Composition, kit, method and use thereof for detecting and typing influenza virus

Abstract

The present invention belongs to the field of molecular biological detection; specifically, it relates to the detection and typing of influenza viruses. More specifically, it relates to the detection and typing of influenza A and B viruses. The present invention provides a composition for detecting and typing influenza viruses, the application of the composition, a kit including the composition, and a method of using the composition. Using the composition of the present invention, there is no need for multiple pairs of primers to amplify influenza A and B. Only one pair of primers is needed to simultaneously detect influenza A and B, the cost is low, the amplification is more uniform, the entire system is more accurate and stable, and the composition of the present invention is more compatible with other virus detection systems.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection; specifically, it relates to the detection and typing of influenza viruses, and more specifically, it relates to the detection and typing of influenza A and B viruses. Background Art

[0002] Acute upper respiratory tract infection is a general term for acute inflammation of the nasal cavity, pharynx or larynx. It is the most common infectious disease of the respiratory tract. Common causes are viruses, accounting for 80%, and a few are caused by bacteria. Patients are regardless of age, gender, occupation and region. Not only is it highly contagious, but it can also cause serious complications, and should be actively prevented and treated. Influenza is the first to be affected, with 5% to 10% of adults and 20% to 30% of children suffering from influenza every year. The influenza virus (influenzavirus), also known as influenza virus for short, is an RNA virus that causes influenza in humans and animals. Human influenza viruses are divided into three types: A, B, and C. They are the pathogens of influenza (flu), among which influenza A and influenza B are more popular.

[0003] The clinical symptoms of influenza and COVID-19 infections are similar, making it difficult to differentiate them. They are also often confused with respiratory infections caused by other pathogens in clinical practice. Therefore, the "Influenza Diagnosis and Treatment Plan" and the "COVID-19 Prevention and Control Plan" both clearly mention the importance of attaching importance to the etiological differentiation of the two viruses to guide precise diagnosis and treatment.

[0004] At present, PCR detection is still more commonly used in laboratory diagnosis. However, its principle is to design primer probes for detecting influenza A virus and primer probes for influenza B virus for the nucleocapsid protein NP gene or the matrix protein M gene respectively. The design is complex and the preparation of reagents is cumbersome, which is not conducive to large-scale production. In addition, with each additional pair of primers in the PCR amplification system, the probability of forming primer dimers increases, the interference between different primer probes will increase to varying degrees, and the mutual influence between all primers will increase, which is bound to affect the amplification effect, thereby affecting the stability and accuracy of the entire detection system. The increase in primers will lead to an increase in cost. At the same time, there are more primer probes in the amplification system, and it is also possible that one amplified fragment inhibits another amplified fragment, which leads to uneven amplification results, thereby affecting the stability and accuracy of the entire detection system.

[0005] Therefore, there is a need in the art for a composition that has a small number of primer pairs and can type influenza A and B, and has high detection stability and accuracy and low cost. Summary of the invention

[0006] In view of this, in a first aspect, the present invention provides a composition for detecting and typing influenza viruses, which also comprises:

[0007] Influenza A and B virus upstream primer: 5'-CCTGGRATGATGATGGG-3' (SEQ ID NO: 1);

[0008] Influenza A and B virus downstream primer: 5'-GATTGSAGYCCATCCCA-3' (SEQ ID NO: 2);

[0009] Influenza A virus probe: 5'-TCGATACTGAATCTTGGACAAAAGAAAT-3' (SEQ ID NO: 3); and

[0010] Influenza B virus probe; 5'-TGGGAGTAGCWGCACTAGGYATC-3' (SEQ ID NO: 4).

[0011] Furthermore, the composition also includes: an internal standard upstream primer, an internal standard downstream primer and an internal standard probe for monitoring.

[0012] By using the composition of the present invention, there is no need to use multiple pairs of primers to amplify influenza A and B respectively. Only one pair of primers is needed to simultaneously detect influenza A and B, which is low in cost, more uniform in amplification, and more accurate and stable in the entire system.

[0013] In a specific embodiment, the composition also includes: a human housekeeping gene internal standard upstream primer as shown in SEQ ID NO:5 (5'-ATCCATGACAACTTTGGTATCGT-3'), a human housekeeping gene internal standard downstream primer as shown in SEQ ID NO:6 (5'-GGGCCATCCACAGTCTTCT-3'), and a human housekeeping gene internal standard probe as shown in SEQ ID NO:7 (5'-AAGGACTCATGACCACAGTCCATGCC-3').

[0014] Furthermore, the fluorescent groups of the influenza A, influenza B and internal standard probes are different from each other and do not interfere with each other.

[0015] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0016] In a specific embodiment, the influenza A probe is labeled with the FAM channel and the influenza B probe is labeled with the HEX channel.

[0017] In a specific embodiment, the internal standard probe is labeled with the CY5 channel.

[0018] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.

[0019] In a specific embodiment, the 3' end of the probe is BHQ1.

[0020] Furthermore, the amount of the primer in the composition is 0.1-0.6 μM, preferably 0.4 μM; the amount of the probe in the composition is 0.05-0.5 μM, preferably 0.2 μM.

[0021] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0022] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0023] In some specific embodiments, the composition of the present invention may also include primers and probes for detecting other viruses.

[0024] In some specific embodiments, the composition of the present invention may also include primers and probes for detecting rhinovirus.

[0025] In a specific embodiment, the present invention includes rhinovirus primers and probe sequences, wherein the upstream primer is SEQ ID NO: 8: 5'-TGACAAGGTGCGAAGAG-3'; the downstream primer is SEQ ID NO: 9: 5'-CAAAGTAGTTGGTCCCATCC-3', and the probe is SEQ ID NO: 10: 5'-CTCCGGCTCCTGAATG TGGCTAAC-3'.

[0026] By using the composition of the present invention, better amplification uniformity is obtained, and therefore, it can be better compatible with other virus detection systems.

[0027] In a second aspect, the present invention provides use of the above-mentioned composition of the present invention in preparing a kit for detecting and typing influenza viruses.

[0028] In a third aspect, the present invention provides a kit for detecting and typing influenza viruses, the kit comprising the composition of the present invention as described above.

[0029] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0030] In a specific embodiment, the negative control product is at least one of DEPC H2O, physiological saline, and internal reference gene pseudovirus. The positive control product is at least one of the target fragment plasmid containing influenza virus target gene and internal reference gene, fragment RNA, and pseudovirus.

[0031] Furthermore, the kit also includes: a nucleic acid release system and a nucleic acid amplification system.

[0032] Furthermore, the kit also includes dNTP, PCR buffer and Mg 2+ At least one of .

[0033] Furthermore, the kit also includes: at least one of a nucleic acid releaser, a nucleic acid extraction reagent, a reverse transcriptase, a uracil glycosylase and a DNA polymerase.

[0034] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, reverse transcriptase, uracil glycosylase, DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0035] Furthermore, the concentration of the reverse transcriptase is 5U / reaction to 15U / reaction, for example, the reverse transcriptase can be mouse leukemia reverse transcriptase (MMLV), Neoscript RT reverse transcriptase or Tth enzyme; the concentration of the DNA polymerase is 3U / reaction to 15U / reaction, for example, the DNA polymerase can be Taq enzyme.

[0036] In a specific embodiment, the kit of the present invention comprises: reverse transcriptase, Taq enzyme, uracil glycosylase, Mg 2+ , Mn 2+ , Rnasin, dNTPs, primers, probes and PCR buffer.

[0037] Common PCR buffers are composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 to 100 μl.

[0038] In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0039] In a fourth aspect, a method for detecting and typing influenza viruses is provided, the method comprising the following steps:

[0040] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0041] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1);

[0042] 3) Obtain and analyze the results.

[0043] In the present invention, the sample used for detection can be a throat swab, an oropharyngeal swab, a nasopharyngeal swab, sputum, alveolar lavage fluid, blood, etc., but is not limited thereto.

[0044] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0045] Reverse transcription, temperature is 50℃~60℃, time is 5~30 minutes, 1 cycle; cDNA pre-denaturation, temperature is 95℃, time is 1~10 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5~30 seconds, annealing, temperature is 55~60℃, time is 20~60 seconds, 40~50 cycles, and fluorescence is collected.

[0046] In a specific embodiment, the reaction conditions of the fluorescent quantitative PCR are: reverse transcription, temperature is 50°C, time is 10 minutes, 1 cycle; cDNA pre-denaturation, temperature is 95°C, time is 1 minute, 1 cycle; denaturation, temperature is 95°C, time is 10 seconds, annealing, temperature is 60°C, time is 30 seconds, 45 cycles, and fluorescence collection.

[0047] In a specific embodiment, a method for detecting and typing influenza virus for non-diagnostic purposes is provided, the method comprising the following steps:

[0048] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0049] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1);

[0050] 3) Obtain and analyze the results.

[0051] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0052] Reverse transcription, temperature is 50℃~60℃, time is 5~30 minutes, 1 cycle; cDNA pre-denaturation, temperature is 95℃, time is 1~10 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5~30 seconds, annealing, temperature is 55~60℃, time is 20~60 seconds, 40~50 cycles, and fluorescence is collected.

[0053] In a specific embodiment, the reaction conditions of the fluorescent quantitative PCR are: reverse transcription, temperature is 50°C, time is 10 minutes, 1 cycle; cDNA pre-denaturation, temperature is 95°C, time is 1 minute, 1 cycle; denaturation, temperature is 95°C, time is 10 seconds, annealing, temperature is 60°C, time is 30 seconds, 45 cycles, and fluorescence collection.

[0054] In this context, the term "non-diagnostic purpose" refers to a method that is not intended to obtain information on whether an individual is infected with influenza virus and has influenza. For example, the method can be used to detect the presence of influenza virus in a test culture in an experiment for scientific research purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a graph showing the test results of the composition of the present invention;

[0056] Figures 2-3 This is a sensitivity test result diagram of the composition of the present invention;

[0057] Figure 4 This is a graph showing the specificity test results of the composition of the present invention;

[0058] Figures 5-6 This is a comparison chart of the compatibility test results of the composition of the present invention;

[0059] Figure 7 This is a graph showing the test results of the composition of the present invention;

[0060] Figure 8 This is a graph showing the test results of the composition of comparative method 1;

[0061] Fig. 9 This is a graph showing the test results of the composition of comparative method 2. DETAILED DESCRIPTION

[0062] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0063] Example 1: Primers and probes used in the present invention

[0064] The primers and probes used in the present invention are shown in Table 1 below:

[0065] Table 1

[0066]

[0067] Example 2: Method for detecting and typing influenza viruses

[0068] Reagent preparation:

[0069] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture according to the proportion (PCR reaction solution 36.0 μL / person + enzyme mixture 4.0 μL / person), mix them thoroughly to form a PCR mixture, and centrifuge at 2000 rpm for 10 seconds before use.

[0070] Sample handling and loading:

[0071] Take 200 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube for nucleic acid extraction.

[0072] Pipette 10 μL of the above-treated samples, negative controls and positive controls and add them to the corresponding 0.2 mL PCR reaction tubes, add 40 μL of PCR mixture to each tube and cover the tube.

[0073] PCR amplification:

[0074] On ABI 7500 fluorescent quantitative PCR instrument, Life Technologies Quant Studio TM 5. On a PCR instrument such as a fluorescent PCR instrument or a SLAN-96P fully automatic medical PCR analysis system, PCR amplification is performed according to a certain temperature and time setting program. The amplification scheme of the present invention is:

[0075]

[0076] Explanation of test results:

[0077] If the sample has an obvious S-shaped amplification curve in the FAM and HEX channels, and the Ct value is ≤40, it is judged as positive; if the sample has no amplification curve (No Ct) or the Ct value is greater than 40 in the FAM and HEX channels, and the CY5 internal standard channel is positive (Ct value ≤40), it is judged as negative. The details are as follows:

[0078]

[0079]

[0080] Example 3. Test results of the positive control of the composition of the present invention

[0081] Using the composition of Table 1 of the present invention, each target positive plasmid was detected according to the method described in Example 2 to simulate clinical samples, and multiple PCR detection was performed on a Macrostone fluorescent quantitative PCR instrument. The results are as follows: Figure 1 Each channel can detect a good amplification curve, indicating that the composition of the present invention can detect and type the virus that causes respiratory tract infection.

[0082] Example 4: Sensitivity of the composition of the present invention

[0083] Experiments were conducted using plasmid templates of different concentrations to detect the sensitivity of the composition of the present invention.

[0084] Using the composition of Table 1 of the present invention, according to the method described in Example 2, each target LOD (sensitivity) positive plasmid was detected to simulate clinical samples, and multiple PCR detection was performed on the ABI7500 fluorescent quantitative PCR instrument. The detection sensitivity of each channel was 200 copies / ml. The results showed that at a concentration of 200 copies / ml, the composition of the present invention can still detect the virus. The results are as follows: Figures 2-3 As shown, it is shown that the sensitivity of the composition of the present invention can reach 200 copies / ml.

[0085] Example 5. Specificity of the composition of the present invention

[0086] Experiments with some other similar viruses showed that the composition of the present invention would not detect other viruses and cause false positives. The results are as follows Figure 4 As shown, the composition of the present invention has no non-specific amplification for common respiratory pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, metapneumovirus, rhinovirus, enterovirus 71, coxsackievirus A, coronavirus 229E, respiratory syncytial virus, adenovirus, human parainfluenza type 1, human parainfluenza type 2, human parainfluenza type 3, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, Streptococcus salivarius, Streptococcus pneumoniae, Mycobacterium tuberculosis, Klebsiella pneumoniae, etc.), indicating that the composition of the present invention has good specificity.

[0087] Example 6: Experiment on compatibility of the composition of the present invention with other viruses

[0088] The composition of the present invention has the advantage of less interference due to the small number of primers and probes. In addition, it has certain compatibility and expansibility. Primers and probes for detecting rhinovirus (wherein the concentrations of primers and probes are 0.4 μM and 0.2 μM, respectively, and their sequences are shown in SEQ ID NOs: 8 to 10, respectively) were added to the composition of the present invention, and no obvious interference was found after comparison. The same concentration of primers and probes for detecting rhinovirus were added to conventional and non-present invention amplification systems, which respectively used conservative sequences of influenza A and B virus genes as detection targets. After comparison, it was found that there was obvious interference. The results are as follows: Figures 5-6 shown.

[0089] Comparative Example 1

[0090] Comparative method 1 (a pair of primers amplification):

[0091] The following primer probes were designed:

[0092] FluA+B-F2: 5'-GGGAATAAACATGAGCAAAAAGA-3' (SEQ ID NO: 11)

[0093] FluA+B-R2:5'-CATTGTTWATCATRTTGTTTCTTTA-3'(SEQ ID NO:12)

[0094] FluA-P2: 5'-ATGGATTTGTGGCTAATTTTAGCATGGAGC-3' (SEQ ID NO: 13)

[0095] FluB-P2: 5'-ATGGATTTGTATCTAACTTTGCAATGGAAATTC-3' (SEQ ID NO: 14)

[0096] Comparative method 2 (two pairs of primers amplified separately):

[0097] Primer probes for detecting influenza A virus and primer probes for detecting influenza B virus were designed for the nucleocapsid protein NP gene or the matrix protein M gene (as shown below):

[0098] Primers for detecting influenza A virus:

[0099] A-F1: 5'-GGTTATAAGAATGATGGAAAGTGC-3' (SEQ ID NO: 15);

[0100] A-R1: 5'-TCTGCATTGTCTCCGAAGAAAT-3' (SEQ ID NO: 16);

[0101] A-P1: 5'-AGTTTTTCGAGCTCTCAGACGAGA-3' (SEQ ID NO: 17);

[0102] Primers for detecting influenza B virus:

[0103] B-F3: 5'-ATGGCTCTTTACAATATGGCAA-3' (SEQ ID NO: 18);

[0104] B-R3: 5'-CTTCCAGGTCTTCATAGGCA-3' (SEQ ID NO: 19);

[0105] B-P3: 5'-CCGAAGCAAGACATGAAGAATAAT-3' (SEQ ID NO: 20);

[0106] The above primers and probes were prepared into a PCR reaction solution for simultaneously detecting influenza A and B viruses, and the same nucleic acid was tested with the detection composition provided by the present invention. The results were as follows (see Table 2 and Table 3). Figures 7 to 9 show):

[0107] Table 2

[0108]

[0109] The results show that the detection effect of the composition of the present invention is better than that of the comparative composition. It further illustrates that the present invention uses the same primers to amplify influenza A virus or influenza B virus, ensuring the uniformity of the detection of the two viruses; without adding too many primers, the interference between primers is reduced, and the sensitivity of the detection is guaranteed. Sequence Listing <110> Shengxiang Biotechnology Co., Ltd. <120> Composition, kit, method and use thereof for detecting and typing influenza virus <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> DNA <213> Artificial sequence <400> 1 cctggratga tgatggg 17 <210> 2 <211> 17 <212> DNA <213> Artificial sequence <400> 2 gattgsagyc catccca 17 <210> 3 <211> 28 <212> DNA <213> Artificial sequence <400> 3 tcgatactga atcttggaca aaagaaat 28 <210> 4 <211> twenty three <212> DNA <213> Artificial sequence <400> 4 tgggagtagc wgcactaggy atc 23 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <400> 5 atccatgaca actttggtat cgt 23 <210> 6 <211> 19 <212> DNA <213> Artificial sequence <400> 6 gggccatcca cagtcttct 19 <210> 7 <211> 26 <212> DNA <213> Artificial sequence <400> 7 aaggactcat gaccacagtc catgcc 26 <210> 8 <211> 17 <212> DNA <213> Artificial sequence <400> 8 tgacaaggtg cgaagag 17 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <400> 9 caaagtagtt ggtcccatcc 20 <210> 10 <211> twenty four <212> DNA <213> Artificial sequence <400> 10 ctccggctcc tgaatgtggc taac 24

Claims

1. A composition for detecting and typing influenza viruses, comprising: Influenza A and B virus upstream primer: 5'-CCTGGRATGATGATGGG-3' (SEQ ID NO: 1); Influenza A and B virus downstream primer: 5'-GATTGSAGYCCATCCCA-3' (SEQ ID NO: 2); Influenza A virus probe: 5'-TCGATACTGAATCTTGGACAAAAGAAAT-3' (SEQ ID NO: 3); and Influenza B virus probe: 5'-TGGGAGTAGCWGCACTAGGYATC-3' (SEQ ID NO: 4).

2. The composition according to claim 1, wherein The composition also includes: a human housekeeping gene internal standard upstream primer as shown in SEQ ID NO: 5 (5'-ATCCATGACAACTTTGGTATCGT-3'), a human housekeeping gene internal standard downstream primer as shown in SEQ ID NO: 6 (5'-GGGCCATCCACAGTCTTCT-3'), and a human housekeeping gene internal standard probe as shown in SEQ ID NO: 7 (5'-AAGGACTCATGACCACAGTCCATGCC-3').

3. The composition according to claim 1, wherein The composition may also include primers and probes for detecting rhinovirus, wherein the upstream primer is SEQ ID NO: 8: 5'-TGACAAGGTGCGAAGAG-3'; the downstream primer is SEQ ID NO: 9: 5'-CAAAGTAGTTGGTCCCATCC-3', and the probe is SEQ ID NO: 10: 5'-CTCCGGCTCCTGAATG TGGCTAAC-3'.

4. The composition according to any one of claims 1 to 3, wherein The components of the composition are present in the package in mixed form.

5. Use of the composition according to any one of claims 1 to 4 in preparing a kit for detecting and typing influenza viruses.

6. A kit for detecting and typing influenza virus, comprising the composition according to any one of claims 1 to 4.

7. The kit according to claim 6, wherein The kit also includes: a nucleic acid release system and a nucleic acid amplification system.

8. A method for detecting and typing influenza viruses for non-diagnostic purposes, the method comprising the following steps: 1) Extracting or releasing nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 4 or the kit according to claim 6 or 7; 3) Obtain and analyze the results.

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