A kit for simultaneous detection of 7 arboviruses and its application

Abstract

This invention discloses a kit for the simultaneous detection of seven arboviruses and its applications, providing primer and probe sets for detecting dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus. Furthermore, this invention utilizes dual reporter group labeling technology to simultaneously achieve qualitative detection of these seven arboviruses in a single reaction tube. The detection results are intuitive and clear, with high accuracy, strong specificity, and a sensitivity reaching 1×10⁻⁶. 2 This invention can meet the practical application needs of simultaneous, rapid, and accurate detection of multiple vector-borne pathogens in scenarios such as clinical disease control screening, rapid on-site detection, port quarantine, and epidemiological monitoring, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the technical field of viral molecular detection, specifically to a kit for simultaneously detecting seven arboviruses and its application. Background Technology

[0002] Vector-borne viruses are a collective term for viruses that infect humans and vertebrates through bites, transmitted by arthropods such as mosquitoes, ticks, midges, and sandflies. Common causative strains include dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus. These viruses are characterized by rapid transmission, wide spread, and a tendency to cause cluster outbreaks and even epidemics. Therefore, accurate and efficient vector-borne virus detection is of irreplaceable importance for preventing outbreaks in advance, optimizing public health decision-making, comprehensively protecting public health, and minimizing socioeconomic losses.

[0003] Vector-borne virus detection is a core support for achieving early detection, early diagnosis, and early intervention. Early symptoms of these viral infections are often nonspecific, highly overlapping with those of the common influenza, sepsis, and other viral infectious diseases. Relying solely on clinical symptoms and physical examinations easily leads to misdiagnosis and missed diagnosis. Furthermore, current methods for detecting single vector-borne pathogens are time-consuming and cannot achieve rapid and accurate detection and identification of multiple pathogens, failing to meet the needs of rapid screening and timely intervention in epidemic prevention and control. Therefore, there is an urgent need to establish a rapid method that can simultaneously achieve early diagnosis of multiple different pathogens to shorten diagnostic time and support the timely implementation of prevention and control measures.

[0004] Currently, the main methods for detecting arboviruses include virus isolation and culture, serological detection, and RT-PCR. Each method has significant limitations and cannot meet the actual needs of clinical and field testing. While virus isolation and culture is the gold standard for infection diagnosis, it is complex, time-consuming, labor-intensive, has poor sensitivity, and a low isolation rate, failing to meet the need for simultaneous detection of multiple pathogens. Serological detection methods have poor specificity, are prone to cross-reactions between different pathogens, making accurate identification of viral types difficult. Furthermore, serum neutralization tests are time-consuming and costly, making them unsuitable for routine clinical laboratory testing and rapid field testing. While RT-PCR offers advantages such as high specificity, high sensitivity, quantitative detection, and type identification, and can currently detect single arboviruses, it suffers from decreased specificity, reduced sensitivity, and insufficient anti-interference capabilities when simultaneously detecting multiple arboviruses. Consequently, there are currently no commercially available RT-PCR test kits that can rapidly, effectively, and accurately detect multiple common arboviruses simultaneously.

[0005] Chinese invention patent CN120350172A can only detect three arboviruses: dengue virus, Zika virus, and chikungunya virus. Its detection throughput is low, making it difficult to cover common pathogenic strains. While Chinese invention patent CN118166168A can detect five arboviruses at once, this technology is based on a microarray-based POCT multiplex PCR detection system. It requires assembling relevant primers and probes into a specific array into a disposable cartridge and relies on specific POCT equipment for detection. This results in high equipment and reagent costs, poor versatility, and limited application in ordinary laboratories. Chinese invention patent CN117721250A can simultaneously detect six arboviruses, but it requires two reaction tubes (A and B), with each tube only capable of detecting three arboviruses. The detection process is relatively cumbersome and time-consuming, failing to achieve truly rapid single-tube multiplex detection.

[0006] Furthermore, in conventional multiplex fluorescent PCR technology, the emission wavelengths of fluorescent dyes are prone to overlap, which can easily cause detection interference and limit the detection throughput. This makes it impossible to achieve simultaneous and accurate detection of multiple pathogens in a single tube, further restricting the efficiency and accuracy of vector-borne virus detection and making it difficult to meet the core requirements of rapid, efficient, and accurate detection in epidemic prevention and control. Summary of the Invention

[0007] To overcome the aforementioned defects and shortcomings in the existing technology, the present invention provides a kit for simultaneously detecting seven arboviruses and its application.

[0008] The first objective of this invention is to provide a primer-probe set for the simultaneous detection of seven arboviruses.

[0009] A second objective of this invention is to provide a composition for the simultaneous detection of seven arboviruses.

[0010] A third objective of this invention is to provide a kit for the simultaneous detection of seven arboviruses.

[0011] A fourth object of the present invention is to provide the use of the above-described primer-probe sets, compositions and / or kits in the preparation of products for the detection of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and / or Sindbis virus.

[0012] This invention claims protection for the following: A primer and probe set for simultaneous detection of seven arboviruses, namely dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus. The primer-probe set consists of a primer set and a fluorescent probe, wherein: The nucleotide sequences of the primer set used for detecting dengue virus are shown in SEQ ID NO: 1-2, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 3; The nucleotide sequences of the primer set used for detecting Chikungunya virus are shown in SEQ ID NO: 4-5, and the nucleotide sequences of the fluorescent probe are shown in SEQ ID NO: 6. The nucleotide sequences of the primer set used for detecting Zika virus are shown in SEQ ID NO: 7-8, and the nucleotide sequences of the fluorescent probe are shown in SEQ ID NO: 9. The nucleotide sequences of the primer set used for detecting yellow fever virus are shown in SEQ ID NO: 10-11, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 12. The nucleotide sequences of the primer set used for detecting West Nile virus are shown in SEQ ID NO: 13-14, and the nucleotide sequences of the fluorescent probe are shown in SEQ ID NO: 15. The nucleotide sequences of the primer set used for detecting Japanese encephalitis virus are shown in SEQ ID NO: 16-17, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 18. The nucleotide sequences of the primer set used to detect Sindbis virus are shown in SEQ ID NO: 19-20, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 21.

[0013] Preferably, the fluorescent probe is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.

[0014] Preferably, of the seven fluorescent probes, four fluorescent probes are each labeled with one fluorescent group, and the fluorescent groups labeled by the four fluorescent probes are different from each other; The remaining three fluorescent probes are each labeled with two fluorescent groups. These two fluorescent groups are selected from any two different fluorescent groups labeled by the four fluorescent probes, and the combination of fluorescent groups labeled by the three fluorescent probes is different from each other.

[0015] As one possible implementation, the fluorescent group includes FAM, ROX, CY5 or VIC, and the quenching group includes BHQ1, BHQ2 or MGB.

[0016] As an implementable method, the nucleotide sequence of the fluorescent probe shown in SEQ ID NO: 3 is labeled with the fluorescent group FAM at the 5' end and BHQ1 at the 3' end; The fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 6 is labeled with the fluorescent group VIC at its 5' end and BHQ1 at its 3' end. The fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 9 is labeled with the fluorescent group ROX at its 5' end and BHQ2 at its 3' end. The fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 12 is labeled with the fluorescent group Cy5 at its 5' end and BHQ2 at its 3' end. The fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 15 is labeled with the fluorescent group FAM at the 5' end, the fluorescent group VIC at the 7th base, and BHQ1 at the 3' end. The fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 18 is labeled with the fluorescent group FAM at the 5' end, the fluorescent group ROX at the 8th base, and BHQ2 at the 3' end. The fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 21 has a 5' end labeled with the fluorescent group FAM, a 9th base labeled with the fluorescent group Cy5, and a 3' end labeled with BHQ2.

[0017] This invention employs dual reporter gene labeling technology, enabling the simultaneous qualitative detection of seven arboviruses in a single reaction tube. This fundamentally solves the problem that conventional single reporter gene labeling technology cannot simultaneously detect more than five pathogens in one reaction tube, effectively overcoming the inherent throughput limitations of traditional fluorescent PCR technology and truly achieving simultaneous multi-target detection of seven pathogens in a single tube.

[0018] Dual reporter group labeled probes can simultaneously generate two independent fluorescence signals during specific amplification and detection. Based on this, a multi-dimensional fluorescence signal discrimination system can be constructed by combining the type, intensity, and characteristic wavelength of the fluorescence signals, enabling accurate differentiation and result determination of different target viruses. Compared to the traditional mode of pathogen differentiation relying solely on a single fluorescence signal by conventional single reporter group probes, this invention significantly reduces the difficulty of system optimization and signal analysis complexity in multiplex fluorescent PCR by utilizing a dual fluorescence signal combination encoding mechanism. Without adding fluorescence detection channels or modifying existing detection instrument hardware, it significantly increases the upper limit of the types of targets that can be simultaneously distinguished, effectively breaking through the technical bottleneck of insufficient throughput in traditional fluorescent PCR technology.

[0019] Based on the aforementioned signal recognition and amplification detection logic, and given the extremely low probability of co-infection of two or more target viruses in a sample, this invention can simultaneously detect seven arboviruses in a single tube. Compared to conventional detection methods that can only detect a single virus at a time, this invention offers significant advantages: for the most common clinical scenario of single-virus infection, a single test can accurately determine the specific virus type infecting the sample; even in rare cases of multiple virus co-infection, this single-tube detection system can still preliminarily screen out the candidate range of infecting viruses. Subsequent tests, combined with a small number of supplementary tests and clinical symptoms, can ultimately clarify all the virus types actually infecting the sample, completely avoiding the cumbersome process of multiple, one-by-one single-virus testing required by conventional methods, greatly improving detection efficiency and simplifying the detection operation.

[0020] A composition for simultaneously detecting seven arboviruses, the composition comprising the primer-probe set described above; The seven arboviruses mentioned are dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus.

[0021] A kit for simultaneous detection of seven arboviruses, the kit comprising the aforementioned primer and probe set; The seven arboviruses mentioned are dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus.

[0022] Preferably, the kit further includes a positive control reference and / or a negative control reference.

[0023] More preferably, the positive control reference is a pseudovirus containing the detection target genes of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and / or Sindbis virus.

[0024] As one feasible approach, the negative control reference is physiological saline.

[0025] Preferably, the kit further includes a real-time PCR detection reagent.

[0026] The above-described primer-probe sets, compositions, and / or kits are used in the preparation of products for the detection of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and / or Sindbis virus.

[0027] A reaction solution for simultaneously detecting seven arboviruses includes an upstream primer, a downstream primer, a fluorescent probe, DNA polymerase, reverse transcriptase, RNase inhibitor, dNTPs, and PCR buffer. The concentrations of the upstream and downstream primers were both 0.32–0.48 µM, the concentration of the fluorescent probe was 0.08–0.16 µM, the concentration of DNA polymerase was 0.3–0.7 U / µL, the concentration of reverse transcriptase was 6–10 U / µL, the concentration of RNase inhibitor was 1–2 U / µL, and the concentration of dNTPs was 0.3–0.7 mM.

[0028] Preferably, the concentrations of the upstream and downstream primers are both 0.32–0.48 µM, the concentration of the fluorescent probe is 0.08–0.16 µM, the concentration of DNA polymerase is 0.5 U / µL, the concentration of reverse transcriptase is 8 U / µL, the concentration of RNase inhibitor is 1.6 U / µL, and the concentration of dNTPs is 0.5 mM.

[0029] Preferably, the reaction conditions for the reaction solution are: reverse transcription at 50℃ for 8 min; pre-denaturation at 95℃ for 2 min; amplification reaction: reaction at 94℃ for 2 s, reaction at 58℃ for 29 s, for 45 cycles. During the cycling steps, fluorescence signals from four channels (FAM, VIC, CY5, and ROX) are collected respectively.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a kit for the simultaneous detection of seven arboviruses and its applications, providing primer and probe sets for detecting dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus. Furthermore, this invention utilizes dual reporter group labeling technology to simultaneously achieve qualitative detection of these seven arboviruses in a single reaction tube. The detection results are intuitive and clear, with high accuracy, strong specificity, and a sensitivity reaching 1×10⁻⁶. 2 This invention can meet the practical application needs of simultaneous, rapid, and accurate detection of multiple vector-borne pathogens in scenarios such as clinical disease control screening, rapid on-site detection, port quarantine, and epidemiological monitoring, and has broad application prospects. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of an RT-PCR amplification procedure.

[0032] Figure 2 The concentration in Example 2 was 1×10 5 Amplification curve of dengue pseudovirus copies / mL.

[0033] Figure 3 The concentration in Example 2 was 1×10 5 Amplification curve of chikungunya pseudovirus at copies / mL.

[0034] Figure 4The concentration in Example 2 was 1×10 5 Amplification curve of Zika pseudovirus copies / mL.

[0035] Figure 5 The concentration in Example 2 was 1×10 5 Amplification curve of yellow fever pseudovirus copies / mL.

[0036] Figure 6 The concentration in Example 2 was 1×10 5 Amplification curve of West Nile pseudovirus copies / mL.

[0037] Figure 7 The concentration in Example 2 was 1×10 5 Amplification curve of Japanese encephalitis pseudovirus at copies / mL.

[0038] Figure 8 The concentration in Example 2 was 1×10 5 Amplification curve of Sindbis pseudovirus at copies / mL.

[0039] Figure 9 The concentration in Example 2 was 1×10 6 Amplification curve of positive control 1 (copies / mL).

[0040] Figure 10 The concentration in Example 2 was 1×10 6 Amplification curve of positive control 2 (copies / mL).

[0041] Figure 11 The concentration in Example 2 was 1×10 6 Amplification curve of positive control 3 (copies / mL).

[0042] Figure 12 This is the amplification curve of the negative control sample in Example 2.

[0043] Figure 13 The results are the sensitivity test results for dengue virus in Example 3.

[0044] Figure 14 The results show the sensitivity detection of Chikungunya virus in Example 3.

[0045] Figure 15 The results show the sensitivity test results for the Zika virus in Example 3.

[0046] Figure 16 The results are the sensitivity test results for yellow fever virus in Example 3.

[0047] Figure 17The results show the sensitivity test results for West Nile virus in Example 3.

[0048] Figure 18 The results show the sensitivity detection of Japanese encephalitis virus in Example 3.

[0049] Figure 19 The results are the sensitivity test results for the Sindbis virus in Example 3.

[0050] Figure 20 This is the amplification result of the virus for fever with thrombocytopenia syndrome in Example 4.

[0051] Figure 21 This is the amplification result of the measles virus in Example 4.

[0052] Figure 22 This is the amplification result of the rubella virus in Example 4.

[0053] Figure 23 This is the amplification result of the EB virus in Example 4.

[0054] Figure 24 This is the amplification result of cytomegalovirus in Example 4.

[0055] Figure 25 This is the amplification result of the varicella-zoster virus culture in Example 4.

[0056] Figure 26 This is the amplification result of dengue virus type 1 in Example 4.

[0057] Figure 27 This is the amplification result of dengue virus type 2 in Example 4.

[0058] Figure 28 This is the amplification result of dengue virus type 3 in Example 4.

[0059] Figure 29 This is the amplification result of dengue virus type 4 in Example 4.

[0060] Figure 30 This is the amplification result of the chikungunya virus in Example 4.

[0061] Figure 31 This is the amplification result of the Zika virus in Example 4.

[0062] Figure 32 This is the amplification result of yellow fever virus in Example 4.

[0063] Figure 33 This is the amplification result of West Nile virus in Example 4.

[0064] Figure 34 This is the amplification result of the Japanese encephalitis virus in Example 4.

[0065] Figure 35 The amplification results are for the simulated Sindbis virus sample in Example 4. Detailed Implementation

[0066] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0067] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0068] Example 1: Primer and probe design The full-length genome sequences of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus were obtained from the NCBI GeneBank database. After sequence alignment analysis, specific primers and fluorescent probes were designed based on conserved regions. The nucleotide sequences are shown in Table 1. The target sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and are shown in Table 2.

[0069] Table 1 Specific primers and fluorescent probes

[0070] Note 1: In the table, the lowercase t bases in the fluorescent probes (P) for West Nile virus, Japanese encephalitis virus, and Sindbis virus represent their marker reporter genes.

[0071] Note 2: S, Y, W, R, and M are degenerate bases, where S represents G or C, Y represents T or C, W represents A or T, R represents A or G, and M represents A or C.

[0072] Table 2 Specific target sequences

[0073] Example 2: Detection of Seven Arboviruses I. Experimental Methods Sequences containing target gene fragments of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus were given to a pseudovirus synthesis company to synthesize corresponding pseudoviruses. After extracting nucleic acids from the pseudoviruses, their concentrations were measured and their copy number concentrations were calculated, then quantified to 1×10⁻⁶. 5 Copies / mL were used to obtain the corresponding pseudovirus samples.

[0074] Preparation of positive control samples: Sequences containing the target gene fragments of West Nile virus, Japanese encephalitis virus, and Sindbis virus were sent to a pseudovirus synthesis company to synthesize corresponding pseudoviruses. After extracting nucleic acids from the pseudoviruses, their concentrations were measured and their copy number concentrations were calculated. Then, they were quantified to 1×10⁻⁶. 6 copies / mL, labeled as positive control 1 (West Nile virus), positive control 2 (Japanese encephalitis virus), and positive control 3 (Sindbis virus).

[0075] Preparation of negative control: Dissolve 9.0 g of sodium chloride in distilled water, then bring the volume to 1000 mL and filter through a 0.22 µm filter membrane to obtain the negative control.

[0076] The corresponding fake virus samples, positive control samples, or negative control samples were used as DNA templates for PCR detection. The preparation of the PCR reaction solution is shown in Table 2.

[0077] Table 2 PCR reaction solution

[0078] RT-PCR amplification was performed using an ABI Prism 7500 real-time PCR instrument: Open the Setup window, set the negative and positive control samples and the test samples in the corresponding order, and set the sample name in the Name field. Select all the sample wells, double-click, select Add Detector, select Reporter as FAM and Quencher as none, then select Reporter as VIC and Quencher as none; then select Reporter as ROX and Quencher as none; then select Reporter as Cy5 and Quencher as none and close the window. Select none in Passive Reference. Open the Instrument window and set the cycling conditions: 50℃ 8 min, 95℃ 2 min; 94℃ 2 s, 58℃ 29 s, 45 cycles (…). Figure 1 Save the file after all settings are complete, then run it.

[0079] After the reaction is complete, save the detection data file. Open the Ampplot window under Results. Select the location of the target sample for analysis. Change the Baseline values ​​to start: 3, stop: 10, and open the manual settings to set Threshold: 1.5 ± 100000. Double-click the value on the Rn coordinate to open the Graph settings window. Change Log to Linear in Post Run Settings, and click OK. Then open the Analysis preferences window and select Analyze under the Analysis menu to automatically analyze the results.

[0080] Result interpretation: Negative result: If there is no obvious amplification curve or Ct value > 38 in any fluorescence detection channel, it is judged as a negative result; Positive result: If the fluorescence detection channel shows a clear amplification curve and the Ct value is ≤38, then the detection channel is judged to be positive. The positive results of the detection channel need to be analyzed in conjunction with Table 3. Table 3. Correspondence between fluorescence detection channel results and viruses.

[0081] Note: If both the FAM and VIC fluorescence channels of a sample are positive, it is interpreted as a positive result for West Nile virus; if both the FAM and ROX fluorescence channels of a sample are positive, it is interpreted as a positive result for Japanese encephalitis virus; if both the FAM and Cy5 fluorescence channels of a sample are positive, it is interpreted as a positive result for Sindbis virus.

[0082] II. Experimental Results As shown in Table 4 and Figures 2-12 As shown, the amplification curves of all pseudoviruses and positive control samples were S-shaped with obvious gradients, while the negative control sample showed no amplification curve. Simultaneously, the detection results of the relevant pseudoviruses were all positive for the corresponding types, and the negative and positive control samples also showed corresponding positive and negative results.

[0083] The above results indicate that the detection method of the present invention has good accuracy.

[0084] Table 4 Virus Detection Results

[0085] Example 3 Sensitivity Detection I. Experimental Methods Nucleic acid was extracted from pseudoviruses containing target gene fragments of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus, and then diluted to a concentration of 1×10⁻⁶. 2 copies / mL, 1×10 3copies / mL, 1×10 4 copies / mL, 1×10 5 copies / mL, and the detection was performed according to Example 2.

[0086] II. Experimental Results The results are as follows Figures 13-19 As shown, 1×10 2 copies / mL~1×10 5 The method of this invention can detect dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus at a concentration of copies / mL, indicating that the detection sensitivity of the method of this invention for dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus is 1×10⁻⁶. 2 copies / mL.

[0087] Example 4 Specificity Detection I. Experimental Methods Following the method in Example 2, dengue virus type 1, dengue virus type 2, dengue virus type 3, dengue virus type 4, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, fever with thrombocytopenia syndrome virus, measles virus, rubella virus, Epstein-Barr virus, cytomegalovirus, varicella-zoster virus culture, Sindbis virus simulant (pseudovirus + negative plasma), positive control and negative control were tested and the results were analyzed.

[0088] II. Experimental Results The results are shown in Table 5 and Figures 20-35 As shown, the test results of the positive and negative control samples met the requirements, indicating that the experiment was effective. Cultures of fever with thrombocytopenia syndrome virus, measles virus, rubella virus, EB virus, cytomegalovirus, and varicella-zoster virus showed no amplification curves, and the test results were negative. Simulated samples of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus showed amplification curves in their respective detection channels, and the test results were positive.

[0089] The above results demonstrate that the method of the present invention has good specificity.

[0090] Table 5 Virus Detection Results

[0091] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A primer probe set for simultaneous detection of 7 arboviruses, characterized in that, The seven arboviruses mentioned are dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus. The primer-probe set consists of a primer set and a fluorescent probe, wherein: The nucleotide sequences of the primer set used for detecting dengue virus are shown in SEQ ID NO: 1-2, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 3; The nucleotide sequences of the primer set used for detecting Chikungunya virus are shown in SEQ ID NO: 4-5, and the nucleotide sequences of the fluorescent probe are shown in SEQ ID NO:

6. The nucleotide sequences of the primer set used for detecting Zika virus are shown in SEQ ID NO: 7-8, and the nucleotide sequences of the fluorescent probe are shown in SEQ ID NO:

9. The nucleotide sequences of the primer set used for detecting yellow fever virus are shown in SEQ ID NO: 10-11, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO:

12. The nucleotide sequences of the primer set used for detecting West Nile virus are shown in SEQ ID NO: 13-14, and the nucleotide sequences of the fluorescent probe are shown in SEQ ID NO:

15. The nucleotide sequences of the primer set used for detecting Japanese encephalitis virus are shown in SEQ ID NO: 16-17, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO:

18. The nucleotide sequences of the primer set used to detect Sindbis virus are shown in SEQ ID NO: 19-20, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO:

21.

2. The primer probe set of claim 1, wherein, The fluorescent probes are labeled with fluorescent groups and quenching groups, respectively.

3. The primer probe set of claim 2, wherein, Of the seven fluorescent probes, four are labeled with a different fluorescent group. The remaining three fluorescent probes are each labeled with two fluorescent groups. These two fluorescent groups are selected from any two different fluorescent groups labeled by the four fluorescent probes, and the combination of fluorescent groups labeled by the three fluorescent probes is different from each other.

4. The primer probe set of claim 2, wherein, The fluorescent group includes FAM, ROX, CY5 or VIC, and the quenching group includes BHQ1, BHQ2 or MGB.

5. A composition for simultaneous detection of 7 arboviruses, characterized in that, The composition comprises the primer-probe set according to any one of claims 1 to 4; The seven arboviruses mentioned are dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus.

6. A kit for simultaneous detection of 7 arboviruses, characterized in that, The kit comprises the primer and probe set as described in any one of claims 1 to 4; The seven arboviruses mentioned are dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Sindbis virus.

7. The kit of claim 6, wherein The kit also includes a positive control reference and / or a negative control reference.

8. The kit of claim 7, wherein The positive control reference is a pseudovirus containing the detection target genes of dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and / or Sindbis virus.

9. The kit of claim 6, wherein The kit also includes a real-time PCR detection reagent.

10. The use of the primer and probe set according to any one of claims 1 to 4, the composition according to claim 5, and / or the kit according to claims 6 to 9 in the preparation of products for detecting dengue virus, chikungunya virus, Zika virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and / or Sindbis virus.

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