Primer probe combination for influenza A virus full-typing detection, kit and application

Abstract

The invention belongs to the technical field of molecular biological detection, and discloses a primer probe combination for influenza A virus full-typing detection, a kit and application. The primer probe combination for influenza A virus full-typing detection comprises a forward primer group, a reverse primer group and a probe group, the nucleotide sequence of the forward primer group comprises at least one of SEQ ID No.1-20; the nucleotide sequence of the reverse primer group comprises at least one of SEQ ID No. 21 to SEQ ID No. 40; and the nucleotide sequence of the probe group comprises at least one of SEQ ID No. 41-60. 20 influenza A viruses can be detected at the same time, rapid detection of all subtypes of influenza A viruses epidemic at home and abroad is achieved, and the defects that in the prior art, influenza A viruses cannot be typed well, and typing detection is not comprehensive enough are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and in particular to a primer-probe combination, a kit and an application thereof for full typing detection of influenza A virus. Background Art

[0002] Influenza viruses are divided into types A, B and C. Influenza A is the most common influenza virus that infects humans, causing seasonal influenza epidemics and other potential epidemics. Influenza A virus belongs to the family Orthomyxoviridae and is a single-stranded segmented negative-strand RNA virus. The viral genome has a total of 8 segments, of which hemagglutinin (HA) and neuraminidase (NA) are two important surface glycoproteins with high genetic diversity. Currently, subtypes that have been found to be able to directly or indirectly infect humans include H1N1, H1N12009, H1N2, H2N3, H3N2, H3N3, H4N6, H5N1, H5N2, H5N6, H5N8, H6N6, H7N4, H7N6, H7N8, H7N9, H9N2, H11N3, H11N7, and H12N8, a total of 20 types. Its rapid mutation and evolutionary characteristics provide the basis for influenza viruses to adapt and then infect humans. Since it caused a sustained epidemic of human influenza in 1878, it is still the main pathogen of large-scale influenza outbreaks. The outbreak and spread of influenza A seriously affect food hygiene and safety and international trade, causing serious public health and safety problems. Influenza A is mainly transmitted through the air (i.e. coughing or sneezing), and the peak of transmission usually occurs in winter. Symptoms usually appear within two days of contact with an infected person, including fever, chills, headache, malaise, cough and sinus congestion. Gastrointestinal symptoms (such as nausea, vomiting or diarrhea) may also occur. Elderly people and people with weakened immune systems may develop pneumonia complications after influenza infection, leading to increased morbidity and mortality. Influenza A viruses can also infect animals such as birds, pigs and horses.

[0003] Therefore, the typing detection of influenza A virus is of great significance for the monitoring and response of influenza virus. Among the existing typing detection methods for influenza A virus, the commonly used methods such as immunofluorescence test and polymerase chain reaction are cumbersome and time-consuming, and cannot meet the requirements of rapid typing detection and diagnosis of influenza A virus on site. Loop-mediated isothermal nucleic acid amplification technology (Loop-Mediated Isothermal Amplification, LAMP) can quickly, efficiently and specifically amplify the target nucleic acid at a constant temperature, simplifying the complexity of nucleic acid detection, but there are disadvantages such as complex primer design, complex product structure and partial quantification difficulties. Competitive complementary annealingmediated isothermal amplification (Competitive annealingmediated isothermal amplification, CAMP) is a method of nucleic acid isothermal amplification, which has similar advantages to LAMP, and the primer design principle of CAMP is simpler, and the required target nucleic acid fragment is shorter, but the reaction conditions are strict, and the specificity and sensitivity are unstable. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a primer-probe combination, a kit and an application for full typing detection of influenza A virus.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a primer-probe combination for full typing detection of influenza A virus, comprising a forward primer set, a reverse primer set and a probe set;

[0007] The nucleotide sequence of the forward primer set includes at least one of SEQ ID No. 1-20; or a nucleotide sequence having at least 95% sequence homology with any of the nucleotide sequences of SEQ ID No. 1-20;

[0008] The nucleotide sequence of the reverse primer set includes at least one of SEQ ID No. 21-40; or a nucleotide sequence having at least 95% sequence homology with any of the nucleotide sequences of SEQ ID No. 21-40;

[0009] The nucleotide sequence of the probe set includes at least one of SEQ ID Nos. 41-60; or a nucleotide sequence having at least 95% sequence homology with any of the nucleotide sequences of SEQ ID Nos. 41-60.

[0010] The primer-probe combination for full typing detection of influenza A virus of the present invention can detect 20 types of influenza A viruses simultaneously, realizes rapid detection of various subtypes of influenza A viruses prevalent at home and abroad, and overcomes the shortcomings of the prior art that influenza A viruses cannot be well typed and typing detection is not comprehensive enough.

[0011] As a preferred embodiment of the primer-probe combination of the present invention, the influenza A virus includes at least one of H1N1, H1N12009, H1N2, H2N3, H3N2, H3N3, H4N6, H5N1, H5N2, H5N6, H5N8, H6N6, H7N4, H7N6, H7N8, H7N9, H9N2, H11N3, H11N7, and H12N8.

[0012] As a preferred embodiment of the primer-probe combination of the present invention, the 5' end of the nucleotide sequence of the probe group is modified with a fluorescence generating group, and the 3' end is modified with a fluorescence quenching group.

[0013] As a further preferred embodiment of the primer-probe combination of the present invention, the fluorescence generating group includes any one of FAM, ROX, VIC or CY5; the fluorescence quenching group includes any one of BHQ1, BHQ2, BHQ3, MGB.

[0014] In a second aspect, the present invention uses the primer-probe combination in the preparation of a reagent and / or a kit for detecting and / or amplifying full typing of influenza A virus.

[0015] In a third aspect, the present invention provides a reagent and / or kit for full typing detection of influenza A virus, comprising the primer-probe combination.

[0016] As a preferred embodiment of the reagent and / or kit of the present invention, it also includes primers and probes for detecting an internal reference gene; preferably, the internal reference gene is RNaseP.

[0017] In a fourth aspect, the present invention provides a method for full typing of influenza A virus for non-diagnostic purposes, comprising the following steps:

[0018] (1) Extracting sample nucleic acid;

[0019] (2) performing PCR amplification on the nucleic acid based on any of the following:

[0020] 1. the primer-probe combination; and / or

[0021] ii. the reagents and / or kits;

[0022] (3) Interpret the results based on the CT value;

[0023] The interpretation criteria are:

[0024] The CT value of the internal reference target is ≤40, and the CT value of other targets is ≤40, which means that the corresponding subtypes of the 20 influenza A viruses are judged to be positive;

[0025] If the CT value of the internal reference target is ≤40 and other targets have no CT value, 20 types of influenza A viruses can be judged to be negative.

[0026] The method of the present invention is targeted at the 20 influenza A virus subtypes that currently mainly infect humans, and can achieve rapid detection of various subtypes of influenza A viruses prevalent at home and abroad. The established detection method does not undergo non-specific amplification with other types, has high sensitivity, and has good application prospects.

[0027] As a preferred embodiment of the influenza A virus full typing detection method described in the present invention, the PCR reaction solution includes: 10×PCR Buffer, 5% DMSO, 0.01U / μL-0.05U / μL reverse transcriptase, 0.01U / μL-0.04U / μL DNA polymerase, 0.01U / μL-0.04U / μL UNG enzyme, 150nM-500nM of the forward primer, 150nM-500nM of the reverse primer, and 125nM-300nM of the probe.

[0028] As a preferred embodiment of the influenza A virus full typing detection method described in the present invention, the PCR reaction program includes: 48°C-50°C for 1min-2min; 90°C-95°C for 30s-1min; the cycle steps are 90°C-95°C for 5s-10s, 60°C-65°C for 20s-30s, for a total of 40-45 cycles.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The primer-probe combination of the present invention can simultaneously detect 20 influenza A viruses based on PCR, covering common influenza A virus subtypes that infect humans, and can quickly help regulatory agencies monitor and respond to influenza viruses, with good specificity, good stability, and low detection limit. The typing detection method of the present invention can be completed within 60 minutes at the fastest, realizing rapid detection of major influenza A virus subtypes prevalent at home and abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the amplification result of the primer probe of system 1;

[0032] Figure 2 is the amplification result of the primer probe of system 2;

[0033] Figure 3is the amplification result of the primer probe of system 3;

[0034] Figure 4 is the amplification result of the primer probe of system 4;

[0035] Figure 5 is the amplification result of the primer probe of system 5;

[0036] Figure 6 is the amplification result of the primer probe of system 6;

[0037] Figure 7 The minimum detection limit test results of the primer-probe combination for H1N1, H1N12009, H1N2, and H2N3;

[0038] Figure 8 The minimum detection limit test results of the primer-probe combination for H3N2, H3N3, H4N6, and H5N1;

[0039] Fig. 9 The minimum detection limit test results of the primer-probe combination for H5N2, H5N6, H5N8, and H6N6;

[0040] Fig.10 The minimum detection limit test results of the primer-probe combination for H7N4, H7N6, H7N8, and H7N9;

[0041] Fig.11 The minimum detection limit test results of the primer-probe combination for H9N2, H11N3, H11N7, and H12N8;

[0042] Fig.12 The results of repeatability tests of primer-probe combinations for H1N1, H1N12009, H1N2, and H2N3;

[0043] Fig.13 The results of repeatability tests of primer-probe combinations for H3N2, H3N3, H4N6, and H5N1;

[0044] Fig.14 The results of repeatability tests of primer-probe combinations for H5N2, H5N6, H5N8, and H6N6;

[0045] Fig.15 The results of repeatability tests of primer-probe combinations for H7N4, H7N6, H7N8, and H7N9;

[0046] Fig.16 The repeatability test results of the primer-probe combination for H9N2, H11N3, H11N7, and H12N8;

[0047] Fig.17The specificity test results of the primer-probe combination. DETAILED DESCRIPTION

[0048] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0049] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0050] According to the characteristics of common fluorescence quantitative PCR instruments on the market (there are 4 channels), in order to make the invention have a wider application range and convenient implementation, 22 targets, including 20 influenza A subtypes, influenza A universal type and internal control, are divided into 6 reaction systems (each system includes all the primer probe combinations of the present invention) for detection, among which H1N1, H1N12009, H1N2 and H2N3 construct reaction system 1, H3N2, H3N3, H4N6 and H5N1 construct reaction system 2, H5N2, H5N6, H5N8 and H6N6 construct reaction system 3, H7N4, H7N6, H7N8 and H7N9 construct reaction system 4, H9N2, H11N3, H11N7 and H12N8 construct reaction system 5, and FluA and RnaseP construct reaction system 6.

[0051] Example 1: A primer-probe combination for full typing detection of influenza A virus

[0052] According to relevant literature reports at home and abroad and influenza virus gene database, the conserved gene sequences of 20 types of influenza A virus were selected. Based on the primer design principles of fluorescent PCR, the gene sequences were brought into the PrimerExplorer V5 website to design primer sequences. The primer probe sequences were designed according to Primer Express5.0.1 software. The primer probe sequences are shown in Table 1.

[0053] Table 1 Primer probe sequences

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Based on the fluorescent PCR technology, the primer probe combination for 20 influenza A virus subtypes is used to qualitatively detect 20 influenza A virus subtypes. During the operation, the sample nucleic acid extract is taken, and then the reaction system is prepared, and amplification is performed on the fluorescent PCR instrument. After the reaction is completed, the result is interpreted according to the CT value.

[0060] The specific methods for detecting 20 types of influenza A viruses are as follows:

[0061] (1) Preparation of PCR reaction solution: RT-PCR reaction solution includes 10×PCR Buffer, 5% DMSO, 0.05 U / μL reverse transcriptase, 0.04 U / μL DNA polymerase, and 0.04 U / μL UNG enzyme;

[0062] (2) placing the above reaction in a fluorescence quantitative PCR instrument for reaction;

[0063] The forward primer F used to detect 20 influenza A virus subtypes is used at a concentration of 150nM to 500nM, the reverse primer R is used at a concentration of 150nM to 500nM, and the probe P is used at a concentration of 125nM to 300nM.

[0064] (3) After the reaction is completed, the CT value is used to identify 20 influenza A virus subtypes.

[0065] The result analysis and judgment criteria are as follows:

[0066] The CT value of the internal reference RnaseP target is ≤40, and the CT value of other targets is ≤40, which means that the corresponding subtypes of 20 influenza A viruses are judged to be positive;

[0067] The CT value of the internal reference RnaseP target is ≤40, and other targets have no CT value, which can determine that 20 types of influenza A viruses are negative.

[0068] Example 2: Screening of primer probes

[0069] In order to screen a better primer-probe combination, 20 influenza A virus subtype nucleic acid samples were selected. The enzymes and primers and probes were purchased from Sangon Biotechnology. The instrument used was ABI's QuantStudio Q5 fluorescent quantitative PCR instrument. The test results were judged by the CT value.

[0070] 1. The samples used were virus culture, pseudovirus and clinical samples. The samples were extracted with TaKaRa MiniBEST ViralRNA / DNAExtraction Kit Ver.5.0. The sample information is shown in Table 2:

[0071] Table 2 Sample information

[0072] Sample name type concentration H1N1 Virus culture 10^4 copies / mL H1N12009 Virus culture 10^4 copies / mL H1N2 Pseudovirus 10^4 copies / mL H2N3 Pseudovirus 10^4 copies / mL H3N2 Virus culture 10^4 copies / mL H3N3 Pseudovirus 10^4 copies / mL H4N6 Pseudovirus 10^4 copies / mL H5N1 Pseudovirus 10^4 copies / mL H5N2 Pseudovirus 10^4 copies / mL H5N6 Pseudovirus 10^4 copies / mL H5N8 Pseudovirus 10^4 copies / mL H6N6 Pseudovirus 10^4 copies / mL H7N4 Pseudovirus 10^4 copies / mL H7N6 Pseudovirus 10^4 copies / mL H7N8 Pseudovirus 10^4 copies / mL H7N9 Pseudovirus 10^4 copies / mL H9N2 Pseudovirus 10^4 copies / mL H11N3 Pseudovirus 10^4 copies / mL H11N7 Pseudovirus 10^4 copies / mL H12N8 Pseudovirus 10^4 copies / mL Negative clinical samples - -

[0073] 2. The PCR amplification system is prepared as shown in Table 3:

[0074] Table 3 PCR amplification system

[0075] Reagents Final concentration 10×PCR Buffer 1× DNA polymerase 0.04U / μL Reverse transcriptase 0.05U / μL UNG enzyme 0.04U / μL DMSO 5％ F 200nM R 200nM Probe 125nM

[0076] 3. The PCR amplification program is shown in Table 4:

[0077] Table 4 PCR amplification program

[0078]

[0079] 4. Results Analysis

[0080] Amplification results are attached. Figure 1-6 , the test results are shown in Tables 5 and 6.

[0081] Table 5 Test results

[0082]

[0083]

[0084] Table 6 Test results

[0085]

[0086]

[0087] According to the above results, by comparing the Ct values, it can be found that combination 1 of system 1 is better than combination 2, combination 1 of system 2 is better than combination 2, combination 2 of system 3 is better than combination 1, combination 2 of system 4 is better than combination 1, combination 1 of system 5 is better than combination 2, and combination 1 of system 6 is better than combination 2. In addition, the corresponding samples of combination 1 of system 1, combination 1 of system 2, combination 2 of system 3, combination 2 of system 4, combination 1 of system 5 and combination 1 of system 1 can all be detected, while other subtypes cannot be detected, indicating that the 20 selected influenza A virus subtype primers and probes have good specificity, and the primer and probe combination screening is qualified, meeting the requirements for influenza A virus typing.

[0088] Example 3: Primer-probe combination minimum detection limit test

[0089] In order to determine the minimum detection limit of the primer-probe combination (combination 1 of system 1, combination 1 of system 2, combination 2 of system 3, combination 2 of system 4, combination 1 of system 5, and combination 1 of system 1), 20 influenza A virus subtype nucleic acid samples were selected, enzymes and primers and probes were purchased from Sangon Biotechnology, and ABI's QuantStudio Q5 fluorescent quantitative PCR instrument was selected. The test results were judged by the CT value.

[0090] 1. The samples used were virus culture, pseudovirus and clinical samples. The samples were extracted with TaKaRa MiniBEST ViralRNA / DNA Extraction Kit Ver.5.0. The sample information is shown in Table 7:

[0091] Table 7 Sample information

[0092]

[0093] 2. The preparation of PCR amplification system is the same as Table 3.

[0094] 3. PCR amplification procedure is the same as Table 4.

[0095] 4. Results Analysis

[0096] Amplification results are attached. Figure 7-11 The test results are shown in Tables 8-10.

[0097] Table 8 Test results

[0098]

[0099] Table 9 Test results

[0100]

[0101]

[0102] Table 10 Test results

[0103]

[0104] According to the above results, the above 20 influenza A virus subtype primer-probe combinations can detect the sample concentrations of 10000 copies / mL and 1000 copies / mL. Compared with the common influenza virus detection kits on the market, the primer-probe combination of the present invention has high sensitivity and meets the requirements for influenza A virus typing.

[0105] Example 4: Primer-probe combination repeatability test

[0106] In order to determine the repeatability of the primer-probe combination (combination 1 of system 1, combination 1 of system 2, combination 2 of system 3, combination 2 of system 4, combination 1 of system 5, and combination 1 of system 1), 20 influenza A virus subtype nucleic acid samples were selected, enzymes and primers and probes were purchased from Sangon Biotechnology, and the instrument was ABI's QuantStudio Q5 model fluorescent quantitative PCR instrument. The test results were judged by the CT value.

[0107] 1. The samples used were virus culture, pseudovirus and clinical samples. The samples were extracted with TaKaRa MiniBEST ViralRNA / DNA Extraction Kit Ver.5.0. The sample information is the same as Table 2.

[0108] 2. The preparation of PCR amplification system is the same as Table 3.

[0109] 3. PCR amplification procedure is the same as Table 4.

[0110] 4. Results Analysis

[0111] Amplification results are attached. Figure 12-16 , the test results are shown in Table 11:

[0112] Table 11 Test results

[0113]

[0114]

[0115] According to the above results, the CV values ​​of the primer-probe combinations for the above 20 influenza A virus subtypes were all less than 5% in 10 repeated tests at a sample concentration of 1000 copies / mL, indicating that the primer-probe combinations for the above 20 influenza A virus subtypes had good repeatability and met the requirements for influenza A virus typing.

[0116] Example 5: Primer-probe combination specificity test

[0117] Other microbial pathogens with high sequence homology to influenza A virus, which are likely to cause the same or similar clinical symptoms and complications, were selected as specific test samples. TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 was used to extract nucleic acid from viral samples, and TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 was used to extract nucleic acid from bacterial samples. Enzymes and primers and probes were purchased from Sangon Biotechnology. ABI's QuantStudio Q5 fluorescent quantitative PCR instrument was selected as the instrument. The test results were judged by the CT value.

[0118] 1. See Table 12 for sample information.

[0119] Table 12 Sample information

[0120]

[0121]

[0122] 2. The preparation of PCR amplification system is shown in Table 13:

[0123] Table 13 PCR amplification system

[0124] Reagents Final concentration 10×PCR Buffer 1× DNA polymerase 0.04U / μL Reverse transcriptase 0.05U / μL UNG enzyme 0.04U / μL DMSO 5％ F 250nM R 250nM Probe 150nM

[0125] 3. PCR amplification program is shown in Table 14:

[0126] Table 14 PCR amplification program

[0127]

[0128] 4. Results Analysis

[0129] Amplification results are attached. Fig.17 , the test results are shown in Table 15:

[0130] Table 15 Test results

[0131]

[0132]

[0133] Influenza B virus Victoria type, influenza B virus Yamagata type, respiratory syncytial virus type A, respiratory syncytial virus type B, human rhinovirus type A, adenovirus type 1, human metapneumovirus type A, human coronavirus, Mycoplasma pneumoniae, Haemophilus influenzae, Bordetella pertussis, Klebsiella pneumoniae, Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae have no CT values, indicating that the primer probe combination of the present invention has good specificity.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A primer-probe combination for full typing detection of influenza A virus, characterized in that: It includes a forward primer set, a reverse primer set and a probe set; The nucleotide sequence of the forward primer set includes at least one of SEQ ID No. 1-20; or a nucleotide sequence having at least 95% sequence homology with any of the nucleotide sequences of SEQ ID No. 1-20; The nucleotide sequence of the reverse primer set includes at least one of SEQ ID No. 21-40; or a nucleotide sequence having at least 95% sequence homology with any of the nucleotide sequences of SEQ ID No. 21-40; The nucleotide sequence of the probe set includes at least one of SEQ ID No. 41-60; or a nucleotide sequence having at least 95% sequence homology with any of the nucleotide sequences of SEQ ID No. 41-60.

2. The primer-probe combination according to claim 1, characterized in that: The influenza A virus includes at least one of H1N1, H1N12009, H1N2, H2N3, H3N2, H3N3, H4N6, H5N1, H5N2, H5N6, H5N8, H6N6, H7N4, H7N6, H7N8, H7N9, H9N2, H11N3, H11N7, and H12N8.

3. The primer-probe combination according to claim 1, characterized in that: The 5' end of the nucleotide sequence of the probe group is modified with a fluorescence generating group, and the 3' end is modified with a fluorescence quenching group.

4. The primer-probe combination according to claim 2, characterized in that: The fluorescence generating group includes any one of FAM, ROX, VIC or CY5; the fluorescence quenching group includes any one of BHQ1, BHQ2, BHQ3 and MGB.

5. Use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a reagent and / or kit for detecting and / or amplifying full typing of influenza A virus.

6. A reagent and / or kit for full typing detection of influenza A virus, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 4.

7. The reagent and / or kit according to claim 6, characterized in that: It also includes primers and probes for detecting an internal reference gene; preferably, the internal reference gene is RNaseP.

8. A method for full typing of influenza A virus for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Extracting sample nucleic acid; (2) performing PCR amplification on the nucleic acid based on any of the following:

1. A primer-probe combination as described in any one of claims 1 to 4; and / or ii. The reagent and / or kit according to claim 6 or 7; (3) Interpret the results based on the CT value; The interpretation criteria are: The CT value of the internal reference target is ≤40, and the CT value of other targets is ≤40, which means that the corresponding subtypes of the 20 influenza A viruses are judged to be positive; If the CT value of the internal reference target is ≤40 and other targets have no CT value, 20 types of influenza A viruses can be judged to be negative.

9. The method for full typing of influenza A virus according to claim 8, characterized in that: The PCR reaction solution includes: 10×PCR Buffer, 5% DMSO, 0.01U / μL-0.05U / μL reverse transcriptase, 0.01U / μL-0.04U / μL DNA polymerase, 0.01U / μL-0.04U / μL UNG enzyme, 150nM-500nM of the forward primer, 150nM-500nM of the reverse primer, and 125nM-300nM of the probe.

10. The method for full typing of influenza A virus according to claim 8, characterized in that: The PCR reaction program includes: 48°C-50°C for 1min-2min; 90°C-95°C for 30s-1min; the cycle steps are 90°C-95°C for 5s-10s, 60°C-65°C for 20s-30s, for a total of 40-45 cycles.

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