Nucleic acid extraction-free SFTSV detection kit, primer probe and method

Abstract

The invention belongs to the technical field of molecular detection of biological medicines, and particularly relates to a nucleic acid extraction-free SFTSV detection kit, primer probes and a method. The primer probe composition comprises an upstream primer SEQ ID NO: 1 for specifically recognizing the SFTSV, a downstream primer SEQ ID NO: 2 of which the 5'end is labeled with biotin, and a probe SEQ ID NO: 3 of which the 5 'end is labeled with a reporter group and contains THF modification. The kit comprises the composition, a nucleic acid releasing agent and a lateral flow chromatography test strip. The method comprises the following steps: treating a serum / plasma sample by using a nucleic acid releasing agent, carrying out RPA isothermal amplification by directly taking a lysate as a template, and detecting a result through a test strip. The RPA-LFD rapid detection of the SFTSV is realized, the sensitivity (200copies / mL) and the specificity are high, the operation is simple, convenient and rapid (about 40 minutes), a complex instrument and a nucleic acid extraction step are not needed, and the method is suitable for base-level field screening.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology in biomedicine, specifically relating to a nucleic acid extraction-free SFTSV detection kit, primers, probes, and method. Background Technology

[0002] Fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by the SFTS virus (SFTSV). Its high mortality rate has become a significant public health threat, necessitating rapid diagnostic methods applicable to primary care and field testing. Recombinase polymerase amplification combined with lateral flow chromatography (RPA-LFD) technology is considered an ideal point-of-care testing (POCT) platform due to its lack of complex instruments and rapid operation. In existing technologies, patent documents CN116042908A and CN108315482A have demonstrated the feasibility of this platform in detecting African swine fever virus (ASFV) and canine distemper virus (CDV), respectively.

[0003] However, applying the RPA-LFD platform to SFTSV detection faces the following two difficulties stemming from existing technologies: Target Specificity Barriers and Technology Non-Transferability: The core protected by CN116042908A and CN108315482A lies in their primer and probe sequences specifically designed for the genomes of ASFV (a large DNA virus) and CDV (Paraxviridae RNA virus). The target of this invention, SFTSV, is a novel RNA virus of the Bunyaviridae family. It differs fundamentally from ASFV and CDV in viral taxonomy, genomic structure, and sequence, lacking homology. Based on the principle of high specificity in molecular detection, recognition elements designed for different viruses are generally not directly interchangeable. Therefore, existing technologies do not provide core recognition tools (primers / probes) that can be directly used for SFTSV detection; those skilled in the art still need to design and screen specific sequences for SFTSV from scratch.

[0004] The sample pretreatment process is mismatched with the convenience requirements of POCT: The sample processing methods in the aforementioned patent literature fail to fully meet the demand for a "minimalist process" in clinical field testing, where serum / plasma is the most common sample. CN116042908A targets tissue samples, and CN108315482A, while detecting viruses, still relies on Trizol or commercial column extraction kits for nucleic acid extraction. These steps involve multiple reagents, specialized equipment, and a considerable amount of time, which falls short of the "sample in, result out" rapid on-site testing concept pursued by RPA-LFD technology, and requires further optimization in terms of operational convenience.

[0005] In summary, while RPA-LFD is a promising rapid detection platform, existing technologies lack core sequence elements directly usable for SFTSV identification, and their sample processing workflows fail to fully meet the simplified operational requirements for rapid on-site testing of clinical serum / plasma samples. Therefore, there is an urgent need in the field for an innovative technical solution to provide a complete rapid detection scheme for SFTSV based on the RPA-LFD platform, incorporating a more streamlined sample processing workflow, while also possessing high sensitivity and specificity. Summary of the Invention

[0006] The objective of this invention is: first, to provide a novel set of primer and probe compositions capable of identifying fever with thrombocytopenia syndrome virus with high specificity and high sensitivity, which is the core material basis for achieving detection.

[0007] Second, a ready-to-use SFTSV detection kit comprising the above-described primer-probe composition is provided.

[0008] Third, a rapid SFTSV detection method using the above-mentioned kit is provided, which eliminates the need for traditional nucleic acid extraction and purification steps. This method is simple and fast to operate and is particularly suitable for field applications.

[0009] The technical solution of the present invention is as follows: Firstly, a set of primer-probe compositions for the specific detection of fever with thrombocytopenia syndrome virus is provided, comprising an upstream primer, a downstream primer, and a probe, wherein: The upstream primer has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer has the nucleotide sequence shown in SEQ ID NO:2 and is labeled with biotin at its 5' end; The probe has the nucleotide sequence shown in SEQ ID NO:3, with a reporter group labeled at its 5' end, a tetrahydrofuran modification site inside the sequence, and a blocking group attached to its 3' end.

[0010] Preferably, the reporter group is fluorescein isothiocyanate or carboxyfluorescein.

[0011] Secondly, a kit for detecting fever with thrombocytopenia syndrome virus is provided, comprising the above-mentioned primer-probe composition, nucleic acid release agent and lateral flow chromatography test strip.

[0012] Preferably, in addition to containing primer and probe compositions, nucleic acid release agents, and lateral flow chromatography strips, the product also contains an enzyme mixture and reaction buffer required for recombinase polymerase amplification reactions.

[0013] Preferably, the enzyme mixture comprises a recombinase, a single-stranded DNA-binding protein, and a strand displacement DNA polymerase.

[0014] Preferably, streptavidin or an antibody against the reporter group is immobilized on the detection line of the lateral flow chromatography test strip.

[0015] Thirdly, a method for detecting fever with thrombocytopenia syndrome virus is provided, comprising the following steps: (1) The test sample containing SFTSV was mixed with a nucleic acid release agent and processed to obtain a lysis buffer; (2) The lysis buffer is directly used as a nucleic acid template and added to a reaction system containing the primer and probe composition of claim 1 or 2 to carry out a recombinase polymerase amplification reaction to obtain the amplification product; (3) The amplification product is detected using a lateral flow chromatography test strip, and the result is determined based on the color development of the test line and the control line.

[0016] Preferably, the sample to be tested is serum or plasma.

[0017] Preferably, step (1) involves mixing the test sample containing SFTSV with the nucleic acid release agent and incubating it at 15-30°C for 2-10 minutes.

[0018] Preferably, the reaction temperature of the recombinase polymerase amplification reaction in step (2) is 38~43℃ and the reaction time is 20~30min.

[0019] Compared with the prior art, the advantages of the present invention are: (1) Solved the problem of rapid RPA-LFD detection of SFTSV: This invention designed and verified an RPA primer-probe combination (SEQ ID NO:1-3) that can specifically recognize SFTSV, and successfully constructed a complete RPA-LFD detection system based on it. This fills the gap in the field of rapid on-site detection technology for this important human pathogen.

[0020] (2) Excellent detection performance, meeting clinical needs: High sensitivity: Experimental verification shows that the limit of detection (LoD) for SFTSV nucleic acid using the method of this invention can reach 200 copies / mL (based on serum samples). This sensitivity is comparable to the current clinical gold standard qPCR method, and it can effectively detect early infection and samples with low viral load.

[0021] High specificity: The primer and probe composition of this invention is designed for the conserved region of the SFTSV genome and has no cross-reactivity with the nucleic acids of eight clinically common pathogens that may cause similar symptoms, such as Salmonella typhi, Anaplasma phagocytophilum, dengue virus, and Hantavirus, thus ensuring the accuracy of the detection results from the source.

[0022] (3) The operation process is significantly simplified, making it fast and convenient: Extraction-free process: This invention uses a simple nucleic acid releasing agent to treat serum / plasma samples, which can complete viral lysis and nucleic acid release within minutes, completely eliminating the time-consuming, material-intensive, and instrument-required nucleic acid extraction and purification steps in traditional nucleic acid testing.

[0023] Extremely low equipment dependence: The entire testing process only requires pipettes, ordinary temperature control devices (such as water baths, metal baths or portable temperature control devices) and test strips, completely eliminating the dependence on large equipment such as expensive and sophisticated PCR instruments and centrifuges.

[0024] The total time is significantly reduced: from sample processing to obtaining visualization results, the total time can be controlled within about 40 minutes, of which the core amplification reaction only takes about 25 minutes, which is much faster than conventional qPCR detection (usually more than 2 hours).

[0025] (4) A stable, reliable, and ready-to-use complete solution has been formed: This invention not only provides the core detection sequence, but also a ready-to-use kit containing all necessary components and a standardized operating procedure. The solution has undergone systematic performance verification and clinical sample testing, and the results are reliable, making it very suitable for promotion and application in scenarios such as disease control centers, primary hospitals, ports, and on-site emergency response. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This is an overall flowchart of the SFTVRPA-LFD detection method provided in the specific embodiments of the present invention; Figure 2 This is a schematic diagram showing representative results of the sensitivity test in Embodiment 4 of the present invention; Figure 3 This is a schematic diagram of the specificity test results in Embodiment 4 of the present invention; Figure 4 This is a schematic diagram comparing the qPCR detection results (Ct values) of some clinical samples in Example 4 of the present invention with the detection results of the LFD-RPA method of the present invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments.

[0028] Example 1

[0029] Design, synthesis and sequence confirmation of specific primers and probes 1. Target sequence selection Complete genome sequences of several representative SFTSV strains (including isolates from China, Korea, and Japan) were downloaded from the NCBi GenBank database. Multiple sequence alignment was performed using bioinformatics software such as MEGA or ClustalW to identify highly conserved regions (sequence identity >99%) as candidate targets. Simultaneously, the conserved region was compared with the genomes of the human genome database and other common pathogens (such as other bunyaviruses, bacteria, and rickettsiae) to ensure sufficient species specificity of the selected target and avoid non-specific amplification. After comprehensive screening, a highly conserved region located in the SFTSV viral genome S segment was identified as the target for RPA detection in this invention.

[0030] 2. Primer and probe design Based on the selected conserved target sequences, primers and probes were designed strictly according to the design principles of RPA technology: Primer design: Design a pair of primers (upstream and downstream) each 31 nucleotides in length. Ensure the GC content of the primers is between 40% and 60%, avoid consecutive single base repetitions, and use software such as Oligo or PrimerPremier to assess their potential for primer dimer formation or stable secondary structures, ensuring they are suitable for efficient RPA reactions.

[0031] Probe Design: Design a 49-nucleotide probe. This probe must be complementary to the internal region of the target sequence. Introduce a tetrahydrofuran (THF) residue in the middle of the probe sequence (typically 34 bases from the 5' end) as a recognition and cleavage site for subsequent exonucleases (such as Exonuclease III). A reporter group (such as FITC) should be attached to the 5' end of the probe, and a blocking group (such as C3Spacer) should be attached to the 3' end to prevent polymerase extension. The downstream primer should be labeled with biotin at its 5' end.

[0032] 3. Sequence synthesis The designed sequences were outsourced to a professional biotechnology service company for synthesis and modification. The mature solid-phase phosphoramidite method was used for synthesis, and all oligonucleotides were purified by high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) to ensure purity and accuracy. After synthesis, the products were identified by mass spectrometry (MS) or capillary electrophoresis (CE).

[0033] The final determined and synthesized core sequence is as follows (all sequences are written in the direction of 5'→3'): Upstream primer (named SFTSV-RPA-F): has the sequence shown in SEQ ID NO:1.

[0034] Downstream primer (named SFTSV-RPA-R): has the sequence shown in SEQ ID NO:2, with biotin covalently labeled at its 5' end.

[0035] The probe (named SFTSV-RPA-P) has the sequence shown in SEQ ID NO:3. Its 5' end is covalently labeled with fluorescein isothiocyanate (FITC); counting from the 5' end, position 34 is a tetrahydrofuran (THF) base analog (i.e., position 34 of the sequence, located between "ACC" and "GAA"); its 3' end is covalently linked with a C3 spacer blocking group. Its base sequence structure is shown in the table below: Table 1: Candidate DNA primers;

[0036] Example 2 This embodiment provides the specific components and assembly method of a ready-to-use SFTSV detection kit. This kit can be stored for extended periods at -20°C.

[0037] 1. Component List The kit contains the following individually packaged components: RPA lyophilized reaction tube (tube A): Contains pre-prepared lyophilized microspheres. Each microsphere contains the following for a single RPA reaction: recombinase, single-stranded DNA-binding protein (SSB), strand displacement DNA polymerase (BsuDNA polymerase), a mixture of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP), reaction stabilizers (such as trehalose and betaine), and buffer salts (such as tris-acetate and CH3COOK). The lyophilized form greatly improves the stability and transport convenience of the reagents.

[0038] Primer-probe mixture (tube B): This is a sterile, nuclease-free aqueous solution containing the three oligonucleotides synthesized in Example 1. The final concentrations of the upstream primer (SEQ ID NO:1) and downstream primer (SEQ ID NO:2) are both 10 μM, and the final concentration of the probe (SEQ ID NO:3) is 10 μM. This mixture is aliquoted into single-use volumes (e.g., 50 μL / tube).

[0039] The reaction initiation solution (tube C) was a 280 mM aqueous solution of magnesium acetate (Mg(OAc)₂). 2+ It is an essential cofactor for the RPA reaction, and its addition formally initiates the amplification reaction.

[0040] Nucleic acid release agent (tube D): Used for rapid processing of serum / plasma samples. This reagent is a mild lysis buffer, which may contain, for example, a nonionic surfactant (such as Triton X-100 or NP-40, concentration 0.1~1%), a weak base (such as NaOH or KOH, concentration 50-200mM), and an RNase inhibitor. Its pH value is approximately 10~12. Its function is to rapidly destroy the viral envelope and capsid at room temperature, releasing viral RNA, while simultaneously inactivating ribonuclease (RNase) in the sample, protecting the RNA from degradation. No heating is required during use.

[0041] Lateral flow chromatography test strip (Component E): Utilizes a double-antigen sandwich principle. The test strip structure consists of, in sequence: a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad. The conjugate pad is coated with colloidal gold-labeled mouse anti-FITC monoclonal antibody. The nitrocellulose membrane has two lines: the detection line (T line) contains streptavidin and goat anti-mouse IgG antibody; the control line (C line) contains goat anti-mouse IgG antibody.

[0042] Test strip buffer (tube F): pH 7.4 phosphate-buffered saline (PBS) containing 0.05–0.2% Tween-20. Used to dilute RPA amplification products to promote their chromatographic flow on the test strip and reduce nonspecific adsorption.

[0043] Positive control (tube G): Contains accurately quantified SFTSV in vitro transcribed RNA fragments (including the target sequence) at a concentration of 1 × 10⁻⁶. 4 Copies / μL, dissolved in nuclease-free water. Used as a validity control for experimental procedures.

[0044] Negative control (tube H): Nuclease-free water (DEPC water), used as a control for contamination monitoring.

[0045] Instruction Manual (Volume I): Describes the experimental procedures, result interpretation methods, and precautions in detail.

[0046] 2. Reagent kit assembly Place the above components (tubes A to H and component E) into their respective compartments within the kit insert, and then place them together with the instructions into the outer packaging box and seal. The entire assembly process should be performed in a clean environment to avoid nuclease contamination.

[0047] Example 3

[0048] Reference Figure 1 The flowchart shown illustrates the procedure for detecting SFTSV using the kit of this invention. The specific detection steps are as follows: (1) Sample pretreatment Number the samples to be tested (e.g., serum, plasma). Take a clean 1.5 mL centrifuge tube and add 50 μL of nucleic acid release agent (tube D). Using a pipette tip with a filter, pipette 50 μL of the sample to be tested and add it to the centrifuge tube containing the release agent. Vortex vigorously for 10-15 seconds and incubate at room temperature (20-25℃) for 5 minutes. At this time, the SFTSV viral particles in the sample will be lysed, and the viral RNA will be released into the solution. (If the sample is initially turbid, centrifuge at 12000 rpm for 1 minute and collect the supernatant). The processed liquid is the "nucleic acid template solution" that can be directly used for amplification.

[0049] Note: This process eliminates the need for phenol-chloroform, column chromatography, and magnetic bead separation, greatly simplifying the operation.

[0050] (2) RPA isothermal amplification reaction Take one RPA lyophilized reaction tube (tube A) and primer-probe mixture (tube B) from the kit. Add 32.9 μL of nuclease-free water to tube A, add 4.6 μL of primer-probe mixture (tube B), and add 10 μL of the "sample nucleic acid template solution" prepared in step (1). For control experiments, add 10 μL of positive control (tube G) or negative control (tube H). Gently pipette and repeatedly blow the tube several times to ensure that the lyophilized microspheres are completely dissolved and the system is well mixed. Finally, add 2.5 μL of reaction starter solution (tube C, 280 mM magnesium acetate). Immediately tighten the tube cap. Briefly and quickly vortex to mix for 3-5 seconds, and then briefly centrifuge to remove the liquid from the tube to the bottom. Place the reaction tube in a preheated 41°C metal bath or water bath and start timing. Incubate at a constant temperature for 25 min.

[0051] (3) Chromatographic test strip detection and result interpretation After the reaction is complete, remove the RPA tube and take a new 0.5 mL centrifuge tube. Add 90 μL of test strip buffer (tube F). Take 10 μL of amplification product from the RPA reaction tube and add it to the centrifuge tube containing the buffer. Mix well by pipetting. This dilution step helps to obtain clear chromatographic bands. Take one side-flow chromatography test strip (component E) from the kit and immerse it, sample pad side down, into the diluted mixture from step 3. Alternatively, add 100 μL of the mixture directly and vertically to the center of the sample pad. Place the test strip horizontally on a dry surface and start chromatography. Wait 8–10 minutes and observe the appearance of bands on the nitrocellulose membrane.

[0052] Result determination: Positive result (+): Two clear red bands are visible on the test strip. One is located in the test line (T line) area, and the other is located in the control line (C line) area. Regardless of the intensity of the T line, as long as it is clearly visible, the result is positive.

[0053] Negative result (-): Only one red band appears in the control line (C line) area, and there is no band in the test line (T line) area.

[0054] Invalid result: No red band appeared in the control line (C line) area. This indicates that the test strip is faulty or the operation was incorrect (e.g., buffer solution was not added). The result is invalid and a new test strip is required.

[0055] Example 4

[0056] Reagent kit performance evaluation 1. Sensitivity Test: Detection results for some representative dilution gradients are as follows: Figure 2 As shown in the figure. N represents the negative control; E1 to E5 represent the detection results of SFTSV RNA standards with progressively increasing viral concentrations (e.g., E1 corresponds to 2.0 × 10⁻⁵). -2 copies / μL, E5 corresponds to 2.0×10 2 (copies / μL or other actual test gradients).

[0057] Objective: To determine the limit of detection (LoD) for SFTSV nucleic acid using this detection method.

[0058] Materials: SFTSV in vitro transcribed RNA standards (containing complete target sequences), accurately quantified using digital PCR technology to a concentration of 1.0 × 10⁻⁶. 3 copies / μL.

[0059] Mixed serum from healthy individuals, confirmed by multiple viral nucleic acid tests to be free of pathogen nucleic acids such as SFTSV, HBV, HCV, and HIV.

[0060] Methods: RNA standards were serially diluted 10-fold using serum from healthy individuals to prepare simulated positive serum at the following concentrations: 1.0 × 10⁻⁶ 2 1.0×10 1 1.0×10 0 1.0×10 -1 1.0×10 -2 copies / μL. Converted to concentration: 10 2 ~10 -2 copies / μL, corresponding to a serum concentration of 10 5 ~10 1 copies / mL.

[0061] Two replicates were set up for each concentration, and each dilution sample was tested according to the complete procedure in Example 3. Parallel assays were performed using the same samples and a SFTSV real-time PCR detection kit approved by the National Medical Products Administration (NMPA) for methodological comparison. The results are as follows: Figure 2 As shown, the control line (C line) of all samples developed normally, and the color intensity of the test line (T line) increased significantly with the increase of virus concentration.

[0062] The RPA-LFD method of this invention: at an RNA concentration of 2.0 × 10⁻⁶ -1 copies / μL (i.e., serum concentration of 2.0 × 10⁻⁶) 2 When the concentration was 2 copies / mL (equivalent to 2 copies / reaction in the reaction system, corresponding to an initial serum concentration of approximately 200 copies / mL), both test strips for repeated testing showed a clear red band at the test line (T line), indicating a positive result. When the concentration decreased to 2.0 × 10⁻⁶, the positive result was confirmed. -2 When the number of copies / μL was 1, only one replicate showed a weak band, while the rest were negative.

[0063] Control qPCR method: at 2.0 × 10 2 At a concentration of copies / mL, only 2 out of 3 replicates were detected, and the Ct values ​​were all greater than 35. The detection rate and stability were slightly lower than those of the method of this invention.

[0064] Conclusion: The RPA-LFD detection method established in this invention has high sensitivity for SFTSV nucleic acid, with a limit of detection of 200 copies / mL (serum). This sensitivity is comparable to that of mainstream clinical qPCR kits, and even shows more stable detection performance, fully meeting the detection needs of clinical samples with low viral load.

[0065] 2. Specificity test Objective: To verify the specificity of the primer-probe composition of the present invention for SFTSV and to investigate its potential for cross-reactivity with other pathogens.

[0066] Materials: Nucleic acid extracts from SFTSV-positive clinical samples (qPCR Ct value known to be 25).

[0067] Nucleic acids from other common pathogens: 1. Novel Bunyavirus; 2. Salmonella Typhi; 3. Anaplasma phagocytophilum; 4. Sandfly virus; 5. Tick-borne encephalitis virus; 6. Orientia tsutsugamushi; 7. Dengue virus; 8. Borrelia burgdorferi; 9. Hantavirus; 10. Nucleic acid extracts from healthy human serum (negative control). All pathogen nucleic acids were validated by sequencing.

[0068] Methods: The nucleic acid concentrations of each pathogen were adjusted to approximately the same level (about 10) using a spectrophotometer or fluorometer. 5 (copies / mL). Using 10 μL of each pathogen's nucleic acid as a template, the RPA amplification and LFD detection procedures described in Example 3 were followed.

[0069] Results and Conclusions: Specificity test results are as follows Figure 3 As shown, number 1 represents the SFTSV nucleic acid template, and both the test line (T line) and control line (C line) on its test strip are colored, indicating a strong positive result. Numbers 2 to 10 represent the nucleic acids of Salmonella typhi, Anaplasma phagocytophilum, sandfly fever virus, tick-borne encephalitis virus, Orientia scrub typhus, dengue virus, Borrelia burgdorferi, Hantavirus, and serum nucleic acid from healthy individuals (negative control), respectively. All test strips numbered 2 to 10 showed only the control line (C line) colored, with no band on the test line (T line), indicating a definitive negative result. This demonstrates that the specific primer-probe composition (SEQ ID NO: 1-3) provided by this invention exhibits high recognition specificity for SFTSV and shows no cross-reactivity with eight clinically important bacteria, rickettsiae, and viral pathogens that may cause fever accompanied by thrombocytopenia. This proves that the detection protocol can effectively distinguish SFTSV infection from other similar diseases, has extremely high diagnostic specificity, and can greatly reduce the risk of false positives.

[0070] 3. Clinical sample validation Objective: To evaluate the actual detection capability of the method of the present invention on real clinical samples.

[0071] Materials: 15 serum samples were collected from patients with acute SFTSV infection from different regions and confirmed by virus isolation or qPCR; 15 serum samples were collected from healthy individuals undergoing physical examinations; and 10 serum samples were collected from patients with fever caused by other etiologies (confirmed as bacterial infection, other viral infection, etc.).

[0072] Methods: All 40 samples were randomly assigned in a blinded manner. A technician unfamiliar with the sample background strictly followed the procedure in Example 3 and used the kit of this invention to test all samples. Simultaneously, another technician performed parallel double-blind testing on all samples using the aforementioned validated commercial qPCR kit, including standard nucleic acid extraction steps. The detection results of the two methods were compared, and concordance rate, sensitivity, specificity, and other indicators were calculated.

[0073] Results: The qPCR gold standard showed that all 15 SFTSV-positive samples were positive; all 15 healthy serum samples were negative; and among the 10 serum samples from other febrile patients, one was positive for dengue fever, while the remaining nine were negative. The RPA-LFD method of this invention showed that all 15 SFTSV-positive samples were positive; all 15 healthy serum samples were negative; and none of the 10 serum samples from other febrile patients were positive for SFTSV (including one qPCR-confirmed dengue-positive sample), consistent with the interpretation results of qPCR for SFTSV.

[0074] To further demonstrate the consistency between the method of this invention and qPCR detection results at the specific data level, 10 representative clinical samples with different viral loads (including positive, negative, and borderline samples) were selected for comparison. The results are as follows: Figure 4 As shown in the figure, the qPCR Ct values ​​of each sample and the corresponding LFD-RPA test strip results are clearly listed. As shown, in the 9 samples where qPCR was clearly detectable (Ct value ≤ 37.66), the LFD-RPA method of this invention gave clear positive results in all of them; for the 1 sample where qPCR was not detectable (No Ct), the LFD-RPA result was negative. This specific data confirms the conclusion of high consistency between the results of the two methods in the aforementioned large-sample validation, and intuitively demonstrates the accurate detection capability of the method of this invention for clinical samples with different viral loads.

[0075] Comparative analysis results For SFTSV detection: the positive and negative concordance rates between the method of this invention and qPCR are both 100%.

[0076] Overall agreement rate: Among the 40 samples, the results of the two methods were completely consistent, with an overall agreement rate of 100%.

[0077] Detection time: The average total time for the method of this invention is about 40 min / batch; the average total time for the qPCR method (including nucleic acid extraction) is about 150 min / batch.

[0078] Conclusion: The rapid RPA-LFD detection method established in this invention exhibits excellent accuracy in detecting real clinical serum samples, and its results are highly consistent with the gold standard qPCR method. Furthermore, this method demonstrates an overwhelming advantage in detection speed and is extremely simple to operate, fully proving its reliability and high efficiency in clinical applications.

[0079] In summary, this invention provides a complete RPA-LFD solution for SFTSV. Compared with existing SFTSV diagnostic technologies, this invention, while maintaining high sensitivity and specificity comparable to quantitative real-time PCR (qPCR), revolutionarily simplifies the operation process (eliminating nucleic acid extraction), reduces reliance on sophisticated instruments, and shortens the detection time from several hours to approximately 40 minutes, thereby enabling a leap from laboratory testing to rapid on-site screening.

[0080] The above embodiments are merely illustrative of the technical concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description, and thus all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention.

Claims

1. A primer-probe composition specifically for detecting fever with thrombocytopenia syndrome virus, characterized in that, It includes upstream primers, downstream primers, and probes, among which: The upstream primer has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer has the nucleotide sequence shown in SEQ ID NO:2 and is labeled with biotin at its 5' end; The probe has the nucleotide sequence shown in SEQ ID NO:3, with a reporter group labeled at its 5' end, a tetrahydrofuran modification site inside the sequence, and a blocking group attached to its 3' end.

2. The primer and probe composition for specific detection of fever with thrombocytopenia syndrome virus according to claim 1, characterized in that, The reporter group is fluorescein isothiocyanate or carboxyfluorescein.

3. A kit for detecting fever with thrombocytopenia syndrome virus, characterized in that, It comprises: the primer and probe composition of claim 1 or 2, the nucleic acid release agent, and the lateral flow chromatography test strip.

4. The kit for detecting fever with thrombocytopenia syndrome virus according to claim 3, characterized in that, It also contains an enzyme mixture and reaction buffer required for the recombinase polymerase amplification reaction.

5. The kit for detecting fever with thrombocytopenia syndrome virus according to claim 4, characterized in that, The enzyme mixture contains recombinant enzymes, single-stranded DNA-binding proteins, and strand displacement DNA polymerases.

6. The kit for detecting fever with thrombocytopenia syndrome virus according to claim 3, characterized in that, The detection line of the lateral flow chromatography test strip is immobilized with streptavidin or an antibody against the reporter group.

7. A method for detecting fever with thrombocytopenia syndrome virus, characterized in that, Includes the following steps: (1) The test sample containing SFTSV was mixed with a nucleic acid release agent and processed to obtain a lysis buffer; (2) The lysis buffer is directly used as a nucleic acid template and added to a reaction system containing the primer and probe composition of claim 1 or 2 to carry out a recombinase polymerase amplification reaction to obtain the amplification product; (3) The amplification product is detected using a lateral flow chromatography test strip, and the result is determined based on the color development of the test line and the control line.

8. The method for detecting fever with thrombocytopenia syndrome virus according to claim 7, characterized in that, The sample to be tested is serum or plasma.

9. The method for detecting fever with thrombocytopenia syndrome virus according to claim 7, characterized in that, Step (1) involves mixing the test sample containing SFTSV with the nucleic acid release agent and incubating it at 15-30°C for 2-10 minutes.

10. The method for detecting fever with thrombocytopenia syndrome virus according to claim 7, characterized in that, The reaction temperature of the recombinase polymerase amplification reaction in step (2) is 38~43℃, and the reaction time is 20~30min.

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