Kit and method for detecting influenza A virus and respiratory syncytial virus

Abstract

The invention discloses a kit for detecting influenza A virus and respiratory syncytial virus and a detection method, relates to virus detection, and aims to solve the problem of lack of an on-site instant detection method which is comprehensive in detection type and convenient and rapid to operate. According to the technical scheme, the kit for detecting the influenza A virus and the respiratory syncytial virus is characterized by comprising a primer and probe combination for detecting genes of the three viruses. According to the kit for detecting the influenza A virus and the respiratory syncytial virus, a nucleic acid extraction step is omitted, operation is convenient and rapid, detection types are comprehensive, and the kit is suitable for on-site instant detection.

Description

Technical Field

[0001] The invention relates to virus detection, and more specifically, to a kit for detecting influenza A and B viruses and respiratory syncytial viruses and a detection method. Background Art

[0002] Influenza A virus and influenza B virus belong to the Orthomyxoviridae family, single-stranded negative-sense RNA viruses. The two have high similarities in virus structure, genome and epidemiology, and can be distinguished by the differences in matrix protein (M protein) and nucleoprotein (NP protein); respiratory syncytial virus belongs to the genus Pneumovirus of the family Paramyxoviridae. The virus is spherical in shape, with a diameter of 120~300nm and an envelope. The genome is a non-segmented single-stranded negative-strand RNA that mainly encodes 10 proteins, namely three transmembrane proteins: fusion protein (F), adhesion protein (G) and small hydrophobic protein (SH), two matrix proteins M1 and M2, three proteins that combine with viral RNA to form the nucleocapsid (N, P and L), and two non-structural proteins (NS1 and NS2). There are spikes composed of glycoproteins on the viral envelope, but no HA, NA and HL.

[0003] Currently, the detection methods for influenza A, influenza B, and respiratory syncytial virus include antigen detection, immunochromatography, and nucleic acid detection. Among them, although the antigen detection method has a short detection time and low cost, the error rate of result interpretation is high and false positives are prone to occur; the immunochromatography method has high requirements for operation mode, sampling location, and disease stage, and the accuracy and sensitivity of the detection are also uncertain; the nucleic acid detection method has become the most common nucleic acid diagnostic method for respiratory pathogen nucleic acid detection due to its advantages of high efficiency, specificity, flexible use, relatively simple operation, and low cost.

[0004] The most commonly used methods on the market are to extract and purify nucleic acid substances based on nasal swabs, throat swabs, and oral swabs, and then conduct tests. The PCR method based on nucleic acid extraction has many experimental steps and a long process, and is prone to contamination during the detection process; therefore, this field needs an on-site instant detection method that is comprehensive in detection types and easy to operate and fast, thereby saving time and cost. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a kit for detecting influenza A and B and respiratory syncytial virus, which eliminates the nucleic acid extraction step, is easy and fast to operate, and has comprehensive detection types, and is suitable for on-site instant detection.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: a kit for detecting influenza A and B viruses and respiratory syncytial viruses, Includes primer and probe combinations for detection of three viral genes: The nucleotide sequence of influenza A virus upstream primer 1 is shown in SEQ ID NO.1; Influenza A virus upstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.2; The nucleotide sequence of influenza A virus downstream primer is shown in SEQ ID NO.3; An influenza A virus probe having a nucleotide sequence as shown in SEQ ID NO.4, wherein the 5' end of the influenza A virus probe sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group; An influenza B virus upstream primer having a nucleotide sequence as shown in SEQ ID NO.5; Influenza B virus downstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.6; Influenza B virus downstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.7; An influenza B virus probe with a nucleotide sequence as shown in SEQ ID NO.8, wherein the 5' end of the influenza B virus probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an MGB SQ quenching fluorescent group; Respiratory syncytial virus upstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.9; Respiratory syncytial virus upstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.10; Respiratory syncytial virus upstream primer 3 having a nucleotide sequence as shown in SEQ ID NO.11; Respiratory syncytial virus downstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.12; Respiratory syncytial virus downstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.13; The nucleotide sequence of the respiratory syncytial virus probe 1 is shown in SEQ ID NO.14, wherein the 5' end of the respiratory syncytial virus probe 1 sequence is labeled with a CY5 fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group; The nucleotide sequence of the respiratory syncytial virus probe 2 is shown in SEQ ID NO. 15, wherein the 5' end of the respiratory syncytial virus probe 2 sequence is labeled with a CY5 fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group.

[0007] By adopting the above technical scheme, the primer and probe combination used can adopt the swab direct amplification combined with fluorescent PCR method to complete the rapid detection of three viruses, namely influenza A virus, influenza B virus and respiratory syncytial virus, eliminating the nucleic acid extraction step, and the operation is convenient and fast, and the detection types are comprehensive, which is suitable for on-site instant detection; at the same time, double upstream primers are used for influenza A virus, double downstream primers are used for influenza B virus, and three upstream primers and double downstream primers are used for respiratory syncytial virus. Double probe design is designed for all different base possibilities encoding a single amino acid, allowing amplification of all nucleic acid sequences encoding known sequences, improving the broad spectrum of detection, and meeting the technical requirements for simultaneous detection of three viruses. By selecting amino acids with small degeneracy and avoiding degeneracy at the 3' end of the primers, the specificity of the PCR product can be improved, and the targeted primers enrich the target sequence, avoiding sequencing of the entire genome, reducing sequencing costs, and improving detection efficiency.

[0008] The present invention is further configured to include a primer-probe combination of an internal reference gene, wherein the internal reference primer-probe combination includes: A β-globin upstream primer having a nucleotide sequence as shown in SEQ ID NO.16; A β-globin downstream primer having a nucleotide sequence as shown in SEQ ID NO.17; The β-globin probe has a nucleotide sequence as shown in SEQ ID NO. 18, wherein the 5' end of the β-globin probe sequence is labeled with a ROX fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group.

[0009] The present invention is further configured to include an amplification enzyme, which is used to amplify the nucleic acid in the nasal swab sample and participate in the fluorescent PCR reaction procedure.

[0010] The present invention is further configured as follows: the final concentrations of the upstream primer 1 and the upstream primer 2 for amplifying the influenza A virus are both 0.1 μmol / L, the final concentration of the downstream primer for amplifying the influenza A virus is 0.2 μmol / L, and the final concentration of the probe for detecting the influenza A virus is 0.1 μmol / L.

[0011] The present invention is further configured as follows: the final concentration of the upstream primer for amplifying the influenza B virus is 0.2 μmol / L, the final concentrations of the downstream primer 1 and the downstream primer 2 for amplifying the influenza B virus are both 0.1 μmol / L, and the final concentration of the probe for detecting the influenza B virus is 0.1 μmol / L.

[0012] The present invention is further configured as follows: the final concentration of the respiratory syncytial virus upstream primer 1 for amplifying is 0.2 μmol / L, the final concentrations of the respiratory syncytial virus upstream primer 2 and the respiratory syncytial virus upstream primer 3 for amplifying are both 0.1 μmol / L, the final concentrations of the respiratory syncytial virus downstream primer 1 and the respiratory syncytial virus downstream primer 2 for amplifying are both 0.2 μmol / L, and the final concentrations of the respiratory syncytial virus probe 1 and the respiratory syncytial virus probe 2 for detecting are both 0.1 μmol / L.

[0013] The present invention is further configured as follows: the final concentrations of the β-globin upstream primer and the β-globin downstream primer for amplification are both 0.1 μmol / L, and the final concentration of the β-globin probe for detection is 0.05 μmol / L.

[0014] The present invention is further configured to include: a positive control substance A1 tube and a negative control substance B1 tube; the positive control substance A1 tube is a pseudovirus control containing influenza A virus, influenza B virus, respiratory syncytial virus and an internal standard sequence; the negative control substance B1 tube is a pseudovirus containing an internal standard sequence.

[0015] The present invention is further configured as follows: the three groups of liquids in the test kit are respectively made into freeze-dried powders through in-situ freeze-drying through a freeze-drying procedure, and the three portions of freeze-dried powders are respectively reagent freeze-dried powder, positive control powder and negative control powder.

[0016] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for using a kit, which effectively improves the uniformity of mixing, ensures the concentration ratio of the amplification template and the detection reagent when the base layer is used, and further improves the convenience of on-site instant detection.

[0017] The above technical purpose of the present invention is achieved through the following technical solution: a method for using a kit, comprising the following steps: S1. Obtain a nasal swab sample to be tested, place it in enzyme-free water, allow the nasal mucosal cells to release nucleic acids, and prepare a swab liquid as an amplification template; S2. The configured primer and probe combination for detecting three viral genes, the primer and probe combination for the internal reference gene, and the amplification enzyme are fully mixed, and the mixed liquid reagent is freeze-dried in situ through a freeze-drying procedure to prepare a reagent freeze-dried powder; S4. Take 25 μL of the amplification template prepared in S1 and directly put it into the lyophilized powder of the reagent prepared in S2 for dissolution, and transfer the solution to a reaction tube for amplification procedure, and judge the result by Ct value.

[0018] By adopting the above technical solution, the detection reagent is freeze-dried through a freeze-drying procedure, so that the liquid amplification template can be used to dissolve the powdered reagent freeze-dried powder to achieve sufficient mixing of the two. Compared with the traditional liquid template mixed liquid reagent, the uniformity of the mixing is effectively improved, the concentration ratio of the amplification template and the detection reagent is guaranteed when the base layer is used, and the convenience of on-site instant detection is further improved.

[0019] The present invention is further configured as follows: the amplification program is: reverse transcription at 50°C for 5 minutes, 1 cycle; pre-denaturation at 95°C for 30 seconds, 1 cycle; denaturation at 95°C for 3 seconds, annealing at 60°C for 15 seconds, alternating for 45 cycles.

[0020] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for using a kit, which can detect the quality of the reagent itself and whether the reagent is contaminated, thereby ensuring the actual detection efficacy of the reagent.

[0021] The above technical purpose of the present invention is achieved through the following technical solution: a method for using a kit, comprising the following steps: S1. Obtain a nasal swab sample to be tested, place it in enzyme-free water, allow the nasal mucosal cells to release nucleic acids, and prepare a swab liquid as an amplification template; S3. Dissolve the positive control powder with 25 μL of enzyme-free water, transfer the solution to a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer to the corresponding reaction tube for amplification; dissolve the negative control powder with 25 μL of enzyme-free water, transfer the solution to a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer to the corresponding reaction tube for amplification; judge the quality control results by the Ct value; S4. Take 25 μL of the amplification template prepared in S1 and directly put it into a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer the solution to a reaction tube for amplification procedure, and judge the result by the Ct value.

[0022] By adopting the above technical solution, step S3 is used to detect the quality of the reagent itself and to detect whether the reagent is contaminated, thereby ensuring the actual detection effectiveness of the reagent.

[0023] In summary, the present invention has the following beneficial effects: it is designed for all different base possibilities encoding a single amino acid, allowing amplification of all nucleic acid sequences encoding known sequences, improving the broad spectrum of detection, and meeting the technical requirements for simultaneous detection of three viruses. By selecting amino acids with small degeneracy and avoiding degeneracy at the 3' end of the primers, the specificity of the PCR product can be improved, and the targeted primers enrich the target sequence, avoiding sequencing of the entire genome, reducing sequencing costs, and improving detection efficiency; it takes only 30 minutes from sample collection to issuing results, which can save a lot of time compared to traditional fluorescent PCR (about 3 hours) and Sanger sequencing (about 9 hours); the step of nucleic acid extraction is omitted, which saves time and reduces the corresponding cost. At the same time, the present invention is simple to operate, the results are fast and accurate, and a lot of labor costs are saved. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of PCR amplification curve of positive control; Figure 2 Schematic diagram of PCR amplification curve of negative control; Figure 3 Schematic diagram of PCR amplification curve of sample 1; Figure 4 is a schematic diagram of the PCR amplification curve of sample 2; Figure 5 is a schematic diagram of the PCR amplification curve of sample 3; Figure 6 is a schematic diagram of the PCR amplification curve of sample 4; Figure 7 is a schematic diagram of the PCR amplification curve of sample 5; Figure 8 is a schematic diagram of the PCR amplification curve of sample 6; Fig. 9 is a schematic diagram of the PCR amplification curve of sample 7; Fig.10 is a schematic diagram of the PCR amplification curve of sample 8; Fig.11 Schematic diagram of the PCR amplification curve of sample 9. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0026] Example 1: A primer and probe combination for detecting influenza A, B and respiratory syncytial virus, comprising: The nucleotide sequence of influenza A virus upstream primer 1 is shown in SEQ ID NO.1; Influenza A virus upstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.2; The nucleotide sequence of influenza A virus downstream primer is shown in SEQ ID NO.3; An influenza A virus probe having a nucleotide sequence as shown in SEQ ID NO.4, wherein the 5' end of the influenza A virus probe sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group; An influenza B virus upstream primer having a nucleotide sequence as shown in SEQ ID NO.5; Influenza B virus downstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.6; Influenza B virus downstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.7; An influenza B virus probe with a nucleotide sequence as shown in SEQ ID NO.8, wherein the 5' end of the influenza B virus probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an MGB SQ quenching fluorescent group; Respiratory syncytial virus upstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.9; Respiratory syncytial virus upstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.10; Respiratory syncytial virus upstream primer 3 having a nucleotide sequence as shown in SEQ ID NO.11; Respiratory syncytial virus downstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.12; Respiratory syncytial virus downstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.13; The nucleotide sequence of the respiratory syncytial virus probe 1 is shown in SEQ ID NO.14, wherein the 5' end of the respiratory syncytial virus probe 1 sequence is labeled with a CY5 fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group; The nucleotide sequence of the respiratory syncytial virus probe 2 is shown in SEQ ID NO. 15, wherein the 5' end of the respiratory syncytial virus probe 2 sequence is labeled with a CY5 fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group.

[0027] Example 2: A kit for detecting influenza A and B and respiratory syncytial virus, comprising the primer and probe combination for detecting the three viruses described in Example 1, the primer and probe combination for the internal reference gene, and an amplification enzyme and enzyme-free water; Among them, the internal reference primer probe combination includes: A β-globin upstream primer having a nucleotide sequence as shown in SEQ ID NO.16; A β-globin downstream primer having a nucleotide sequence as shown in SEQ ID NO.17; A β-globin probe having a nucleotide sequence as shown in SEQ ID NO.18, wherein the 5' end of the β-globin probe sequence is labeled with a ROX fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group; The amplification enzyme is used to amplify the nucleic acid in the nasal swab sample and participate in the fluorescent PCR reaction procedure; The final concentrations of upstream primer 1 and upstream primer 2 for amplifying influenza A virus were both 0.1 μmol / L, the final concentration of downstream primer for amplifying influenza A virus was 0.2 μmol / L, and the final concentration of probe for detecting influenza A virus was 0.1 μmol / L.

[0028] The final concentration of the upstream primer for amplifying influenza B virus was 0.2 μmol / L, the final concentrations of the downstream primer 1 for amplifying influenza B virus and the downstream primer 2 for amplifying influenza B virus were both 0.1 μmol / L, and the final concentration of the probe for detecting influenza B virus was 0.1 μmol / L.

[0029] The final concentration of the upstream primer 1 for amplifying respiratory syncytial virus was 0.2 μmol / L, the final concentrations of the upstream primer 2 and the upstream primer 3 for amplifying respiratory syncytial virus were both 0.1 μmol / L, the final concentrations of the downstream primer 1 and the downstream primer 2 for amplifying respiratory syncytial virus were both 0.2 μmol / L, and the final concentrations of the probe 1 and the probe 2 for detecting respiratory syncytial virus were both 0.1 μmol / L.

[0030] The final concentrations of the β-globin upstream primer and the β-globin downstream primer were both 0.1 μmol / L, and the final concentration of the β-globin detection probe was 0.05 μmol / L.

[0031] Table 1: Reagent composition

[0032] Embodiment 3, a kit for detecting influenza A, influenza B and respiratory syncytial virus. Based on Embodiment 2, this embodiment further comprises a positive control tube A1 and a negative control tube B1; the positive control tube A1 is a pseudovirus control containing influenza A virus, influenza B virus, respiratory syncytial virus and an internal standard sequence; the negative control tube B1 is a pseudovirus containing an internal standard sequence.

[0033] This embodiment additionally introduces a positive control substance A1 tube and a negative control substance B1 tube to realize the verification of the reagent itself, including the quality of the reagent and whether contamination occurs.

[0034] Example 4: A kit for detecting influenza A, B and respiratory syncytial virus. The three groups of liquids involved in Example 3 are made into freeze-dried powders by in situ freeze-drying using the same freeze-drying procedure. The three freeze-dried powders are reagent freeze-dried powder, positive control powder and negative control powder.

[0035] Table 2: Freeze-drying process sequence

[0036] The kit provided in this embodiment prepares the liquid reagent into a lyophilized powder, so that the product is in the form of three groups of independently packaged powders when it leaves the factory, wherein Group 1 is 3 portions of reagent lyophilized powder, Group 2 is 1 portion of positive control powder, and Group 3 is 1 portion of negative control powder. Compared with traditional liquid substances, not only is the packaging simpler, but it also solves the problem of insufficient reagent storage conditions in grassroots applications, and is more suitable for grassroots detection applications.

[0037] Embodiment 5: This embodiment is a method for using the kit disclosed in Embodiment 4 to detect a sample.

[0038] A method for using a kit comprises the following steps: S1. Obtain a nasal swab sample to be tested, place it in enzyme-free water, allow the nasal mucosal cells to release nucleic acids, and prepare a swab liquid as an amplification template; S3. Dissolve the positive control powder with 25 μL of enzyme-free water, transfer the solution to a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer to the corresponding reaction tube (marked as tube B1) for amplification; dissolve the negative control powder with 25 μL of enzyme-free water, transfer the solution to a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer to the corresponding reaction tube (marked as tube B2) for amplification; S4. Take 25 μL of the amplification template prepared in S1 and directly put it into a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer the solution to a reaction tube for amplification procedure; The amplification program of steps S3 and S4 is: reverse transcription at 50°C for 5 minutes, 1 cycle; pre-denaturation at 95°C for 30 seconds, 1 cycle; denaturation at 95°C for 3 seconds, annealing at 60°C for 15 seconds, alternating for 45 cycles.

[0039] Table 3: Amplification Procedure

[0040] S5. Make quality control judgment based on the Ct value obtained by the S3 amplification procedure. The quality control judgment conditions are as follows: If the B1 tube shows a typical S-shaped curve in the four channels of FAM, VIC, CY5 and ROX and Ct (FAM) ≤ 40, Ct (VIC) ≤ 40, Ct (CY5) ≤ 40, Ct (ROX) ≤ 40, the reagent is considered qualified, otherwise it needs to be re-amplified; If the B2 tube has no Ct value in the FAM, VIC and CY5 channels and the ROX channel presents a typical S-shaped curve and Ct (ROX) ≤ 40, it is considered that the reagent is not contaminated and the reagent results are credible.

[0041] Table 4: Quality control result judgment table

[0042] like Figure 1 The Ct value of the amplification curve of the positive control shown meets the quality control judgment conditions, and the reagent is determined to be qualified.

[0043] Table 5: Figure 1 Curve Ct value calculation table

[0044] like Figure 2 The Ct value of the amplification curve of the negative control shown meets the quality control judgment conditions, and it is determined that the reagent is not contaminated.

[0045] Table 6: Figure 2 Curve Ct value calculation table

[0046] ( Figure 1 , Figure 2 In the figure, the horizontal axis is the cycle number, the vertical axis is the fluorescence intensity, the blue curve is the FAM channel, the green curve is the VIC channel, the orange curve is the ROX channel, and the red curve is the CY5 channel).

[0047] S6. According to the Ct value obtained by the S4 amplification procedure, the test results are judged. The result judgment comparison table is as follows: Table 7: Test result judgment table

[0048] like Figure 3 The amplification curve of sample 1 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are positive for influenza A virus, positive for influenza B virus, and positive for respiratory syncytial virus.

[0049] Table 8: Figure 3 Curve Ct value calculation table

[0050] like Figure 4The amplification curve of sample 2 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are positive for influenza A virus, positive for influenza B virus, and negative for respiratory syncytial virus.

[0051] Table 9: Figure 4 Curve Ct value calculation table

[0052] like Figure 5 The amplification curve of sample 3 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are positive for influenza A virus, negative for influenza B virus, and positive for respiratory syncytial virus.

[0053] Table 10: Figure 5 Curve Ct value calculation table

[0054] like Figure 6 The amplification curve of sample 4 shown in the figure is compared with Table 7 based on its Ct value, and the results are interpreted as negative for influenza A virus, positive for influenza B virus, and positive for respiratory syncytial virus.

[0055] Table 11: Figure 6 Curve Ct value calculation table

[0056] like Figure 7 The amplification curve of sample 5 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are positive for influenza A virus, negative for influenza B virus, and negative for respiratory syncytial virus.

[0057] Table 12: Figure 7 Curve Ct value calculation table

[0058] like Figure 8 The amplification curve of sample 6 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are negative for influenza A virus, positive for influenza B virus, and negative for respiratory syncytial virus.

[0059] Table 13: Figure 8 Curve Ct value calculation table

[0060] like Fig. 9 The amplification curve of sample 7 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are negative for influenza A virus, negative for influenza B virus, and positive for respiratory syncytial virus.

[0061] Table 14: Fig. 9 Curve Ct value calculation table

[0062] like Fig.10 The amplification curve of sample 8 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are negative for influenza A virus, negative for influenza B virus, and negative for respiratory syncytial virus.

[0063] Table 15: Fig.10 Curve Ct value calculation table

[0064] like Fig.11 The amplification curve of sample 9 shown in the figure shows that, after comparing its Ct value with that in Table 7, the results are negative for influenza A virus, positive for influenza B virus, and positive for respiratory syncytial virus.

[0065] Table 16: Fig.11 Curve Ct value calculation table

[0066] The interpretation results of the above 9 samples are completely consistent with the hospital's diagnosis results. The present invention has the technical effect of accurate detection.

[0067] In this embodiment, it takes only 30 minutes from sample collection to result issuance, which can save a lot of time compared to traditional fluorescent PCR method (about 3 hours) and Sanger sequencing method (about 9 hours).

[0068] This embodiment omits the step of nucleic acid extraction, which saves time and reduces corresponding costs. At the same time, the present invention is simple to operate, produces fast and accurate results, and saves a lot of labor costs.

[0069] The specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A kit for detecting influenza A and B viruses and respiratory syncytial virus, characterized in that: Includes primer and probe combinations for detection of three viral genes: The nucleotide sequence of influenza A virus upstream primer 1 is shown in SEQ ID NO.1; Influenza A virus upstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.2; The nucleotide sequence of influenza A virus downstream primer is shown in SEQ ID NO.3; An influenza A virus probe having a nucleotide sequence as shown in SEQ ID NO.4, wherein the 5' end of the influenza A virus probe sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group; An influenza B virus upstream primer having a nucleotide sequence as shown in SEQ ID NO.5; Influenza B virus downstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.6; Influenza B virus downstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.7; An influenza B virus probe with a nucleotide sequence as shown in SEQ ID NO.8, wherein the 5' end of the influenza B virus probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an MGB SQ quenching fluorescent group; Respiratory syncytial virus upstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.9; Respiratory syncytial virus upstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.10; Respiratory syncytial virus upstream primer 3 having a nucleotide sequence as shown in SEQ ID NO.11; Respiratory syncytial virus downstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.12; Respiratory syncytial virus downstream primer 2 having a nucleotide sequence as shown in SEQ ID NO.13; The nucleotide sequence of the respiratory syncytial virus probe 1 is shown in SEQ ID NO.14, wherein the 5' end of the respiratory syncytial virus probe 1 sequence is labeled with a CY5 fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group; The nucleotide sequence of the respiratory syncytial virus probe 2 is shown in SEQ ID NO. 15, wherein the 5' end of the respiratory syncytial virus probe 2 sequence is labeled with a CY5 fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group.

2. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 1, characterized in that: Also included is a primer-probe combination of an internal reference gene, wherein the internal reference primer-probe combination includes: A β-globin upstream primer having a nucleotide sequence as shown in SEQ ID NO.16; A β-globin downstream primer having a nucleotide sequence as shown in SEQ ID NO.17; The β-globin probe has a nucleotide sequence as shown in SEQ ID NO. 18, wherein the 5' end of the β-globin probe sequence is labeled with a ROX fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group.

3. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 2, characterized in that: Also included is an amplification enzyme, which is used to amplify the nucleic acid in the nasal swab sample and participate in the fluorescent PCR reaction procedure.

4. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 3, characterized in that: The final concentrations of the influenza A virus upstream primer 1 and the influenza A virus upstream primer 2 for amplifying the influenza A virus were both 0.1 μmol / L, the final concentration of the influenza A virus downstream primer was 0.2 μmol / L, and the final concentration of the influenza A virus probe for detecting the influenza A virus was 0.1 μmol / L.

5. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 3, characterized in that: The final concentration of the upstream primer for amplifying the influenza B virus is 0.2 μmol / L, the final concentrations of the downstream primer 1 and the downstream primer 2 for amplifying the influenza B virus are both 0.1 μmol / L, and the final concentration of the probe for detecting the influenza B virus is 0.1 μmol / L.

6. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 3, characterized in that: The final concentration of the respiratory syncytial virus upstream primer 1 for amplification is 0.2 μmol / L, the final concentrations of the respiratory syncytial virus upstream primer 2 and the respiratory syncytial virus upstream primer 3 are both 0.1 μmol / L, the final concentrations of the respiratory syncytial virus downstream primer 1 and the respiratory syncytial virus downstream primer 2 are both 0.2 μmol / L, and the final concentrations of the respiratory syncytial virus probe 1 and the respiratory syncytial virus probe 2 for detection are both 0.1 μmol / L.

7. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 3, characterized in that: The final concentrations of the β-globin upstream primer and the β-globin downstream primer for amplification were both 0.1 μmol / L, and the final concentration of the β-globin probe for detection was 0.05 μmol / L.

8. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 3, characterized in that: It also includes positive control tube A1 and negative control tube B1; The positive control A1 tube is a pseudovirus control containing influenza A virus, influenza B virus, respiratory syncytial virus and an internal standard sequence; The negative control substance B1 tube is a pseudovirus containing an internal standard sequence.

9. A kit for detecting influenza A and B and respiratory syncytial virus according to claim 8, characterized in that: The three groups of liquids in the kit are made into freeze-dried powders through in-situ freeze-drying through a freeze-drying procedure. The three freeze-dried powders are reagent freeze-dried powder, positive control powder and negative control powder.

10. A method for using the kit according to any one of claims 3 to 7, characterized in that: The steps include: S1. Obtain a nasal swab sample to be tested, place it in enzyme-free water, allow the nasal mucosal cells to release nucleic acids, and prepare a swab liquid as an amplification template; S2. The configured primer and probe combination for detecting three viral genes, the primer and probe combination for the internal reference gene, and the amplification enzyme are fully mixed, and the mixed liquid reagent is freeze-dried in situ through a freeze-drying procedure to prepare a reagent freeze-dried powder; S4. Take 25 μL of the amplification template prepared in S1 and directly put it into the lyophilized powder of the reagent prepared in S2 for dissolution, and transfer the solution to a reaction tube for amplification procedure, and judge the result by Ct value.

11. The method for using the kit according to claim 10, characterized in that: The amplification program is: reverse transcription at 50°C for 5 minutes, 1 cycle; pre-denaturation at 95°C for 30 seconds, 1 cycle; denaturation at 95°C for 3 seconds, annealing at 60°C for 15 seconds, alternating for 45 cycles.

12. A method for using the kit according to claim 9, characterized in that: The steps include: S1. Obtain a nasal swab sample to be tested, place it in enzyme-free water, allow the nasal mucosal cells to release nucleic acids, and prepare a swab liquid as an amplification template; S3. Dissolve the positive control powder with 25 μL of enzyme-free water, transfer the solution to a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer to the corresponding reaction tube for amplification; dissolve the negative control powder with 25 μL of enzyme-free water, transfer the solution to a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer to the corresponding reaction tube for amplification; judge the quality control results by the Ct value; S4. Take 25 μL of the amplification template prepared in S1 and directly put it into a portion of the reagent freeze-dried powder for dissolution and mixing, and transfer the solution to a reaction tube for amplification procedure, and judge the result by the Ct value.

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