin antibody, and the control line is coated with biotin.

[0008] Preferably, the 3' end of the hairpin probe H1 is modified with digoxigenin, and the 3' end of the hairpin probe H2 is modified with biotin.

[0009] The time-resolved fluorescence immunochromatographic test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the conjugate pad is immobilized with streptavidin-labeled time-resolved fluorescent microspheres; the nitrocellulose membrane has a detection line and a control line, the detection line is coated with anti-digoxigenin antibody, and the control line is coated with biotin.

[0010] A second aspect of the present invention relates to a Chikungunya virus nucleic acid detection kit, comprising hairpin probe H1, hairpin probe H2, time-resolved fluorescence immunochromatographic strip, and reaction buffer. The nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO:2, and the 3' end of the hairpin probe H1 is modified with digoxigenin; the nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO:3, and the 3' end of the hairpin probe H2 is modified with biotin. The time-resolved fluorescence immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad connected in sequence; the conjugate pad is immobilized with streptavidin-labeled time-resolved fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with anti-digoxigenin antibody, and the control line is coated with biotin; The reaction buffer contains 20 mM Tris, 140 mM NaCl, 5 mM KCl and 2% guanidine hydrochloride, with a pH of 7.5.

[0011] During the detection process, when the chikungunya virus target nucleic acid is present in the sample, the target sequence first binds to hairpin probe H1 and opens its hairpin structure, subsequently promoting the participation of hairpin probe H2 in the reaction, forming an H1-H2 complex dual-labeled with digoxigenin and biotin. Simultaneously, the target sequence is released and triggers the next round of reaction, thus achieving cyclic signal amplification. The generated complex binds to streptavidin-labeled time-resolved fluorescent microspheres during chromatography and is specifically captured by anti-digoxigenin antibody at the detection line, generating a fluorescent signal, thereby achieving the detection of the target nucleic acid.

[0012] Preferably, the molar concentration ratio of the hairpin probe H1 to the hairpin probe H2 is 2:1.

[0013] Preferably, the detection system performs a catalytic hairpin self-assembly reaction at 35 °C for 20 min, and the chromatography detection time is 10 min.

[0014] A third aspect of the present invention relates to the application of a hairpin probe composition in the preparation of a Chikungunya virus nucleic acid detection kit, the hairpin probe composition comprising hairpin probe H1 and hairpin probe H2; the nucleotide sequence of hairpin probe H1 is shown in SEQ ID NO:2, and the 3' end of hairpin probe H1 is modified with digoxigenin; the nucleotide sequence of hairpin probe H2 is shown in SEQ ID NO:3, and the 3' end of hairpin probe H2 is modified with biotin.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention organically combines the catalytic hairpin self-assembly reaction with time-resolved fluorescence immunochromatographic test strips, achieving signal amplification and detection of target nucleic acids without the need for complex enzyme amplification systems. The operation steps are simple, the detection time is short, and it is suitable for rapid detection and on-site applications.

[0016] 2. The detection system constructed in this invention has high detection sensitivity. Under optimized conditions, its limit of detection can reach 100 fM, and its linear detection range is 100 fM to 1 μM, which can meet the detection requirements of low-abundance target nucleic acids.

[0017] 3. The detection system constructed in this invention has good specificity. It can effectively distinguish between the chikungunya virus target sequence and double-base mismatch, insertion, and deletion mutation sequences, as well as related viral sequences such as dengue virus, Zika virus, Mayaro virus, Ross River virus, and Sindbis virus, thereby helping to reduce the risk of false positives caused by non-target sequences.

[0018] 4. The detection system constructed in this invention has good repeatability. Experimental results show that the system has good intra-batch and inter-batch precision and exhibits relatively stable detection performance. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram illustrating the catalytic hairpin self-assembly reaction principle of the present invention; Figure 2 This is a schematic diagram illustrating the principle of the time-resolved fluorescence immunochromatographic test strip of the present invention for detecting digoxigenin and biotin-modified H1+H2 hybrid double strands. Figure 3 The feasibility analysis results of using the catalytic hairpin self-assembly reaction to detect Chikungunya virus RNA were verified by 12% non-denaturing polyacrylamide gel electrophoresis. Figure 4 The results of the feasibility analysis for using the fluorescence quenching system of this invention to verify the catalytic hairpin self-assembly reaction for detecting Chikungunya virus RNA were obtained. Figure 5 This is a schematic diagram showing the sensitivity analysis results of the catalytic hairpin self-assembly combined with time-resolved fluorescence immunochromatographic test strip detection system of the present invention. Figure 6 This is a schematic diagram of the specific analysis results of the catalytic hairpin self-assembly combined with time-resolved fluorescence immunochromatographic test strip detection system of the present invention; Figure 7 This is a schematic diagram of the repeatability analysis results of the catalytic hairpin self-assembly combined time-resolved fluorescence immunochromatographic test strip detection system of the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] Example 1 This embodiment describes the preparation process of the CHA detection probe.

[0023] Catalytic hairpin assembly (CHA) is a typical enzyme-free isothermal nucleic acid amplification technique. This technique relies on the specific recognition ability of target molecules to induce cyclic self-assembly between hairpin structures, thereby achieving signal amplification. The reaction principle of CHA is as follows: Figure 1 As shown, the CHA system consists of two partially complementary hairpin probes, H1 and H2, each composed of a toe region, a stem region, and a loop region. The complementary sequences of both probes are stably sealed by the stem region, ensuring the probes are in a relatively stable state and preventing non-specific spontaneous hybridization. When the target T is present, it first specifically binds to the toe region of H1, opening the hairpin structure of H1, exposing the complementary region, and pairing with H2 to form a stable H1-H2 double-stranded complex. Subsequently, the T molecule is released through a chain displacement mechanism, triggering a new reaction cycle until either H1 or H2 is depleted.

[0024] Based on the publicly available conserved RNA sequence of Chikungunya virus, a specific target sequence of 24 bases in length was screened as the detection target, as shown in SEQ ID NO:1. Two hairpin probes, denoted as H1 and H2, were designed based on the target sequence. The sequence of hairpin probe H1 is shown in SEQ ID NO:2, and the sequence of hairpin probe H2 is shown in SEQ ID NO:3; wherein, the 3' end of hairpin probe H1 is modified with digoxigenin, and the 3' end of hairpin probe H2 is modified with biotin. The sequences are shown in Table 1 below.

[0025] Table 1: Sequences used in this invention The two prepared probes, H1 and H2, were dissolved in TNaK buffer and then annealed to maintain their hairpin structure. The TNaK buffer consisted of 20 mM Tris, 140 mM NaCl, 5 mM KCl, and 2% guanidine hydrochloride, with a pH of 7.5. Annealing was performed at 95 °C for 5 min, followed by gradual cooling to 25 °C and holding for 90 min. After annealing, the probes were stored at -20 °C for later use.

[0026] The hairpin probes H1 and H2 prepared in this embodiment can be used for subsequent electrophoresis verification, fluorescence quenching verification, and detection and analysis using combined time-resolved fluorescence immunochromatographic test strips.

[0027] Example 2 In this embodiment, the feasibility of the catalytic hairpin self-assembly system is verified by electrophoresis.

[0028] The catalytic hairpin self-assembly system was validated using 12% non-denaturing polyacrylamide gel electrophoresis. Annealed hairpin probes H1 and H2, along with the target detection sequence, were diluted to 1 μM, thoroughly mixed in TNaK buffer, and reacted at 37 °C for 30 min. Subsequently, the resulting reaction solution was electrophoresed in 1×TAE buffer at 110 V for 90 min. After electrophoresis, the solution was stained with 10 mg / mL 4S GelRed for 15 min, and then analyzed using a gel imaging system.

[0029] Gel imaging showed bands 1-7 in lanes, namely: hairpin probe H1, hairpin probe H2, target detection sequence, a mixture of hairpin probe H1 and target detection sequence, a mixture of hairpin probe H2 and target detection sequence, a mixture of hairpin probe H1 and hairpin probe H2, and a mixture of hairpin probe H1, hairpin probe H2 and target detection sequence.

[0030] like Figure 3As shown, in the absence of the target detection sequence, there is only a low background reaction between hairpin probe H1 and hairpin probe H2; when the target detection sequence is added, the target detection sequence can open the hairpin structure of hairpin probe H1 and further promote the formation of a hybrid double-stranded complex between hairpin probe H1 and hairpin probe H2.

[0031] Example 3 In this embodiment, the feasibility of the catalytic hairpin self-assembly system is verified by using a fluorescence quenching system.

[0032] The probe H2, labeled with the fluorescent group 6-FAM and the quencher group BHQ1, was synthesized and purified by a biotechnology company. The fluorescence quenching system is constructed based on the principle of fluorescence resonance energy transfer. When H2 is in a complete hairpin structure, 6-FAM and BHQ1 are close together, and the fluorescence signal is quenched. When the target detection sequence triggers the catalytic hairpin self-assembly reaction, the H2 hairpin structure is opened, 6-FAM and BHQ1 separate, and the fluorescence signal is restored.

[0033] The 90 μL reaction system was prepared as follows: 30 μL of hairpin probe H1, 30 μL of hairpin probe H2 labeled with 6-FAM and BHQ1, and 30 μL of the target detection sequence were taken, thoroughly mixed, and then reacted. The concentrations of hairpin probe H1, H2, and the target detection sequence were 500 nM and 1 μM respectively. A control group without the target detection sequence was also included. Fluorescence signals were monitored in real time using a 7500 Fast Real-Time PCR System.

[0034] like Figure 4 As shown, real-time fluorescence detection results indicate that when the target detection sequence is absent, the system only produces a low background fluorescence signal; when the target detection sequence is present, hairpin probes H1 and H2 open sequentially under target triggering and form H1+H2 hybrid double strands, thereby producing a significantly enhanced fluorescence signal.

[0035] Example 4 This embodiment describes a time-resolved fluorescence immunochromatographic test strip.

[0036] The time-resolved fluorescence immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, which are sequentially connected to form a chromatographic flow path. The sample pad is located at the beginning of the test strip and is used to receive the sample; the conjugate pad is adjacent to the sample pad and is used to immobilize streptavidin-labeled time-resolved fluorescent microspheres; the nitrocellulose membrane connects to the conjugate pad and is used to set the detection line and control line; the absorbent pad is located at the end of the test strip and is used to drive unidirectional liquid flow.

[0037] The time-resolved fluorescent microspheres were prepared as follows: the microspheres were mixed with 50 mM MES buffer, centrifuged and washed, the supernatant was discarded and the microspheres were resuspended by sonication; then 6 μL (20 mg / mL) of freshly prepared NHS solution and 3 μL (20 mg / mL) of EDC solution were added, and the microspheres were incubated for 20 min. The microspheres were washed twice with MES buffer. Then streptavidin was added and the microspheres were incubated at room temperature for 2 h. After adding blocking solution and blocking for 1 h, the microspheres were washed and resuspended.

[0038] The obtained microsphere conjugate was sprayed onto a glass fiber membrane at a rate of 5 μL / cm² to form a conjugation pad, which was then dried at 34±3 ℃ and humidity not exceeding 40% for 16 h. The nitrocellulose membrane was then fixed onto a PVC substrate and equilibrated for 30 min. Detection and control lines were then set on the substrate, and the membrane was dried further at 34±3 ℃ and humidity not exceeding 40% for 16–20 h. Finally, the sample pad, conjugation pad, nitrocellulose membrane, and absorbent pad were assembled sequentially, cut into strips, and sealed for storage to obtain the time-resolved fluorescence immunochromatographic test strip.

[0039] Example 5 This embodiment describes the sensitivity analysis of the catalytic hairpin self-assembly combined with time-resolved fluorescence immunochromatographic test strip detection system.

[0040] A DNA target strand was synthesized based on the target detection sequence and then serially diluted to a concentration range of 1 fM to 1 μM. Different concentrations of the DNA target strand were thoroughly mixed with appropriate concentrations of hairpin probes H1 and H2, with hairpin probe H1 at 5 nM and hairpin probe H2 at 2.5 nM. The mixture was reacted at 35 °C for 20 min to obtain the reaction solution. This reaction solution was then added dropwise to a time-resolved fluorescence immunochromatographic test strip. After 10 min, the color development of the detection line was observed under a darkroom UV analyzer, and the fluorescence signal value at the detection line was read using a dry fluorescence immunoassay analyzer. Only the detection line signal was used as the analytical basis; the control line was only used to determine the effectiveness of the test strip.

[0041] The cutoff value is calculated by adding three times the standard deviation to the average value of the negative control signal, i.e.: Cutoff value = Average value of negative control + 3 × standard deviation. In this embodiment, the cutoff value is 65.

[0042] Test results as follows Figure 5As shown, the fluorescence signal at the detection line gradually decreases as the target concentration decreases. A clear detection signal can be observed when the target concentration is not lower than 100 fM. The detection limit of this system is 100 fM, the linear detection range is 100 fM to 1 μM, and there is a good linear relationship between the fluorescence signal value Y and the target concentration X, with the linear equation: Y = 885.79X - 986.07, R 2 =0.976.

[0043] Example 6 This embodiment describes the specificity analysis of the catalytic hairpin self-assembly combined with time-resolved fluorescence immunochromatographic test strip detection system.

[0044] Chikungunya virus target sequences were selected as positive controls. Conserved sequences of dengue virus (DENV), Zika virus (ZIKV), Mayaro virus (MAYV), Ross River virus (RRV), and Sindbis virus (SINV), as well as double-base insertion targets (TDI), double-based deletion targets (TDD), and double-base mismatch targets (TDM) were selected as interfering sequences for specificity analysis. The sequences are shown in Table 2. Table 2: Sequences used for specific detection The different sequences were thoroughly mixed with hairpin probes H1 and H2 at appropriate concentrations, with hairpin probe H1 at 5 nM and hairpin probe H2 at 2.5 nM. The mixture was reacted at 35 °C for 20 min to obtain the reaction solution. The reaction solution was then added dropwise to a time-resolved fluorescence immunochromatographic test strip. After 10 min, the color development of the detection line was observed under a darkroom UV analyzer, and the fluorescence signal value at the detection line was read using a dry fluorescence immunoassay analyzer for specific analysis.

[0045] Test results as follows Figure 6As shown, the positive control produced a clear detection signal, while DENV, ZIKV, MAYV, RRV, SINV, TDM, TDI, and TDD did not produce clear detection signals, and their fluorescence signals were significantly lower than those of the positive control (P < 0.0001). The results indicate that the catalytic hairpin self-assembly combined with time-resolved fluorescence immunochromatographic test strip detection system described in this invention has good specificity for the chikungunya virus target sequence and can effectively distinguish related viral sequences and mutant sequences.

[0046] Example 7 This embodiment describes the repeatability analysis of a catalytic hairpin self-assembly combined with time-resolved fluorescence immunochromatographic test strip detection system.

[0047] The repeatability analysis includes intra-batch repeatability analysis and inter-batch repeatability analysis. In intra-batch repeatability analysis, a 100 nM target detection sequence is thoroughly mixed with hairpin probes H1 and H2, where the concentration of hairpin probe H1 is 5 nM and the concentration of hairpin probe H2 is 2.5 nM. After reacting at 35 ℃ for 20 min, the resulting reaction solution is added to a time-resolved fluorescence immunochromatographic test strip. After 10 min, the fluorescence signal value at the detection line is read using a dry fluorescence immunoassay analyzer. The test is repeated 30 times, and the relative standard deviation (RSD) is calculated.

[0048] In the inter-batch repeatability analysis, target detection sequences at high, medium, and low concentrations were used for detection. The high, medium, and low concentrations were 100 nM, 1 nM, and 10 pM, respectively. Detection was carried out over 5 independent detection days, with 3 replicates set for each concentration per day. Fluorescence signal values ​​at the detection line were read according to the above method, and the relative standard deviation (RSD) was calculated based on the average signal values ​​of each detection day.

[0049] Test results as follows Figure 7 As shown, in the intra-batch repeatability analysis, the relative standard deviation of the 100 nM target detection sequence after 30 repeated detections was 5.611%; in the inter-batch repeatability analysis, the relative standard deviations of the 100 nM, 1 nM, and 10 pM target detection sequences were 2.801%, 4.076%, and 4.456%, respectively. The results indicate that the catalytic hairpin self-assembly combined with time-resolved fluorescence immunochromatographic test strip detection system described in this invention has good repeatability.

[0050] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0051] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A chikungunya virus detection system based on time-resolved fluorescence immunochromatographic test strips, characterized in that, Includes nucleic acid probes and time-resolved fluorescence immunochromatographic test strips; The nucleic acid probes include hairpin probe H1 and hairpin probe H2; the nucleotide sequence of hairpin probe H1 is shown in SEQ ID NO:2, and the 3' end of hairpin probe H1 is modified with digoxigenin; the nucleotide sequence of hairpin probe H2 is shown in SEQ ID NO:3, and the 3' end of hairpin probe H2 is modified with biotin. The time-resolved fluorescence immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad connected in sequence; the conjugate pad is immobilized with streptavidin-labeled time-resolved fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with anti-digoxigenin antibody, and the control line is coated with biotin.

2. The chikungunya virus detection system according to claim 1, characterized in that, The molar concentration ratio of the hairpin probe H1 to the hairpin probe H2 is 2:

1.

3. The chikungunya virus detection system according to claim 1, characterized in that, The target sequences for the chikungunya virus detection targeted by the hairpin probes H1 and H2 are shown in SEQ ID NO:

1.

4. A chikungunya virus nucleic acid detection kit, characterized in that, Includes hairpin probe H1, hairpin probe H2, time-resolved fluorescence immunochromatographic test strip, and reaction buffer; The nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO:2, and the 3' end of the hairpin probe H1 is modified with digoxigenin; the nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO:3, and the 3' end of the hairpin probe H2 is modified with biotin. The time-resolved fluorescence immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad connected in sequence; the conjugate pad is immobilized with streptavidin-labeled time-resolved fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with anti-digoxigenin antibody, and the control line is coated with biotin; The reaction buffer contains 20 mM Tris, 140 mM NaCl, 5 mM KCl and 2% guanidine hydrochloride, with a pH of 7.

5.

5. The chikungunya virus nucleic acid detection kit according to claim 4, characterized in that, The concentration of hairpin probe H1 in the kit is 5 nM, and the concentration of hairpin probe H2 is 2.5 nM.

6. The application of a hairpin probe composition in the preparation of a Chikungunya virus nucleic acid detection kit, characterized in that, The hairpin probe composition includes hairpin probe H1 and hairpin probe H2; the nucleotide sequence of hairpin probe H1 is shown in SEQ ID NO:2, and the 3' end of hairpin probe H1 is modified with digoxigenin; the nucleotide sequence of hairpin probe H2 is shown in SEQ ID NO:3, and the 3' end of hairpin probe H2 is modified with biotin.

7. The application according to claim 6, characterized in that, The Chikungunya virus nucleic acid detection kit also includes time-resolved fluorescence immunochromatographic strips.

8. The application according to claim 6, characterized in that, The time-resolved fluorescence immunochromatographic test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad connected in sequence.

9. The application according to claim 8, characterized in that, The conjugation pad is immobilized with streptavidin-labeled time-resolved fluorescent microspheres.

10. The application according to claim 8, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with anti-digoxin antibody, and the control line is coated with biotin.

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