A rapid typing kit for influenza virus A and B

Abstract

The application discloses a rapid typing detection kit for influenza virus A and B. The application is based on the sequences of influenza virus A and B, and a primer and probe combination for simultaneously detecting multiple influenza virus A or B subtypes and distinguishing influenza virus A and B is designed. The application also provides a rapid typing detection kit for influenza virus A and B, which has the advantages of good detection specificity, high sensitivity and good accuracy, and the detection limit of influenza virus A / B nucleic acid can be as low as 50 copies / mL. The kit can be used for rapidly identifying whether a subject carries influenza virus A or B, and can distinguish influenza A patients, influenza B patients and uninfected people, and can also distinguish the rest of the virus infected patients, which is beneficial to the treatment of influenza patients and the prevention of influenza.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of virus detection. More particularly, it relates to a rapid typing detection kit for influenza A and B viruses. BACKGROUND

[0002] Influenza viruses are a class of RNA viruses that can cause respiratory diseases, belong to the Orthomyxoviridae family, have high infectivity, and are mainly transmitted through air routes such as droplets and aerosols, and can be divided into four types: A, B, C and D. Due to the complexity of the pathogen infection of the respiratory system, the early clinical symptoms of the disease are atypical, and it is difficult to effectively diagnose through clinical symptoms, etc., and it is necessary to combine the results of clinical laboratory tests for judgment. Therefore, it is of great significance to provide a kit for detecting influenza viruses for the prevention and treatment of influenza.

[0003] At present, influenza A and B viruses are more common in clinical practice. Among them, influenza A virus can spread in a variety of animals, has strong variability, and has a wide range of transmission, and is the main pathogen leading to large-scale outbreaks of influenza. Subtypes of influenza A virus such as H1N1, H3N2, H5N1 and H7N9 have caused serious public health and safety problems. Influenza B virus is mainly spread in a small range, and the main infection of humans is the influenza B Yamagata line or the influenza B Victoria line. In the 15 countries in the Asia-Pacific region, the confirmed rate of influenza B virus in monthly confirmed cases of influenza is 0-92%. Unlike influenza A virus, children, the elderly and other specific populations are susceptible to influenza B virus and are prone to complications, and the burden on society caused by influenza B virus may even exceed that of influenza A virus. The detection rate of influenza C and D viruses in the population is low, and their impact on public health and human health is small. Therefore, it is necessary to rapidly type and detect influenza A and B viruses.

[0004] Common detection methods for influenza viruses include pathogen isolation and culture detection, antigen-antibody detection, and PCR detection, etc. Among them, although pathogen isolation and culture detection is the gold standard for identification of influenza viruses, this method has problems such as long culture time, low sensitivity, and easy false negatives. The antigen-antibody detection is also fast, but it also has the problem of low specificity. In addition, the detection limit of existing PCR detection products for influenza viruses is about 500 copies / mL, and the detection time is long, which cannot sensitively detect viruses in the early stage of infection, and cannot achieve rapid detection of viruses, which is not conducive to the monitoring, prevention and treatment of influenza. SUMMARY

[0005] The present application provides a rapid typing detection kit for influenza A and B viruses with good specificity, high sensitivity and good accuracy.

[0006] The first object of the present application is to provide a primer and probe combination for typing detection of influenza A and B viruses.

[0007] The second object of the present application is to provide an application of the primer and probe combination in preparing a product for typing detection of influenza A and B viruses.

[0008] The third object of the present application is to provide a rapid typing detection kit for influenza A and B viruses.

[0009] The above objects of the present application are achieved by the following technical solutions.

[0010] The present application provides a primer and probe combination for typing detection of influenza A and B viruses, comprising a primer pair and a probe for detecting influenza A and B viruses; wherein the nucleotide sequence of the primer pair for detecting influenza A virus is shown in SEQ ID NO. 1-2, the nucleotide sequence of the probe for detecting influenza A virus is shown in SEQ ID NO. 3; the nucleotide sequence of the primer pair for detecting influenza B virus is shown in SEQ ID NO. 4-5, the nucleotide sequence of the probe for detecting influenza B virus is shown in SEQ ID NO. 6; the 5' end of the probe is labeled with different fluorescent groups, and the 3' end is labeled with a quenching group.

[0011] Specifically, the primer and probe combination further comprises an internal control primer pair and an internal control probe; the nucleotide sequence of the internal control primer pair is shown in SEQ ID NO. 7-8, and the nucleotide sequence of the internal control probe is shown in SEQ ID NO. 9.

[0012] Specifically, the 5' end of the probe for detecting influenza A virus is labeled with a fluorescent group FAM, and the 3' end is labeled with Eclipse; the 5' end of the probe for detecting influenza B virus is labeled with a fluorescent group Texas Red, and the 3' end is labeled with Eclipse; the 5' end of the internal control probe is labeled with a fluorescent group VIC, and the 3' end is labeled with BHQ1.

[0013] The primer and probe combination of the present application can simultaneously detect multiple influenza A or B virus subtypes and distinguish influenza A and B viruses, achieving rapid typing detection of influenza A and B viruses. Therefore, the present application also claims the application of the primer and probe combination in preparing a product for typing detection of influenza A and B viruses.

[0014] The present application also provides a rapid typing detection kit for influenza A and B viruses, which contains the primer and probe combination of the present application.

[0015] The primer and probe combination can exist in the form of a primer and probe mixture containing the primer and probe combination and a solvent.

[0016] Specifically, the solvent is TE solution.

[0017] Specifically, the kit further contains positive and negative quality controls.

[0018] Specifically, the positive quality control is a pseudo virus of influenza A virus, a pseudo virus of influenza B virus and a pseudo virus containing the detected internal standard fragment.

[0019] Specifically, the negative quality control is TE solution and a pseudo virus containing the detected internal standard fragment.

[0020] In contrast, the pseudo virus is selected as the negative / positive quality control, which is superior to using other alternative materials.

[0021] Specifically, the kit further contains reagents required for the fluorescent PCR reaction.

[0022] Specifically, the reagents required for the fluorescent PCR reaction include IVA / IVB PCR Buffer.

[0023] Specifically, the PCR Buffer contains Tris-HCl, MgCl2, (NH4)2SO4, KCl, Tween-20 and a solvent.

[0024] Specifically, the solvent is DEPC H2O.

[0025] Specifically, in the PCR Buffer, the concentration of Tris-HCl ranges from 0.45 to 0.55 mol / L, the concentration of MgCl2 ranges from 0.004 to 0.008 mol / L, the concentration of (NH4)2SO4 ranges from 0.8 to 0.9 mol / L, the concentration of KCl ranges from 0.35 to 0.55 mol / L, and the concentration of Tween-20 ranges from 0.075 to 0.09%.

[0026] Specifically, the reagents required for the fluorescent PCR reaction include a PCR reaction enzyme system containing heat-start Taq antibody enzyme, Taq enzyme and UDG enzyme.

[0027] Specifically, the PCR reaction enzyme system further contains dATP, dGTP, dCTP, dUTP, dTTP, RNasin, KCl, NaCl, Tween-20, NP-40, DTT, xylitol and a solvent.

[0028] Specifically, the solvent is sterilized water.

[0029] Specifically, in the PCR reaction enzyme system, the concentration of the hot start Taq antibody enzyme is 1.1-1.3 U / μL, the concentration of the Taq enzyme is 0.4-0.43 U / μL, the concentration of the UDG enzyme is 0.008-0.009 U / μL, the concentration of dATP / dGTP / dCTP is 2.5-2.7 mmol / L, the concentration of dUTP is 3.8-4.2 mmol / L, the concentration of dTTP is 1.2-1.4 mmol / L, the concentration of RNasin is 5.1-5.4 U / μL, the concentration of KCl is 0.035-0.045 mol / L, the concentration of NaCl is 0.05-0.065 mol / L, the concentration of Tween-20 is 0.035-0.04%, the concentration of NP-40 is 0.17-0.24%, the concentration of DTT is 0.001-0.003 mol / L, and the concentration of xylitol is 0.12-0.15 g / mL.

[0030] The present application has the following advantages:

[0031] Based on the sequences of influenza A and B viruses, the present application designs a primer and probe combination which can be used to simultaneously detect multiple influenza A or B virus subtypes and distinguish influenza A and B viruses. The present application also provides a rapid typing detection kit for influenza A and B viruses, which can realize rapid typing detection of influenza A and B viruses, has good detection specificity, high sensitivity and good accuracy, and the detection limit of influenza A / B virus nucleic acid can be as low as 50 copies / mL. The kit can be used to quickly identify whether a subject carries influenza A / B virus, and can distinguish influenza A patients, influenza B patients and uninfected people, and can also distinguish them from other virus infected patients, which is beneficial to the treatment of influenza patients and the prevention of influenza. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Table 1 shows the detection results of IVA-1 primers and probes on positive samples of influenza A pseudovirus.

[0033] Figure 2 Table 1 shows the detection results of IVA-2 primers and probes on positive samples of influenza A pseudovirus.

[0034] Figure 3 Table 1 shows the detection results of IVA-3 primers and probes on positive samples of influenza A pseudovirus.

[0035] Figure 4 Table 1 shows the detection results of IVB-1 primers and probes on positive samples of influenza B pseudovirus.

[0036] Figure 5 Detection results of IVB-1 primer and probe shown in Table 1 for positive sample of influenza B pseudovirus.

[0037] Figure 6 Detection results of IVB-1 primer and probe shown in Table 1 for positive sample of influenza B pseudovirus.

[0038] Figure 7 Specific detection results of IVA-1 primer and probe shown in Table 1.

[0039] Figure 8 Specific detection results of IVA-2 primer and probe shown in Table 1.

[0040] Figure 9 Specific detection results of IVB-2 primer and probe shown in Table 1.

[0041] Figure 10 Specific detection results of IVB-3 primer and probe shown in Table 1.

[0042] Figure 11 Detection results of the kit for 100000 copies / mL positive sample of influenza A virus.

[0043] Figure 12 Detection results of the kit for 1000 copies / mL positive sample of influenza A virus.

[0044] Figure 13 Detection results of the kit for 200 copies / mL positive sample of influenza A virus.

[0045] Figure 14 Detection results of the kit for 100 copies / mL positive sample of influenza A virus.

[0046] Figure 15 Detection results of the kit for 100000 copies / mL positive sample of influenza B virus.

[0047] Figure 16 Detection results of the kit for 1000 copies / mL positive sample of influenza B virus.

[0048] Figure 17 Detection results of the kit for 200 copies / mL positive sample of influenza B virus.

[0049] Figure 18 Detection results of the kit for 100 copies / mL positive sample of influenza B virus.

[0050] Figure 19 The detection of Guo-Shen S1 (50 copies / mL) by the kit.

[0051] Figure 20 The detection of Guo-Shen S2 (50 copies / mL) by the kit.

[0052] Figure 21 The detection of Guo-Shen S3 (50 copies / mL) by the kit.

[0053] Figure 22 The detection of Guo-Shen S4 (50 copies / mL) by the kit.

[0054] Figure 23 The detection of Guo-Shen S5 (50 copies / mL) by the kit.

[0055] Figure 24 The detection of positive clinical samples by the kit. DETAILED DESCRIPTION

[0056] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0057] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0058] Example 1 Primers and probes for detecting influenza A and B viruses

[0059] 1. Design of primers and probes for detecting influenza A, B viruses and internal control sequence

[0060] The present application is based on the reference sequences of influenza A virus M region and influenza B virus M region in the Gene database of NCBI (sequence numbers are NC_002016.1 and NC_002210.1, respectively), and multiple sequence alignment is performed on multiple influenza A M region sequences and influenza B M region sequences found in the Nucleotide database of NCBI (103345 and 12406, respectively, and 3000 of each are randomly selected for alignment). According to the alignment results, the conserved region of the sequence is found as the reference sequence for primer and probe design. According to the reference sequences of influenza A and B viruses after alignment, multiple sets of primers and probes are designed, and the PPIA sequence in the Gene database of NCBI (sequence number is NC_000007.14) is used as the reference sequence for internal standard primer and probe design, and multiple sets of primers and probes are designed.

[0061] 2. NTC test of primers and probes

[0062] The designed multiple sets of primers and probes were respectively prepared into single RT-PCR reaction system for NTC (No Template Control) test, and 1 x 10 5 copies / mL of influenza A pseudovirus and 1 x 10 5 copies / mL of influenza B pseudovirus were used as positive controls for detection on a real-time fluorescent quantitative PCR instrument.

[0063] Among them, the nucleotide sequences of several groups of primers and probes with relatively good detection effect are shown in Table 1. The primers and probes in the table will not appear fluorescence curve in the absence of template.

[0064] Table 1

[0065]

[0066]

[0067] 3. Amplification efficiency test of primers and probes

[0068] The primers and probes of IVA-1, IVA-2, IVA-3, IVB-1, IVB-2 and IVB-3 were respectively prepared into single RT-PCR reaction system by using the same method as 2, and 1 x 10 5 copies / mL, 1 x 10 4 copies / mL and 1 x 10 3 copies / mL of influenza A pseudovirus or influenza B virus positive samples were used for detection on a real-time fluorescent quantitative PCR instrument.

[0069] The detection results of IVA-1, IVA-2 and IVA-3 on influenza A pseudovirus positive samples are shown in Table 3 in turn. Figures 1 to 3 The detection results of IVB-1, IVB-2 and IVB-3 on influenza B pseudovirus positive samples are shown in Table 4 in turn. Figures 4 to 6 It can be seen from Table 3 and Table 4 that IVA-3 and IVB-1 have no obvious gradient compared with other combinations, the Ct value is obviously later, and the fluorescence intensity is relatively low. Therefore, IVA-1, IVA-2, IVB-2 and IVB-3 were further adjusted and optimized. Figures 1 to 6

[0070] 4. Specificity test of primers and probes

[0071] ​The primers and probes of IVA-1, IVA-2, IVB-2 and IVB-3 were respectively prepared into single RT-PCR reaction systems, and diluted with physiological saline to 1×10 5 copies / mL of influenza A virus (H1N1), influenza B virus (Victoria), and physiological saline (virus diluted to 1×10 6 pfu / mL, bacteria diluted to 1×10 5 cfu / mL) of Staphylococcus epidermidis, Epstein-Barr virus, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Bordetella pertussis, mumps virus, Neisseria meningitidis, Adenovirus 3, Adenovirus 7, Respiratory syncytial virus B, Human parainfluenza virus 2, and negative samples were detected on a real-time fluorescent quantitative PCR instrument.

[0072] The specific detection results of IVA-1, IVA-2, IVB-2 and IVB-3 are shown in Figures 7 to 10 It can be seen from Figures 7 to 10 that IVB-3 has non-specific amplification, and IVA-1, IVA-2 and IVB-2 are further adjusted and optimized.

[0073] 5. Combination test of primers and probes

[0074] The primers and probes of IVA-1, IVA-2 and IVB-2 are respectively combined with internal standard gene detection primers and probes (PPIA-1 and PPIA-2) to obtain four different composite systems. The obtained composite systems are respectively used for NTC test, positive sample test, amplification efficiency test and specificity test. It is found that the four composite systems have some deficiencies in NTC test, amplification efficiency test or specificity test, and cannot realize specific and accurate detection. After continuous adjustment and optimization, the finally obtained nucleotide sequences of the primer and probe combination for typing detection of influenza A and B viruses are shown in Table 2:

[0075] Table 2

[0076]

[0077]

[0078] For typing detection, the 5' end of probe IVA-P is labeled with fluorescent group FAM, and the 3' end is labeled with Eclipse; the 5' end of probe IVB-P is labeled with fluorescent group Texas Red, and the 3' end is labeled with Eclipse; the 5' end of internal control probe PPIA-P is labeled with fluorescent group VIC, and the 3' end is labeled with BHQ1. According to whether the fluorescent signal appears in the reaction tube and the type of the fluorescent signal, it can be judged whether the sample contains influenza A virus or influenza B virus.

[0079] Example 2 Construction of rapid typing detection kit for influenza A and B viruses

[0080] Based on the primer and probe combination obtained in Example 1 (shown in Table 2), the present application optimizes the reaction system to construct a rapid typing detection kit for influenza A and B viruses and its use method, so that the constructed kit has the advantages of good detection specificity and high sensitivity.

[0081] 1. A rapid typing detection kit for influenza A and B viruses

[0082] The rapid typing detection kit for influenza A and B viruses constructed by optimizing the reaction system of the present application comprises the following components, as shown in Table 3:

[0083] Table 3

[0084]

[0085] The concentration ranges of IVA / IVB PCR Buffer and each component in the PCR reaction enzyme system are shown in Table 4:

[0086] Table 4

[0087] Component Concentration range Component Concentration range Tris-HCl 0.45 to 0.55 mol / L UDG enzyme 0.008 to 0.009 U / μL MgCl2 0.004 to 0.008 mol / L dATP / dGTP / dCTP 2.5 to 2.7 mmol / L (NH4)2SO4 0.8 to 0.9 mol / L dUTP 3.8 to 4.2 mmol / L KCl (buffer) 0.35 to 0.55 mol / L dTTP 1.2 to 1.4 mmol / L Tween-20 (buffer) 0.075～0.09％ RNasin 5.1 to 5.4 U / μL Hot start Taq antibody enzyme 1.1 to 1.3 U / μL KCl (reaction enzyme system) 0.035 to 0.045 mol / L Taq enzyme 0.4 to 0.43 U / μL NaCl 0.05 to 0.065 mol / L DTT 0.001 to 0.003 mol / L Tween-20 (reaction enzyme system) 0.035～0.04％ Xylitol 0.12 to 0.15 g / mL NP-40 0.17～0.24％

[0088] 2. A method for rapid typing detection of influenza A and B viruses using the kit

[0089] The present application also provides a method for rapid typing detection of influenza A and B viruses using the kit. Taking a human throat swab sample as an example, the method comprises the following steps:

[0090] S1. Sample extraction; extract nucleic acid from the collected human throat swab sample using a human throat swab extraction kit; the nucleic acid extraction and purification reagent (Yue Sui Machine No. 20170583) produced by Daan Gene Co., Ltd. is used to extract and purify the nucleic acid of the throat swab sample collected in this embodiment;

[0091] S2. Configure the reaction system; take out the primer probe mixture, IVA / IVB PCR Buffer and PCR reaction enzyme system from the kit, melt at room temperature, shake and mix, centrifuge at 8,000 rpm for a few seconds, and then use; take N (N = the number of samples to be tested + IVA / IVB negative quality control + IVA / IVB positive quality control) PCR reaction tubes, prepare N amplification systems, and the amplification system for a single person is shown in Table 5:

[0092] Table 5 Single-person amplification system

[0093]

[0094] S3. Sample addition; add 20 μL of the extracted sample nucleic acid, IVA / IVB negative quality control, and IVA / IVB positive quality control to the above-mentioned PCR reaction tube, and tightly cover the tube cap; after instantaneous centrifugation for 15 seconds, transfer to the amplification detection area, place the reaction tube into the instrument sample slot and record the placement order;

[0095] After adding the sample nucleic acid, the final concentration of the IVA primer in the amplification system is 0.2-0.25 μmol / L; the final concentration of the IVA probe is 0.06-0.09 μmol / L; the final concentration of the IVB primer is 0.7-0.9 μmol / L; the final concentration of the IVB probe is 0.3-0.4 μmol / L; the final concentration of the PPIA primer is 0.4-0.45 μmol / L; and the final concentration of the PPIA probe is 0.2-0.25 μmol / L.

[0096] S4. Fluorescence channel selection; open the "Setup" window, set the negative quality control, positive quality control and unknown sample (Unknown) according to the sample placement order, select the FAM channel to detect influenza A virus nucleic acid, select the Texas Red channel to detect influenza B virus nucleic acid, and select the VIC channel to detect the internal standard;

[0097] S5. Set the cycle conditions; the cycle conditions are shown in Table 6, save the file after setting, and run the program;

[0098] Table 6 Cycle conditions

[0099]

[0100] After the reaction is finished, the result is automatically saved, the Start value, the End value and the Threshold value of the Baseline are adjusted according to the image after the analysis (the user can adjust them according to the actual situation, the Start value can be set at 3-15, the End value can be set at 5-20, and the Value value of the Threshold in the Log map window is set so that the threshold line is located at the exponential period of the amplification curve), and the analysis result is automatically obtained by clicking Analysis, and the detection result is read in the Report window.

[0101] S6. Result judgment; the standard for judging the detection effectiveness is that a negative control group and a positive control group are set for each detection, when the positive control group of the detection result is positive for the influenza A virus and the negative control group is negative, it is indicated that the experimental result is effective; after the reaction is finished, the threshold line (Threshold value) is set according to the amplification curve of the positive control, the threshold line is located at the exponential period of the amplification curve, and the instrument is automatically analyzed to obtain the Ct value by clicking Analysis (or Analysis, different fluorescence PCR instruments or inconsistent); when the Ct value of the FAM channel of the measured sample is less than 40, the sample is determined to be positive for the influenza A virus, and vice versa; when the Ct value of the Texas Red channel of the measured sample is less than 40, the sample is determined to be positive for the influenza B virus, and vice versa.

[0102] 3. Optimization of the reaction system

[0103] The kit is used for preparing Mg 2+ The reaction liquid with a final concentration (the final concentration of the reaction system after sample addition) of 2, 4, 6, 8 and 9 mM is used for detecting the influenza A virus clinical sample, the influenza B virus clinical sample and the negative throat swab. The positive sample is diluted into the concentration of 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL and 1×10 2 copies / mL of the influenza A and B mixed sample by using the negative throat swab, the mixed sample and the negative sample are used as the sample to be detected, and the detection is performed on the real-time fluorescence quantitative PCR instrument, and each concentration is detected twice. Through the test, when the final concentration of Mg 2+ in the system is 2 mM, the system has no obvious platform period and the amplification efficiency is poor; when the final concentration of Mg 2+ in the system is 9 mM, the reaction system has non-specific amplification; when the final concentration of Mg 2+ in the system is 4, 6 or 8 mM, the amplification curve of each channel is normal, and the amplification efficiency is relatively optimal, so the optimal final concentration of Mg 2+ in the reaction system is 4-8 mM.

[0104] Example 3 Detection range and sensitivity test

[0105] The present application selected 20 cases of throat swab samples of influenza A and B virus infection, and 20 cases of negative throat swab samples, numbered A1-A20, B1-B20, C1-C20. The nucleic acid of the samples was extracted and determined by digital PCR. The positive samples were diluted with negative throat swab to obtain sample dilutions with concentrations of 10000 copies / mL, 1000 copies / mL, 200 copies / mL and 100 copies / mL. The diluted positive samples and negative samples were used as test samples to test the detection range and sensitivity of the kit described in Example 2.

[0106] The test results of the positive samples of influenza A and B virus are shown in Table 7. The detection of positive samples of influenza A virus with concentrations of 100000, 1000, 200 and 100 copies / mL is shown in Table 7. Figures 11 to 14 The detection of positive samples of influenza B virus with concentrations of 100000, 1000, 200 and 100 copies / mL is shown in Table 7. Figures 15 to 18

[0107] Table 7

[0108]

[0109]

[0110] The test results of the negative samples are shown in Table 8.

[0111] Table 8

[0112]

[0113] Another "second generation influenza A / B virus nucleic acid detection reagent national reference" S1-S5 was taken according to the instruction concentration and diluted to 50 copies / mL. The diluted national reference S1-S5 was used as a test sample, and the nucleic acid extraction or purification reagent (Yue Sui Machine No. 20170665) produced by Guangzhou Daan Gene Co., Ltd. was used to extract and purify the nucleic acid of the test sample. The detection sensitivity of the kit described in Example 2 was tested. The sensitivity test results of the kit on the national reference S1-S5 are shown in Table 9, and the detection of the kit on the national reference S1-S5 is shown in Table 9. Figures 19 to 23

[0114] Table 9

[0115]

[0116] From Tables 7-9, Figures 11 to 18 and​​Figures 19 to 23 The results show that the kit can detect the throat swab sample with a concentration range of 100-10000 copies / mL, and the sample concentration can be detected at 200 copies / mL or more, with a detection rate of 100%, meeting the clinical detection requirements; and when detecting Guo Can S1-S5, the detection concentration is as low as 50 copies / mL, and the detection rate is 100%.

[0117] Accuracy and repeatability test of Example 4

[0118] In this embodiment, the kit is used to detect positive reference P1 containing influenza A virus nucleic acid, positive reference P2 containing influenza B virus nucleic acid, and negative reference N1 containing human genome, each 20 times, to test the detection accuracy and repeatability of the kit. The reaction system and method are the same as those of Example 2.

[0119] The accuracy test results are shown in Table 10 as follows:

[0120] Table 10 Accuracy and repeatability test results

[0121]

[0122]

[0123] As shown by the results in Table 10, the positive rate of the detection results of the accuracy of each quality control product is 100%, and the CV value is 0.47% and 1.4%, both of which are <5%, indicating that the detection accuracy and repeatability of the kit are good.

[0124] Actual sample detection of Example 5

[0125] 20 cases of clinical positive samples of influenza A and influenza B were selected respectively, the nucleic acid was extracted, the sample label was marked and the label information was ensured to be correct, and it was stored at -80℃. In the experiment, 20 μL of each sample was taken, and the detection was carried out according to the method described in Example 2.

[0126] The actual sample detection results are shown in Table 11. The detection results of the positive samples by the kit are shown in Table 11. Figure 24

[0127] Table 11

[0128]

[0129]

[0130] As shown by the results in Table 11 and Figure 24 As shown by the results in Table 11 and ​

[0131] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A primer and probe combination for typing detection of influenza virus A and B, characterized by, The invention includes primer pairs and probes for detecting influenza A and B viruses, as well as internal control primer pairs and internal control probes. The nucleotide sequences of the primer pairs for detecting influenza A virus are shown in SEQ ID NO. 1–2, and the nucleotide sequences of the probes are shown in SEQ ID NO.

3. The nucleotide sequences of the primer pairs for detecting influenza B virus are shown in SEQ ID NO. 4–5, and the nucleotide sequences of the probes are shown in SEQ ID NO.

6. The nucleotide sequences of the internal control primer pairs are shown in SEQ ID NO. 7–8, and the nucleotide sequences of the internal control probes are shown in SEQ ID NO.

9. The 5' end of the probe is labeled with different fluorescent groups, and the 3' end is labeled with a quenching group.

2. The combination of primers and probes according to claim 1, characterized in that, The probes used to detect influenza A virus are labeled with the fluorescent group FAM at the 5' end and Eclipse at the 3' end; the probes used to detect influenza B virus are labeled with the fluorescent group Texas Red at the 5' end and Eclipse at the 3' end; the internal control probes are labeled with the fluorescent group VIC at the 5' end and BHQ1 at the 3' end.

3. The use of the primer and probe combination of claim 1 or 2 in the preparation of products for typing detection of influenza A and B viruses.

4. A rapid typing test kit for influenza A and B viruses, characterized by, The kit contains the primer and probe combination as described in claim 1 or 2.

5. The reagent kit according to claim 4, characterized in that, The kit also contains positive and negative controls.

6. The reagent kit according to claim 4, characterized in that, The kit also contains reagents required for fluorescent PCR reactions.

7. The reagent kit according to claim 6, characterized in that, The reagents required for the fluorescent PCR reaction include PCR Buffer, which contains Tris-HCl, MgCl2, (NH4)2SO4, KCl and Tween-20.

8. The reagent kit according to claim 6, characterized in that, The reagents required for the fluorescent PCR reaction include a PCR reaction enzyme system, which contains a hot-start Taq antibody enzyme, a Taq enzyme, and an UDG enzyme.

9. The reagent kit according to claim 8, characterized in that, The PCR reaction enzyme system also contains dATP, dGTP, dCTP, dUTP, dTTP, RNasin, KCl, NaCl, Tween-20, NP-40, DTT, and xylitol.

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