Kit for detecting respiratory syncytial virus and typing thereof based on microfluidics method, and application and method thereof

Abstract

The present invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting and typing respiratory syncytial virus based on microfluidics, and its application and method. The kit provided by the present invention includes a microfluidic chip, and reaction holes are provided on the microfluidic chip, and a PCR enhancing freeze-dried mixture is filled in the reaction holes; the PCR enhancing freeze-dried mixture is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. By optimizing the components of the PCR enhancing freeze-dried mixture and combining the microfluidic method to detect and type respiratory syncytial virus, the present invention can significantly improve the detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting and typing respiratory syncytial virus based on microfluidics, and its application and method. Background Art

[0002] Respiratory syncytial virus (RSV), as a pathogen that has drawn significant attention in the global public health field, plays a crucial role in respiratory infectious diseases. Especially for infants, the elderly, and immunocompromised individuals, RSV infection can pose a serious health threat.

[0003] RSV belongs to the genus Pneumovirus of the Paramyxoviridae family. Its virions are spherical or filamentous, have an envelope, and the genome is single-stranded negative-sense RNA. During natural infection, the virus spreads through airborne droplets and close contact, infects respiratory epithelial cells, triggers an immune response, and causes typical symptoms such as fever, cough, and wheezing. In severe cases, it can also cause bronchiolitis and pneumonia, even endangering life.

[0004] RSV is divided into subtype A (RSV-A) and subtype B (RSV-B) according to the antigenicity of its attachment protein G. Clinically, typing RSV is of great significance. Accurate typing results can guide the formulation of clinical treatment plans. Since different subtypes of RSV have different sensitivities to drugs, knowing the specific type allows doctors to more specifically select drugs, thereby improving the treatment effect. From the perspective of disease prevention and control, monitoring the prevalence of different subtypes of RSV in different regions and seasons helps public health departments plan prevention and control strategies in advance and rationally allocate medical resources. In addition, the severity of the disease and prognosis caused by different subtypes of RSV infection are different. RSV-A infection may lead to more severe conditions, such as a relatively high incidence of complications such as respiratory failure, while RSV-B infection is relatively mild; accurate typing can more accurately assess the prognosis of patients and provide more reliable rehabilitation advice. In the field of vaccine research and development, understanding the main epidemic strains can develop more targeted vaccines and improve the protective effect of vaccines.

[0005] However, RSV typing faces many challenges. RSV has a high mutation rate, its antigenicity is constantly changing, and new variants continue to emerge, making traditional typing methods difficult to cope with. At the same time, mixed infections between different RSV types and RSV with other pathogens are not uncommon, which further interferes with the accuracy of typing. In addition, existing detection technologies have shortcomings in sensitivity, specificity, and ease of operation, making it difficult to meet the needs of rapid and accurate clinical typing. At present, commonly used RSV typing methods mainly include serological methods, virus culture methods, and genotyping methods. Serological methods, such as immunofluorescence and enzyme-linked immunosorbent assay, detect viral antigens or antibodies in patient serum through specific antibodies, but this method is easily affected by cross-reactions, has limited sensitivity, and is not effective for detecting samples with low viral loads. The virus culture method requires the virus to be inoculated into cell culture, and the type is determined by observing the cytopathic characteristics and combining it with serological methods. This method is time-consuming, has a low positive rate, and has strict requirements for sample collection and transportation conditions, which limits its application in clinical rapid detection. The genotyping method uses reverse transcription polymerase chain reaction (RT-PCR) and sequencing technology to analyze the differences in viral gene sequences for typing. Although it has high accuracy, the operation process is complicated and costly, and it has strict requirements on laboratory conditions and personnel technical level. In addition, unreasonable primer design may lead to missed detection.

[0006] Against the above technical background, the emergence of microfluidics has brought new hope for RSV typing. Microfluidics has significant advantages. The first is integration and miniaturization. It can integrate multiple experimental steps on a tiny chip, greatly reducing the amount of samples and reagents used. At the same time, it is easy to carry, making on-site testing possible. Secondly, microfluidics has the characteristics of high throughput and can process multiple samples at the same time, greatly improving detection efficiency and enabling rapid typing of a large number of samples. Furthermore, its high sensitivity can effectively enrich and detect low concentrations of viruses and reduce the missed detection rate. In addition, microfluidics can also shorten the reaction time and achieve rapid detection, which is conducive to rapid clinical diagnosis and treatment. Moreover, this technology has the advantage of being customizable and can flexibly design chip structures and detection processes according to different detection needs.

[0007] Currently, the technology for RSV typing using microfluidics is extremely scarce. Therefore, developing this technology has become a key focus in current scientific research. Summary of the Invention

[0008] To address the above shortcomings, the present invention provides a kit for detecting and typing respiratory syncytial virus based on a microfluidic method, as well as its application and method. The kit provided by the present invention includes a microfluidic chip, the microfluidic chip is provided with reaction wells, and the reaction wells are filled with a PCR-enhancing lyophilized mixture; the PCR-enhancing lyophilized mixture is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA, and nuclease-free water. By optimizing the components of the PCR-enhancing lyophilized mixture and combining it with microfluidics for detecting and typing respiratory syncytial virus, the present invention can significantly improve detection sensitivity.

[0009] The technical solution of the present invention is:

[0010] In the first aspect, the present invention provides a kit for detecting and typing respiratory syncytial virus based on the microfluidic method, the kit comprising a microfluidic chip, the microfluidic chip being provided with reaction wells, the reaction wells being filled with a PCR-enhancing lyophilized mixture; the PCR-enhancing lyophilized mixture is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water.

[0011] Specifically, the PCR enhancement freeze-dried mixture includes 0.5-1.5M trimethylglycine, 0.1-0.3M tetramethylammonium chloride, 2-5U / mL bovine thrombin, 0.1-0.5% v / v NP-40, 10-15% w / v trehalose and 0.02-0.05% w / v BSA.

[0012] Preferably, the PCR enhancing lyophilized mixture comprises 1.0 M trimethylglycine, 0.2 M tetramethylammonium chloride, 5 U / mL bovine thrombin, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0013] Specifically, the reaction wells are also filled with PCR premix and primer-probe combination.

[0014] Preferably, the PCR premix comprises PCR buffer, MgSO4, dNTPs and Taq enzyme.

[0015] Specifically, the reaction wells include an RSV-A reaction well, an RSV-B reaction well and an internal reference reaction well.

[0016] Preferably, the primer-probe combination in the RSV-A reaction well is a RSV-A primer-probe combination, consisting of an RSV-A upstream primer, an RSV-A downstream primer and an RSV-A probe.

[0017] In certain embodiments, the RSV-A upstream primer has a nucleotide sequence as shown in SEQ ID NO.1; the RSV-A downstream primer has a nucleotide sequence as shown in SEQ ID NO.2; and the RSV-A probe has a nucleotide sequence as shown in SEQ ID NO.3.

[0018] Preferably, the primer-probe combination in the RSV-B reaction well is an RSV-B primer-probe combination, consisting of an RSV-B upstream primer, an RSV-B downstream primer and an RSV-B probe;

[0019] In certain embodiments, the RSV-B upstream primer has a nucleotide sequence as shown in SEQ ID NO.4; the RSV-B downstream primer has a nucleotide sequence as shown in SEQ ID NO.5; and the RSV-B probe has a nucleotide sequence as shown in SEQ ID NO.6.

[0020] Preferably, the primer-probe combination in the internal reference reaction well is an internal reference gene primer-probe combination, consisting of a B2M upstream primer, a B2M downstream primer and a B2M probe.

[0021] In certain embodiments, the B2M upstream primer has a nucleotide sequence as shown in SEQ ID NO.7; the B2M downstream primer has a nucleotide sequence as shown in SEQ ID NO.8; and the B2M probe has a nucleotide sequence as shown in SEQ ID NO.9.

[0022] Specifically, the kit also includes a positive control substance or a negative control substance.

[0023] Preferably, the positive control substance is a positive control plasmid containing RSV-A type G gene, RSV-B type G gene and B2M gene.

[0024] Preferably, the negative control substance is nucleic acid-free plum water.

[0025] Specifically, the microfluidic chip in the kit is obtained by filling a PCR enhancement freeze-dried mixture, a PCR premix solution and a primer probe combination into reaction wells and then freeze-drying the mixture.

[0026] Preferably, the volume ratio of the PCR synergistic lyophilized mixture, PCR premix and primer-probe combination is 6.5:6.5:2.

[0027] In a second aspect, the present invention provides use of the above-mentioned kit in detecting respiratory syncytial virus or typing respiratory syncytial virus.

[0028] Preferably, the respiratory syncytial virus is classified into RSV-A and RSV-B.

[0029] Third aspect, a method for detecting respiratory syncytial virus or typing respiratory syncytial virus according to the present invention, the method comprising using the above-mentioned kit.

[0030] Specifically, the method comprises the following steps:

[0031] S1. Take the sample to be detected, extract genomic DNA to obtain the nucleic acid sample to be detected;

[0032] S2. Inject the nucleic acid sample to be detected into the injection hole, and flow it through the injection channel into each reaction hole, so that the nucleic acid sample to be detected reacts with the filler in the reaction hole;

[0033] S3. Perform nucleic acid amplification;

[0034] S4. Read and determine the result.

[0035] Preferably, the filler in the reaction hole is a freeze-dried product of a PCR enhancing freeze-dried mixture, a PCR premix and a primer-probe combination.

[0036] Preferably, the nucleic acid amplification conditions include: reacting at 50 °C for 20 min; pre-denaturing at 95 °C for 5 min; reacting at 95 °C for 15 s and at 60 °C for 30 s for 45 cycles.

[0037] Preferably, the conditions for result determination include:

[0038] Conditions for determining RSV-A type: Ct value in the FAM channel ≤ 35, and the amplification curve is a typical S shape; Ct value in the VIC channel ≤ 35, and the amplification curve is a typical S shape; Ct value in the ROX channel ≥ 35 or no Ct value.

[0039] Conditions for determining RSV-B type: Ct value in the FAM channel ≥ 35 or no Ct value; Ct value in the VIC channel ≤ 35, and the amplification curve is a typical S shape; Ct value in the ROX channel ≤ 35, and the amplification curve is a typical S shape.

[0040] Conditions for determining a negative result: Ct value in the FAM channel ≥ 35 or no Ct value; Ct value in the VIC channel ≤ 35, and the amplification curve is a typical S shape; Ct value in the ROX channel ≥ 35 or no Ct value.

[0041] Conditions for determining an invalid result: Ct value in the VIC channel ≥ 35 or no Ct value.

[0042] The beneficial effects of the present invention are: by optimizing the components of the PCR enhancing freeze-dried mixture and combining with the microfluidic method to detect and type respiratory syncytial virus, the detection sensitivity can be significantly improved. Description of the Drawings

[0043] Figure 1 It is a positive test result for RSV-A type.

[0044] Figure 2 It is a positive test result for RSV-B type. Detailed implementation manners

[0045] The present invention will be further clearly and completely described below through embodiments. The following embodiments are only a part of the embodiments of the present invention, and are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are all conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified. The microfluidic chip and the real-time detector are both products of Beijing Baikangxin Biotechnology Co., Ltd.

[0046] Embodiment 1 A kit for detecting respiratory syncytial virus and typing based on microfluidics

[0047] 1. Filling and lyophilization of the microfluidic chip

[0048] 1.1 PCR enhancing lyophilized mixture

[0049] The PCR enhancing lyophilized mixture is composed of betaine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. The PCR enhancing lyophilized mixture is nuclease-free water containing 1.0 M betaine, 0.2 M tetramethylammonium chloride, 5 U / mL bovine thrombin, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0050] 1.2 Preparation of the PCR premix

[0051] Mix Tris-HCl buffer, MgSO4, dNTPs, Taq enzyme and nuclease-free water evenly according to the volumes described in Table 1 to obtain the PCR premix.

[0052] Table 1 PCR premix

[0053]

[0054] 1.3 Primer-probe combination

[0055] For the specific conserved regions of the G genes of RSV-A and RSV-B in the GenBank database, primers and probes for two subtypes of RSV-A and RSV-B were designed; human β-2 microglobulin (B2M) was selected as the internal reference gene, and primers and probes for B2M were designed. The primer and probe sequence information is shown in Table 2.

[0056] Table 2 Primer and probe sequence information

[0057]

[0058] Note: "F" in the table stands for forward primer, "R" stands for reverse primer, and "P" stands for probe.

[0059] The present invention is based on the optimization of a PCR-enhancing lyophilized mixture for the detection of respiratory syncytial virus and its typing based on the aforementioned primer probes. The aforementioned primer probes can be replaced with commonly used primer probes in the prior art. The aforementioned primer probe combination is not intended to limit the composition of the primer probes in the microfluidic chip or kit of the present invention.

[0060] Based on the above primer and probe sequences, RSV-A primer probe combination, RSV-B primer probe combination and B2M primer probe combination were prepared according to the compositions shown in Tables 3-5.

[0061] Table 3 Composition of RSV-A primer-probe combinations

[0062]

[0063] Table 4 Composition of RSV-B primer-probe combinations

[0064]

[0065] Table 5 Composition of B2M primer-probe combination

[0066]

[0067] 1.4 Reaction well filling and freeze-drying

[0068] (1) Add 6.5 μL of PCR booster freeze-dried mixture, 6.5 μL of PCR premix, and 2 μL of RSV-A primer-probe combination to the RSV-A reaction well; add 6.5 μL of PCR booster freeze-dried mixture, 6.5 μL of PCR premix, and 2 μL of RSV-B primer-probe combination to the RSV-B reaction well; add 6.5 μL of PCR booster freeze-dried mixture, 6.5 μL of PCR premix, and 2 μL of B2M primer-probe combination to the internal control reaction well.

[0069] (2) Place the microfluidic chip in a freeze dryer with a pre-freezing temperature of -40°C to -80°C. After pre-freezing, vacuumize the chip and raise the temperature to -20°C to 0°C to allow the ice crystals to sublime directly into water vapor. After sublimation drying is completed, further raise the temperature to 20°C-30°C to remove the remaining bound water. After freeze-drying, package the chip to obtain the freeze-dried microfluidic chip.

[0070] 2. Positive and negative controls

[0071] The kit also includes a positive control and a negative control. A positive control plasmid containing the RSV-A G gene, RSV-B G gene, and B2M gene was synthesized at Shanghai Bioengineering Co., Ltd. and used as the positive control; nucleic acid-free plum water was used as the negative control.

[0072] Example 2: Method for using a kit for detecting and typing respiratory syncytial virus

[0073] 1. Sample collection and purification

[0074] Extraction of nucleic acid samples: DNA / RNA extraction was performed using a DNA / RNA extraction kit (Yisheng Biotechnology, 19321ES50) according to the manufacturer's instructions. The extracted DNA / RNA could be tested immediately or stored at -80°C for subsequent testing.

[0075] 2. Microfluidic chip loading

[0076] Take the freeze-dried microfluidic chip prepared in Example 1, inject the nucleic acid sample to be tested into the injection hole, and flow it into each reaction well through the injection channel (10 μL of nucleic acid sample to be tested per well). Then, seal the entire chip with a sealing layer to ensure that there are no bubbles.

[0077] 3. Nucleic Acid Amplification

[0078] After the sample addition is completed, nucleic acid amplification is performed. The amplification reaction procedure is shown in Table 6.

[0079] Table 6 Amplification reaction procedure

[0080]

[0081] 4. Result determination

[0082] The result determination method is shown in Table 7.

[0083] Table 7 Result determination method

[0084]

[0085] Example 3 Microfluidics-based detection and typing kit for RSV-A and RSV-B viral genomic DNA determination

[0086] RSV-A and RSV-B positive samples were selected, and genomic DNA was extracted from the two positive samples. The two nucleic acid samples to be tested were assayed using the kit described in Example 1 of the present invention and according to the method described in Example 2.

[0087] RSV-A positive test results Figure 1As shown, the positive test result of RSV-B type is as Figure 2 shown. The test result shows that the kit provided by the present invention can be successfully used for the determination and differentiation of RSV-A type and RSV-B type.

[0088] Example 4 Sensitivity of the Kit for Detecting Respiratory Syncytial Virus and Typing Based on Microfluidic Method

[0089] The positive control plasmids containing the G gene of RSV-A type, the G gene of RSV-B type and the B2M gene were serially diluted to samples equivalent to 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 copies / mL, 10 copies / mL. The kit described in Example 1 of the present invention was used and the measurement was carried out according to the usage method described in Example 2. The test results are shown in Table 8.

[0090] Table 8 Test Results of Sensitivity

[0091]

[0092] Note: In the table, "+" represents positive and "-" represents negative. [[ID=?]]

[0093] The results show that the lowest detection limit of the kit of the present invention for the two detection targets can reach 10 2 copies / mL, and the sensitivity is good.

[0094] Example 5 Detection of Clinical Samples by the Kit for Detecting Respiratory Syncytial Virus and Typing Based on Microfluidic Method

[0095] Samples from clinically diagnosed patients with RSV-A type (30 cases), patients with RSV-B type (26 cases) and healthy volunteers (20 cases) were used as the samples to be tested. The kit prepared in Example 1 was used and the detection was carried out with reference to the method described in Example 2. The test results are shown in Table 9.

[0096] Table 9 Test Results of Clinical Samples

[0097]

[0098] Comparative Example 1 A Kit for Detecting Respiratory Syncytial Virus and Typing Based on Microfluidic Method

[0099] The difference between the kit of Comparative Example 1 and the kit of Example 1 of the present invention is only that: 1.1 The PCR enhancing freeze-dried mixture is different.

[0100] It should be noted that there seems to be a formatting issue with the "? " in the original text at line 27 which is retained as is in the translation. If this is an error, it may need to be corrected in the original source for a more accurate translation.The PCR enhancing freeze-dried mixture of Comparative Example 1 is composed of trimethylglycine, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. The PCR enhancing freeze-dried mixture is nuclease-free water containing 1.2 M trimethylglycine, 5 U / mL bovine thrombin, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0101] Comparative Example 2 A kit for detecting and typing respiratory syncytial virus based on microfluidics

[0102] The difference between the kit of Comparative Example 2 and the kit of Example 1 of the present invention is only that: 1.1 The PCR enhancing freeze-dried mixtures are different.

[0103] The PCR enhancing freeze-dried mixture of Comparative Example 2 is composed of tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. The PCR enhancing freeze-dried mixture is nuclease-free water containing 1.2 M tetramethylammonium chloride, 5 U / mL bovine thrombin, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0104] Comparative Example 3 A kit for detecting and typing respiratory syncytial virus based on microfluidics

[0105] The difference between the kit of Comparative Example 3 and the kit of Example 1 of the present invention is only that: 1.1 The PCR enhancing freeze-dried mixtures are different.

[0106] The PCR enhancing freeze-dried mixture of Comparative Example 3 is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, Triton X-100, trehalose, BSA and nuclease-free water. The PCR enhancing freeze-dried mixture is nuclease-free water containing 1.0 M trimethylglycine, 0.2 M tetramethylammonium chloride, 5 U / mL bovine thrombin, 0.3% v / v Triton X-100, 12% w / v trehalose and 0.04% w / v BSA.

[0107] Comparative Example 4 A kit for detecting and typing respiratory syncytial virus based on microfluidics

[0108] The difference between the kit of Comparative Example 4 and the kit of Example 1 of the present invention is only that: 1.1 The PCR enhancing freeze-dried mixtures are different.

[0109] The PCR enhancing freeze-dried mixture of Comparative Example 4 is composed of trimethylglycine, tetramethylammonium chloride, NP-40, trehalose, BSA and nuclease-free water. The PCR enhancing freeze-dried mixture is nuclease-free water containing 1.0 M trimethylglycine, 0.2 M tetramethylammonium chloride, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0110] Comparative Example 5 A kit for detecting and typing respiratory syncytial virus based on microfluidics

[0111] The difference between the kit of Comparative Example 5 and the kit of Example 1 of the present invention is only that: 1.1 The PCR enhancing lyophilized mixture is different.

[0112] The PCR enhancing lyophilized mixture of Comparative Example 5 is composed of trimethylglycine, NP-40, trehalose, BSA and nuclease-free water. The PCR enhancing lyophilized mixture is nuclease-free water containing 1.2 M trimethylglycine, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0113] Experimental Example 1 Sensitivity verification of the kits of Comparative Examples 1-5

[0114] The positive control plasmid containing the RSV-A type G gene, RSV-B type G gene and B2M gene was serially diluted to samples equivalent to 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 copies / mL, 10 copies / mL sample. The kits of Comparative Examples 1-5 were used and measured according to the usage method described in Example 2. The measurement results are shown in Table 10.

[0115] Table 10 Sensitivity test results of the kits of Comparative Examples 1-5

[0116]

[0117] Note: In the table, "+" represents positive and "-" represents negative.

[0118] The results showed that the sensitivity of the kits prepared in Comparative Examples 1-5 was lower than that of Example 1. Among them, the sensitivity of Comparative Example 5 was the lowest, and the lowest detection limits of RSV-A type and RSV-B type were only 10 5 copies / mL.

[0119] The above detailed description is a specific description of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A kit for detecting respiratory syncytial virus and typing based on microfluidics method, characterized in that, The described kit includes a microfluidic chip, on which reaction holes are provided, and a PCR enhancement freeze-dried mixture is filled in the reaction holes; The PCR enhancement freeze-dried mixture is composed of 0.5 - 1.5 M trimethylglycine, 0.1 - 0.3 M tetramethylammonium chloride, 2 - 5 U / mL bovine thrombin, 0.1 - 0.5% v / v NP-40, 10 - 15% w / v trehalose, 0.02 - 0.05% w / v BSA and nuclease-free water.

2. The kit according to claim 1, wherein The PCR enhancement freeze-dried mixture is composed of 1.0 M trimethylglycine, 0.2 M tetramethylammonium chloride, 5 U / mL bovine thrombin, 0.3% v / v NP-40, 12% w / v trehalose, 0.04% w / v BSA and nuclease-free water.

3. The kit according to claim 1, characterized in that, The reaction holes are further filled with a PCR premix and a primer-probe combination.

4. The kit according to claim 3, wherein The PCR premix includes a PCR buffer, MgSO4, dNTPs and Taq enzyme.

5. The kit according to claim 3, characterized in that, The reaction holes include an RSV-A reaction hole, an RSV-B reaction hole and an internal reference reaction hole; The primer-probe combination in the RSV-A reaction hole is an RSV-A primer-probe combination, which is composed of an RSV-A upstream primer, an RSV-A downstream primer and an RSV-A probe; The primer-probe combination in the RSV-B reaction hole is an RSV-B primer-probe combination, which is composed of an RSV-B upstream primer, an RSV-B downstream primer and an RSV-B probe; The primer-probe combination in the internal reference reaction hole is an internal reference gene primer-probe combination, which is composed of a B2M upstream primer, a B2M downstream primer and a B2M probe.

6. The kit according to claim 1, wherein The kit further includes a positive control or a negative control.

7. Use of the kit according to any one of claims 1-6 in the preparation of a reagent for detecting respiratory syncytial virus or typing respiratory syncytial virus, characterized in that, The described application is for non-disease diagnosis purposes.

8. The application according to claim 7, characterized in that, The typing of the respiratory syncytial virus is RSV-A type and RSV-B type.

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