Primer pair, composition, kit and detection method for identifying H1 and H3 influenza A virus replication period active viruses

Abstract

The invention discloses a primer pair, a composition, a kit and a detection method for identifying H1 and H3 influenza A virus replication period active viruses, and belongs to the technical field of biology. The primer pair is a primer pair H1 and / or a primer pair H3, the primer pair H1 is composed of a primer H1-subgene-F and a primer H1-subgene-R, the primer pair H3 is composed of a primer H3-subgene-F and a primer H3-subgene-R, and the primer pair H3 is a single-stranded DNA molecule as shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.6 and SEQ ID No.7 in sequence. The primer composition provided by the invention can be used for detecting the seasonal live influenza A virus subjected to cell replication, the lowest detection sensitivity to H1N1 can reach 10 <-6 > copies / mu L, and the lowest detection sensitivity to H3N2 can reach 10 <-5 > copies / mu L.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer pair, a composition, a kit and a detection method for identifying influenza A H1 and H3 viruses in active replication phase. Background Art

[0002] Influenza is a respiratory infectious disease caused by influenza virus (IV) that can be transmitted between humans and a variety of animals (To J, Torres J.Viroporins in the influenza virus[J].Cells,2019,8(7):654.). IV belongs to the family of Orthomyxoviridae, and its genome is a single-stranded negative-stranded segmented RNA; according to the degree and scale of prevalence, influenza epidemics can be divided into pandemic influenza and seasonal influenza epidemics. According to the differences in nucleoprotein (NP) and matrix protein (M), they are divided into four categories: influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV) and influenza D virus (IDV) (Velislava NP, Colin A R.The evolution of seasonal influenza viruses[J].Nat Rev Microbiol,2018,16(1):47-60.).

[0003] Influenza A virus can cause influenza pandemics, and both influenza A virus and influenza B virus can cause seasonal influenza epidemics. Influenza A virus is a respiratory pathogen that seriously affects human health worldwide and has a significant impact on human health worldwide (Kirsty RS, Edwin JB, Ron AM, et al. Pathogenesis of influenza-induced acute respiratory distress syndrome [J]. Lancet Infect Dis, 2014, 14 (1): 57–69.).

[0004] During IAV replication, the gene encoding NS produces two mRNAs: mRNA 1 is an unspliced ​​transcript encoding NS1 protein. mRNA 2 is composed of NS mRNA alternative splicing, encoding NS2 protein (nuclear export protein, NEP) (Nongluk S, Yasuo S. Molecular basis of the structure and function of H1 hemagglutinin of influenza virus [J]. Proc Jpn Acad Ser B Phys Biol Sci, 2012, 88 (6): 226-49.). The gene encoding MP can also use the same alternative splicing mechanism as the NS gene to encode M1 and M2 proteins (Aartjan JW, Ervin F. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis [J]. Nat Rev Microbiol, 2016, 14 (8): 479-93.).

[0005] According to the latest report from WHO, the influenza A(H3N2) virus will be the main virus in 2022; in 2023, the virus will change from being dominated by influenza A(H3N2) to being dominated by H1N1 and co-circulating with H3N2 (https: / / app.powerbi.com / view?r=eyJrIjoiZTkyODcyOTEtZjA5YS00ZmI0LWFkZGUtODIxNGI5OTE3YjM0IiwidCI6ImY2MTBjMGI3LWJkMjQtNGIzOS04MTBiLTNkYzI4MGFmYjU5MCIsImMiOjh9).

[0006] Rapid and accurate diagnosis of seasonal influenza viruses is essential. PCR technology has been widely used in the diagnosis of pathogens, and PCR detection methods for influenza viruses have been studied extensively. However, the current influenza virus nucleic acid PCR detection method cannot distinguish whether it is a live virus or viral nucleic acid residues, and distinguishing between the two is the key to determining whether the virus is infectious. Summary of the invention

[0007] The technical problem to be solved by the present invention is to identify seasonal live influenza A virus. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The present invention provides a primer pair for identifying influenza A virus, wherein the primer pair is an H1 primer pair and / or an H3 primer pair, wherein the H1 primer pair consists of a primer H1-subgene-F and a primer H1-subgene-R, and the H3 primer pair consists of a primer H3-subgene-F and a primer H3-subgene-R;

[0010] A1) the primer H1-subgene-F is a single-stranded DNA molecule shown in SEQ ID No.3; the primer H1-subgene-R is a single-stranded DNA molecule shown in SEQ ID No.4;

[0011] A2) The primer H3-subgene-F is a single-stranded DNA molecule shown in SEQ ID No.6; the primer H3-subgene-R is a single-stranded DNA molecule shown in SEQ ID No.7.

[0012] The primers H1-subgene-F and H3-subgene-F are forward primers, and the primers H1-subgene-R and H3-subgene-R are reverse primers.

[0013] The H1 primer pair is a primer pair for specifically amplifying the NS gene of influenza virus A / Brisbane / 02 / 2018 (H1N1), i.e., used to detect H1N1, and the full sequence of the NS gene is shown in SEQ ID No. 1; the H3 primer pair is a primer pair for specifically amplifying the M gene of influenza virus A / Kansas / 14 / 2017 (H3N2), i.e., used to detect H3N2, and the full sequence of the M gene of A / Kansas / 14 / 2017 (H3N2) is shown in SEQ ID No. 2.

[0014] The present invention also provides a primer composition for identifying influenza A virus, wherein the composition is any one of the following:

[0015] B1) The composition comprises the H1 primer pair and the probe H1-subgene-P described in A1) above, wherein the nucleotide sequence of the probe H1-subgene-P is SEQ ID No. 5;

[0016] B2) The composition comprises the H3 primer pair and probe H3-subgene-P described in A2) above, and the nucleotide sequence of the probe H3-subgene-P is SEQ ID No.8.

[0017] Furthermore, the 5' end of the probe H1-subgene-P and / or the probe H3-subgene-P is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0018] The fluorescent group is selected from at least one of FAM, 6-FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 and Texas Red.

[0019] The quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1.

[0020] Furthermore, the 5' end of the probe H1-subgene-P and / or the probe H3-subgene-P is labeled with 6-FAM, and the 3' end is labeled with BHQ1.

[0021] The present invention also provides a kit for detecting influenza A virus, wherein the kit comprises the aforementioned primer pair.

[0022] Furthermore, the above-mentioned kit also contains the aforementioned probe H1-subgene-P and / or probe H3-subgene-P.

[0023] The fluorescent group is selected from at least one of FAM, 6-FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 and Texas Red.

[0024] The quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB and Dabcy1.

[0025] The present invention also provides a method for detecting influenza A virus, which comprises using the above-mentioned composition to perform a PCR amplification reaction, and determining whether the sample to be tested contains live influenza A virus according to the amplification result.

[0026] Furthermore, the PCR amplification reaction in the above method is a real-time fluorescence quantitative PCR reaction, and the method comprises the following steps:

[0027] A1) extracting RNA from the sample to be tested;

[0028] A2) reverse transcribing the RNA into cDNA, using the cDNA as a template, and performing real-time fluorescence quantitative PCR reaction detection with the aforementioned primer combination, and determining whether the sample to be tested contains live influenza A virus based on the amplification result.

[0029] If the product contains a DNA molecule whose nucleotide is SEQ ID No.12 or has a fluorescent signal, it indicates that the NS2 gene of A / Brisbane / 02 / 2018(H1N1) is present in the sample to be tested, that is, it indicates that A / Brisbane / 02 / 2018(H1N1) that can replicate is present. If the product does not contain a DNA molecule whose nucleotide is SEQ ID No.2 or has no fluorescent signal, it indicates that the NS2 gene of A / Brisbane / 02 / 2018(H1N1) is not present in the sample to be tested, that is, it indicates that A / Brisbane / 02 / 2018(H1N1) that can replicate is not present in the sample to be tested.

[0030] If the product contains a DNA molecule whose nucleotide is SEQ ID No.13 or has a fluorescent signal, it indicates that the M2 gene of A / Kansas / 14 / 2017(H3N2) exists in the sample to be tested, that is, it indicates that A / Kansas / 14 / 2017(H3N2) that can replicate exists. If the product does not contain a DNA molecule whose nucleotide is SEQ ID No.4 or has no fluorescent signal, it indicates that the M2 gene of A / Kansas / 14 / 2017(H3N2) does not exist in the sample to be tested, that is, it indicates that A / Kansas / 14 / 2017(H3N2) that can replicate does not exist in the sample to be tested.

[0031] The sample to be tested may be a sample obtained from a nasal swab, a throat swab, sputum or a nasal wash of a subject, or may be a blood or tissue sample.

[0032] The use of the aforementioned primer pair and / or composition in the preparation of a product for detecting live influenza A virus also falls within the scope of protection of the present invention.

[0033] The present invention is based on the fact that when influenza virus replicates in cells, the two separated segments of NS2 gene mRNA sequences in the NS gene (the nucleotide sequences are the 1st to 30th bases of SEQ ID No.1 and the 503rd to 838th bases of SEQ ID No.1, respectively) will be connected together; the two separated segments of M2 gene mRNA sequences in the M gene (the nucleotide sequences are the 1st to 26th bases of SEQ ID No.2 and the 715th to 982th bases of SEQ ID No.2, respectively) will be connected together, so the probe is designed at the connection of the two segments, and during qRT-PCR detection, only viruses active in the replication phase will be detected. The primer combination provided by the present invention has a typical amplification curve, with a good slope and CT value, and can detect live influenza A viruses that have undergone cell replication, and can effectively distinguish whether influenza viruses are infectious. At the same time, the detection method provided by the present invention is fast and accurate, sensitive and reliable, and simple to operate. The detection sensitivity for H1N1 can reach a minimum of 10 -6 copies / μL, the lowest sensitivity for H3N2 detection can reach 10 -5 copies / μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 2 is the qRT-PCR amplification curve for identifying influenza A virus H1N1 using genomic primers and subgenomic primers; 1 represents the amplification curve of three samples (sample 3, sample 4 and sample 5) of influenza A virus H1N1 using genomic primers; 2 represents the amplification curve of three samples (sample 3, sample 4 and sample 5) of influenza A virus H1N1 using subgenomic primers.

[0035] Figure 2 Figure 3 is the qRT-PCR amplification curve for identifying influenza A virus H3N2 using genomic primers and subgenomic primers; wherein 3 represents the amplification curve of three samples (sample 6, sample 7 and sample 8) of influenza A virus H3N2 using genomic primers; 4 represents the amplification curve of three samples (sample 6, sample 7 and sample 8) of influenza A virus H3N2 using subgenomic primers.

[0036] Figure 3 The supernatant of the lungs of mice infected with H1N1 virus was ground and diluted directly to extract RNA, and the CT-Value and virus copy number copies / μL were detected by qRT-PCR.

[0037] Figure 4 The supernatant of the lungs of mice infected with H3N2 virus was ground and diluted directly to extract RNA, and the CT-Value and virus copy number copies / μL were detected by qRT-PCR. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0040] The experiments were conducted in the BSL-2 laboratory of the Institute of Microbiology, Chinese Academy of Sciences.

[0041] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0042] The following examples used GraphPad Prism 8 statistical software to process the data, and the test results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference.

[0043] Example 1. Primer probe design

[0044] The full sequence of the NS gene of A / Brisbane / 02 / 2018 (H1N1) is shown in SEQ ID No. 1. The mRNA1 produced by the NS gene encodes the NS1 protein, and the nucleotide sequence of the gene encoding the NS1 protein, mRNA1 (660 bp), is shown in the bases 1-660 bp (bases indicated by lowercase letters) of SEQ ID No. 1. The mRNA2 produced by the NS gene encodes the NS2 protein, and the nucleotide sequence of the gene encoding the NS2 protein, mRNA2 (366 bp), consists of the bases 1-30 and 503-838 in SEQ ID No. 1 (bases indicated by underline), i.e., SEQ ID No. 12. In SEQ ID No. 1, bases 1-18 are the sequence of primer H1-subgene-F (bold lowercase letters), the reverse complementary sequence of bases 600-617 is the sequence of primer H1-subgene-R (bold lowercase letters), and the nucleotide sequence composed of bases 19-30 and 503-514 is the sequence of probe H1-subgene-P (italic lowercase letters).

[0045] SEQ ID No.1(838bp)

[0046]

[0047] SEQ ID No.12 (366 bp)

[0048]

[0049] The full sequence of the M gene of A / Kansas / 14 / 2017 (H3N2) is shown in SEQ ID No. 2. The mRNA1 produced by the M gene encodes the M1 protein, and the nucleotide sequence of the gene encoding the M1 protein (759 bp) is shown in bases 1-759 of SEQ ID No. 2 (bases indicated by lowercase letters). The mRNA2 produced by the NS gene encodes the NS2 protein, and the nucleotide sequence of the gene encoding the NS2 protein (294 bp) is shown in bases 1-26 and 715-982 of SEQ ID No. 2 (bases indicated by underlined letters), i.e., SEQ ID No. 13. In SEQ ID No. 2, bases 1-18 are the sequence of primer H3-subgene-F (bold lowercase letters), the reverse complementary sequence of bases 770-788 is the sequence of primer H3-subgene-R (bold uppercase letters), and the nucleotide sequence composed of bases 15-26 and 715-723 is the sequence of probe H3-subgene-P (italic lowercase letters).

[0050] SEQ ID No.2 (982bp)

[0051]

[0052] SEQ ID No.13 (294 bp)

[0053]

[0054] The primers designed according to the above sequences are shown in Table 1. Among them, H1-subgene-F, H1-subgene-R and H1-subgene-P are subgenomic primer pairs for detecting H1N1 viruses, H3-subgene-F, H3-subgene-R and H3-subgene-P are subgenomic primer pairs for detecting H3N2 viruses, and IFA-genome-F, IFA-genome-R and IFA-genome-P are genomic primer pairs that can be used to detect both H1N1 and H3N2 viruses.

[0055] Table 1 Primers and probes for H1N1 and H3N2

[0056]

[0057]

[0058] Among them, 6-FAM modification was added to the 5-end of H1-subgene-P, H3-subgene-P and IFA-genome-P, and BHQ1 modification was added to the 3-end. I is represented by N in the sequence table, N represents hypoxanthine nucleotide, R, Y and W are annexed bases, R represents a or g, Y represents c or t (or u), and W represents a or t (or u).

[0059] Example 2: qRT-PCR verification of the amplification efficiency of the primer-probe combination

[0060] Influenza virus A / Brisbane / 02 / 2018 (H1N1): recorded in the document "Li YL, Liu PP, Hao TJ, et al. Rational design of an influenza-COVID-19 chimeric protective vaccine with HA-stalk and S-RBD [J]. Emerg Microbes Infect, 2023 Dec; 12 (2): 2231573", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0061] Influenza virus A / Kansas / 14 / 2017 (H3N2): Derived from the Chinese Center for Disease Control and Prevention, recorded in the document "Ge J, Lin XJ, Guo JL, et al. The Antibody Response Against Neuraminidase in Human Influenza A (H3N2) Virus Infections During 2018 / 2019 Flu Season: Focusing on the Epitopes of 329-N-Glycosylation and E344 in N2. Front Microbiol. 2022 Mar 21; 13: 845088.", the public can obtain it from the Chinese Center for Disease Control and Prevention. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0062] 1. Extract viral RNA using MagaBio plus viral RNA purification kit

[0063] 1. Place the 96-well reagent plate at room temperature and turn it upside down 3 times. After removing the plastic film, centrifuge it briefly in a 96-well plate centrifuge (or shake it by hand) to avoid liquid hanging. Tear off the aluminum foil on the 96-well reagent plate and confirm the direction of the plate (the magnetic beads are in the 6th / 12th column).

[0064] 2. Sample processing methods from the same source:

[0065] Place each sample into columns 1 and 7 of a 96-well reagent plate to avoid cross contamination.

[0066] The specific information and sources of each sample are as follows:

[0067] Sample 1: Allantoic fluid harvested from chicken embryos after H1N1 influenza virus was propagated in chicken embryos (but not through cell propagation).

[0068] Sample 2: Allantoic fluid harvested from chicken embryos after H3N2 influenza virus was propagated in chicken embryos (but not through cell propagation).

[0069] Sample 3, Sample 4 and Sample 5: Three mice were infected with H1N1 influenza virus intranasally, and the lungs were collected after being killed on the fifth day post-infection (5 days post-infection, 5 dpi). Sample 3, Sample 4 and Sample 5 were repeated three times.

[0070] Sample 6, Sample 7 and Sample 8: Three mice infected with H3N2 influenza virus intranasally, lungs collected after being killed at 5 dpi. Sample 6, Sample 7 and Sample 8 were repeated three times.

[0071] 3. Place the 96-well reagent plate into the NPA-32+ nucleic acid extraction and purification instrument and install the magnetic rod sheath.

[0072] 4. Carry out the automated extraction experiment according to the program table shown in Table 2:

[0073] Table 2 Procedure for automated extraction of viral RNA

[0074]

[0075] Heating settings: Elution temperature: 70°C, elution start heating step: 7.

[0076] 5. After the automated program is completed, transfer the eluate from columns 5 and 11 to a clean nuclease-free centrifuge tube or transfer the eluate to a new nuclease-free centrifuge tube. The resulting eluate is the RNA of each sample and is stored at -80°C.

[0077] 2. qRT-PCR detection of influenza-related samples

[0078] (1) Using HiScript U +One Step qRT-PCR Probe Kit (Vazyme, Q223) kit for reaction system configuration

[0079] Table 3 qRT-PCR reaction system

[0080] Reagents Dosage <![CDATA[RNase Free dH2O]]> 6μL <![CDATA[2×One Step U + Mix]]> 10μL <![CDATA[One Step U + Enzyme Mix]]> 1μL 50×ROX Reference Dye 2 0.4μL Upstream primer F (20 μM) 0.2μL Downstream primer R (20 μM) 0.2μL Probe P (20 μM) 0.2μL RNA from samples 2μL Total 20μL

[0081] Table 4 qRT-PCR reaction conditions

[0082]

[0083] 3. Test results

[0084] 1. Comparison of CT value results of each primer combination

[0085] Table 5 Comparison of CT values ​​of two groups of compositions of H1N1 virus

[0086] serial number Genomic primers and probes to detect CT values Subgenomic primers and probes to detect CT values Sample 1 13.72 U Sample 3 13.86 19.68 Sample 4 13.85 18.58 Sample 5 13.63 19.39

[0087] Table 6 Comparison of CT values ​​of two groups of compositions of H3N2 virus

[0088] serial number Genomic primers and probes to detect CT values Subgenomic primers and probes to detect CT values Sample 2 18.13 U Sample 6 18.86 23.54 Sample 7 18.11 22.70 Sample 8 18.18 23.24

[0089] Note: U stands for Undetermined, which means the qRT-PCR test is negative.

[0090] Samples 1 and 2 are the results of chicken embryo allantoic fluid harvested after influenza virus was propagated with chicken embryos (but not cell propagated), RNA was extracted, and qRT-PCR was performed. Samples 3, 4, and 5 are the results of qRT-PCR testing of 3 mice infected with H1N1 influenza virus by intranasal drip, lungs were taken after being killed at 5 dpi, and viral RNA was extracted; Samples 6, 7, and 8 are the results of qRT-PCR testing of 3 mice infected with H3N2 influenza virus by intranasal drip, lungs were taken after being killed at 5 dpi, and viral RNA was extracted.

[0091] It can be seen from Tables 5 and 6 that the subgenomic primers cannot detect the viruses in sample 1 and sample 2, but can detect the viruses in sample 3 to sample 8. Because when the influenza virus replicates in the cell, the two separate NS2 gene DNA sequences in the NS gene will be connected together, and the two separate M2 gene DNA sequences in the M gene will be connected together. Therefore, the probe designed at the junction of the two segments (H1-subgene-P or H3-subgene-P) will only detect active viruses in the replication period during qRT-PCR detection. It can be seen that there are no active viruses in the replication period among the viruses harvested from the amniotic fluid, and it also shows that the primer probe provided by the present invention can detect active viruses that have undergone cell replication, so as to distinguish whether the virus is infectious.

[0092] 2. Amplification curves of each primer combination

[0093] qRT-PCR amplification curves for identifying the genomic primer combination and subgenomic primer combination of influenza A virus H1N1 Figure 1 As shown. 1 represents the amplification curve of three samples (sample 3, sample 4 and sample 5) of H1N1 identified by the genomic primer combination; 2 represents the amplification curve of three samples (sample 3, sample 4 and sample 5) of H1N1 identified by the subgenomic primer combination. The qRT-PCR amplification curves of the genomic primer combination and the subgenomic primer combination for identifying influenza A virus H3N2 are shown in Figure 2 As shown. 3 represents the amplification curves of three samples (sample 6, sample 7 and sample 8) of H3N2 identified by the genomic primer combination; 4 represents the amplification curves of three samples (sample 6, sample 7 and sample 8) of H3N2 identified by the subgenomic primer combination.

[0094] Depend on Figure 1 and Figure 2 From the qRT-PCR amplification curve results, it can be seen that although the sensitivity of using subgenomic primers and probes is relatively weak, it can be shown that the use of subgenomic primers and probes can detect live viruses, and the amplification curve is better than the amplification curve using genomic primers and probes, with a better slope and CT value.

[0095] Example 3: Method for detecting live influenza A virus, verification of the effect of primer-probe combination

[0096] 1. Test methods

[0097] Influenza virus A / Brisbane / 02 / 2018 (H1N1): recorded in the document "Li YL, Liu PP, Hao TJ, et al. Rational design of an influenza-COVID-19 chimeric protective vaccine with HA-stalk and S-RBD [J]. Emerg Microbes Infect, 2023 Dec; 12 (2): 2231573", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0098] Influenza virus A / Kansas / 14 / 2017 (H3N2): Derived from the Chinese Center for Disease Control and Prevention, recorded in the document "Ge J, Lin XJ, Guo JL, et al. The Antibody Response Against Neuraminidase in Human Influenza A (H3N2) Virus Infections During 2018 / 2019 Flu Season: Focusing on the Epitopes of 329-N-Glycosylation and E344 in N2. Front Microbiol. 2022 Mar 21; 13: 845088.", the public can obtain it from the Chinese Center for Disease Control and Prevention. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0099] 1. Preparation of original poison

[0100] Influenza virus A / Brisbane / 02 / 2018 (H1N1) and influenza virus A / Kansas / 14 / 2017 (H3N2) were inoculated into chicken embryos and the allantoic fluid was harvested as follows:

[0101] (1) Egg test

[0102] ① Use an egg-illuminating lamp to detect the chicken embryo, mark the boundary between the air chamber and the allantois, and the position of the embryo.

[0103] ② If the chicken embryo is dead, unfertilized, cracked, underdeveloped, or has many water seepage holes on the surface, it should be discarded.

[0104] ③How to judge the status of chicken embryo:

[0105] 1) Blood vessels: The blood vessels of living embryos are clear, while those of dead embryos are blurred, forming congestion bands or blood clots.

[0106] 2) Fetal movement: A live embryo has obvious natural movement, while a dead embryo has no fetal movement.

[0107] 3) Developmental boundary of the chorioallantoic membrane: The chorioallantoic membrane, which is densely covered with blood vessels, forms a clear boundary with the other side of the chicken embryo.

[0108] (2) Chicken embryo inoculation

[0109] ① Place the embryos on the egg tray with the air chamber facing up and mark each embryo.

[0110] ② Disinfect the chicken embryo with 75% alcohol, drill a hole at the end of the air chamber, and make a 1×1mm hole. Drill a 1×1mm hole about 5mm to 15mm below the air chamber in a place without blood vessels or with few blood vessels.

[0111] ③ Use a 1 mL syringe to draw 100 μL of influenza virus A / Brisbane / 02 / 2018 (H1N1) or influenza virus A / Kansas / 14 / 2017 (H3N2) and attach the needle.

[0112] ④ Inject 100 μL of sample into the allantoic cavity of the chicken embryo from the hole under the air chamber.

[0113] ⑤ Use a syringe to repeatedly draw up 84 disinfectant 2-3 times, then place the needle in a sharps box, sterilize it at 121℃, and then hand the sharps box over to a company that specializes in handling medical waste. Discard the syringe barrel in a high-pressure bag, sterilize it at 121℃, and then discard it.

[0114] ⑥Seal with wax.

[0115] ⑦ Cultivate the chicken embryos in a 37℃ incubator (mammalian influenza virus is cultured at 35℃) for 2-3 days. Starting from 24 hours, check the growth of the chicken embryos every 12 hours. Chicken embryos that die within 24 hours are considered to be non-specific deaths and should be discarded.

[0116] (3) Harvesting of chicken embryo allantoic fluid (harvesting virus)

[0117] Use a 2mL or 5mL syringe to draw the chicken embryo allantoic fluid and place it in the corresponding collection tube. Centrifuge at 3000rpm for 5min to remove blood and cells to obtain A / Brisbane / 02 / 2018 (H1N1) and A / Kansas / 14 / 2017 (H3N2) original viruses. Subsequent tests can be carried out immediately or frozen in a -80℃ refrigerator for later use.

[0118] 2. Preparation of mouse lung grinding supernatant

[0119] ① Influenza virus (H1N1 and H3N2) infected mice

[0120] A1. First, SPF-grade C57BL / 6J experimental mice were anesthetized with isoflurane inhalation;

[0121] A2. Grasp the anesthetized mouse and turn its head upward. Use a 200 μL pipette to drip the virus stock solution (A / Brisbane / 02 / 2018 (H1N1) original virus or A / Kansas / 14 / 2017 (H3N2) original virus) slowly into the nasal cavity along the side wall of one side. Drip 50 μL into the nose without blocking the nasal cavity.

[0122] A3. After nasal drops, the mouse head should be placed upward for 30 to 60 seconds to ensure that the virus does not flow out.

[0123] ② Killing and sampling of mice

[0124] Five days after infection, the H1N1-infected mice and H3N2-infected mice were anesthetized and their eyeballs were removed to collect blood. The mice were then killed by dissection. The first set of scissors and tweezers were used to cut the mouse fur until the mouth, and attention was paid to blunt separation. After completion, the scissors and tweezers were placed in a disinfection box for soaking, or a small incision was made in the middle of the mouse abdomen, and then both hands were used to tear open the mouse skin. The second set of scissors and tweezers were used to cut the mouse abdominal cavity and chest cavity, and the field of vision was as clear as possible. It was advisable not to touch the mouse fur during the process. After completion, the scissors and tweezers were placed in a disinfection box for soaking. The third set of scissors and tweezers was used to peel off the mouse trachea from the larynx, cut it off from the top of the larynx and clamp it with tweezers. The scissors were used to bluntly separate the entire lung (with the heart) from the chest cavity along the trachea, and the heart was continued to be peeled off in a flat dish. The esophagus was removed, the entire lung was weighed, and it was placed in a tissue grinding tube. After grinding (4000 rpm, working 10 s, interval 10 s) three times, centrifuged at 7000 rpm for 10 min, and the supernatant was collected to obtain A / Brisbane / 02 / 2018 (H1N1) lung grinding supernatant and A / Kansas / 14 / 2017 (H3N2) lung grinding supernatant.

[0125] 3. Lung grinding supernatant inoculated into chicken embryos

[0126] According to the method shown in 1, the A / Brisbane / 02 / 2018 (H1N1) lung supernatant and A / Kansas / 14 / 2017 (H3N2) lung supernatant were inoculated into chicken embryos, and the allantoic fluid was collected to obtain A / Brisbane / 02 / 2018 (H1N1) allantoic fluid and A / Kansas / 14 / 2017 (H3N2) allantoic fluid.

[0127] 4. Determination of HA titer in chicken embryo allantoic fluid

[0128] A. Prepare 1% chicken red blood cell suspension or 2% guinea pig red blood cell suspension.

[0129] A1. Mix the red blood cells (chicken red blood cells or guinea pig red blood cells) stored in Alsever's solution, and use a graduated pipette to aspirate an appropriate amount of red blood cell suspension and transfer it to a centrifuge tube. The volume of each tube should not exceed 1 / 3 of the total volume. Centrifuge at 1500rpm for 5 minutes and discard the supernatant.

[0130] A2. Add at least twice the volume of sterile saline or PBS and wash three times, then mix well.

[0131] A3. Centrifuge at 1500rpm for 5 minutes for the first two times and discard the supernatant. Centrifuge at 1500rpm for 10 minutes for the last time and discard the supernatant. The deposited red blood cells are concentrated chicken red blood cells or concentrated guinea pig red blood cells.

[0132] A4. Aspirate chicken red blood cells at 1 / 100 of the final volume of the required solution or aspirate guinea pig red blood cells at 2 / 100 of the final volume of the solution, and repeatedly pipette with sterile saline or PBS for more than 10 times to prepare 1% chicken red blood cell suspension and 2% guinea pig red blood cell suspension. Label them and place them at 4℃ for use.

[0133] A5.1% chicken red blood cell suspension and 2% guinea pig red blood cell suspension can be stored at 4℃ for about 7 days. If hemolysis occurs, it should be discarded and re-prepared. It is best to prepare fresh and prepare it before use.

[0134] B. Potency Determination

[0135] B1. Use a pipette tip with a filter to pipette 25 μL of the allantoic fluid collected in step 3 into the first column (rows AH) of a V-shaped microtiter plate.

[0136] B2. Use a 12-channel adjustable pipette to pipette 25 μL of PBS into all wells.

[0137] B3.8 pipette equipped with 200μL filter tip, pipette 5-10 times to mix column 1. Pipette 25μL of mixed solution from each well of column 1 to column 2, pipette 10 times to mix column 2, dilute to column 11 in sequence, and discard 25μL of liquid from column 11.

[0138] B4. Add 25 μL of the 1% red blood cell suspension or 2% guinea pig red blood cell suspension prepared in step A to each well and flick the V-shaped plate to fully mix the red blood cells and the virus.

[0139] B5. Incubate at room temperature for 20 to 40 minutes, observe the red blood cell agglutination phenomenon and record the results.

[0140] B6. Result determination: The determination of red blood cell agglutination titer takes the highest dilution at which complete agglutination occurs as the end point, and the reciprocal of the dilution is the red blood cell agglutination titer of the virus. Complete agglutination of red blood cells is recorded as "+"; no agglutination or partial agglutination is recorded as "-".

[0141] 5. RNA extraction and qRT-PCR detection

[0142] The A / Brisbane / 02 / 2018 (H1N1) lung grinding supernatant and A / Kansas / 14 / 2017 (H3N2) lung grinding supernatant collected in step 2, and the A / Brisbane / 02 / 2018 (H1N1) allantoic fluid and A / Kansas / 14 / 2017 (H3N2) allantoic fluid collected in step 3 were used as samples, and RNA was extracted and qRT-PCR was performed according to the method in Example 1.

[0143] 2. Test results

[0144] 1. qRT-PCR detection of viral load after gradient dilution of the supernatant of lungs of mice infected with H1N1

[0145] Table 7 HA titer and qRT-PCR results of chicken embryo allantoic fluid harvested from chicken embryos inoculated with H1N1 lung grind supernatant

[0146]

[0147] Note: "+" represents positive hemagglutination titer, "-" represents negative hemagglutination titer. U represents negative result of qRT-PCR test.

[0148] A / Brisbane / 02 / 2018(H1N1) infected mice were killed to obtain the ground lung supernatant. The qRT-PCR test results after gradient dilution were as follows: Figure 3 As shown, the left picture is CT-Value, and the right picture is the number of viral copies / μL. Figure 3 It can be seen from the CT value results that the use of genomic primers and probes in 10 -8 The H1N1 virus can still be detected in the dilution gradient, but only 10 -6 Dilution gradient. The trend of viral copy number results is the same as that of CT value.

[0149] After the lung grinding supernatant was diluted in gradients, the chicken embryo allantoic fluid was harvested and the HA titer was detected. The viral RNA of the chicken embryo allantoic fluid was harvested after the lung grinding supernatant was diluted in gradients and inoculated into the chicken embryo, and the genomic primers and probes were used for qRT-PCR detection. The results are shown in Table 7. Wherein "+" is a positive HA result, "-" is a negative HA result, and U is Undetermined, i.e., the qRT-PCR test is negative. The test was repeated three times. The chicken embryo allantoic fluid with a negative hemagglutination titer but a CT value continued to be inoculated into the chicken embryo until the test results after three times.

[0150] From the results of HA titer and qRT-PCR test after RNA extraction, it can be seen that-6 The dilution gradient test was positive, 10 -7 Only one chicken embryo was positive in the dilution gradient, 10 -8 All three chick embryos were negative. The results obtained after inoculation of chick embryos were consistent with the results of qRT-PCR detection by extracting RNA directly from the supernatant of H1N1 mouse lungs after gradient dilution.

[0151] Therefore, the calibration is ≤10 -6 The CT value and virus copy number detected by the dilution gradient are the positive standard, 10 -6 to 10 -7 The CT value and virus copy number detected by the dilution gradient are suspected. If this happens, repeat it once. If it is still suspected, it will be judged as positive, and if it is negative, it will be judged as negative. ≥10 -8 The CT value and viral copy number detected by the dilution gradient of 10 μg / mL were negative standards. The results corresponded to the CT-Value and viral copy number detected using subgenomic primers.

[0152] 2. qRT-PCR detection of viral load after gradient dilution of the supernatant of lungs of mice infected with H3N2

[0153] Table 8 HA titer and qRT-PCR results of chicken embryo allantoic fluid harvested from chicken embryos inoculated with H3N2 lung grinding supernatant

[0154]

[0155] Note: "+" represents positive hemagglutination titer, "-" represents negative hemagglutination titer. U represents negative result of qRT-PCR test.

[0156] Mice infected with A / Kansas / 14 / 2017(H3N2) were killed to obtain the supernatant of the mouse lungs. The qRT-PCR test results after gradient dilution were as follows: Figure 4 As shown in the left figure, the left figure is CT-Value, and the right figure is the number of viral copies / μL. Figure 4 It can be seen from the CT value results that the use of genomic primers and probes in 10 -8 It can still be detected in the dilution gradient, but only 10 -5 The trend of the viral copy number results was the same as that of the CT value.

[0157] After the lung grinding supernatant was diluted in gradient, the chicken embryo was harvested and the chicken embryo allantoic fluid was inoculated to detect the HA titer, and the viral RNA of the chicken embryo allantoic fluid was extracted and inoculated in the chicken embryo after gradient dilution of the lung grinding supernatant, and the genomic primers and probes were used for qRT-PCR detection, and the results are shown in Table 8. Wherein "+" is a positive HA result, "-" is a negative HA result, and U is Undetermined, that is, the qRT-PCR test is negative. The test was repeated three times. The chicken embryo allantoic fluid with a negative hemagglutination titer but a CT value continued to be inoculated into the chicken embryo until the test result after three times.

[0158] From the results of HA titer and qRT-PCR test after RNA extraction, it can be seen that -3 The dilution gradient test was positive, 10 -4 Only two chicken embryos were detected positive in the dilution gradient, 10 -5 Only one chicken embryo was positive in the dilution gradient, 10 -6 All three chicken embryos were negative. The results obtained after inoculation of chicken embryos were consistent with the results of qRT-PCR detection by extracting RNA directly after gradient dilution of H3N2 mouse lung supernatant.

[0159] Therefore, the calibration is ≤10 -3 The CT value and virus copy number detected by the dilution gradient are the positive standard, 10 -4 to 10 -5 The CT value and virus copy number detected by the dilution gradient are suspected. If this happens, repeat it once. If it is still suspected, it will be judged as positive, and if it is negative, it will be judged as negative. ≥10 -6 The CT value and viral copy number detected by the dilution gradient of 10 μg / mL were negative standards. The results corresponded to the CT-Value and viral copy number detected using subgenomic primers.

[0160] In summary, the primer probe provided by the present invention can detect the replicating influenza viruses H1N1 and H3N2, distinguish whether the viruses are infectious, provide a certain basis for the clinical accurate detection of virus samples, and also lay a foundation for the future research on live viruses, and can well monitor the proliferation of viruses in cells.

[0161] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A primer pair for identifying influenza A H1 and H3 viruses in the active replication phase, characterized in that: The primer pair is an H1 primer pair and / or an H3 primer pair, wherein the H1 primer pair consists of a primer H1-subgene-F and a primer H1-subgene-R, and the H3 primer pair consists of a primer H3-subgene-F and a primer H3-subgene-R; A1) the primer H1-subgene-F is a single-stranded DNA molecule shown in SEQ ID No.3; the primer H1-subgene-R is a single-stranded DNA molecule shown in SEQ ID No.4; A2) The primer H3-subgene-F is a single-stranded DNA molecule shown in SEQ ID No.6; the primer H3-subgene-R is a single-stranded DNA molecule shown in SEQ ID No.

7.

2. A primer composition for identifying influenza A virus, characterized in that: The composition is any of the following: B1) The composition comprises the H1 primer pair and the probe H1-subgene-P in A1) of claim 1, wherein the nucleotide sequence of the probe H1-subgene-P is SEQ ID No. 5; B2) The composition comprises the H3 primer pair and the probe H3-subgene-P described in A2) of claim 1, wherein the nucleotide sequence of the probe H3-subgene-P is SEQ ID No.

8.

3. The composition according to claim 2, characterized in that The 5' end of the probe H1-subgene-P and / or the probe H3-subgene-P is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

4. The composition according to claim 3, characterized in that The 5' end of the probe H1-subgene-P and / or the probe H3-subgene-P is labeled with 6-FAM, and the 3' end is labeled with BHQ1.

5. A kit for detecting influenza A H1 and H3 viruses in the active replication phase, characterized in that: The kit comprises the primer pair according to claim 1.

6. The kit according to claim 5, characterized in that The kit further comprises the probe H1-subgene-P and / or the probe H3-subgene-P as described in any one of claims 2 to 4.

7. A method for detecting influenza A H1 and H3 viruses in the active replication phase, characterized in that: The method comprises using the composition described in any one of claims 2 to 4 to perform a PCR amplification reaction, and determining whether the sample to be tested contains live influenza A virus according to the amplification result.

8. The method according to claim 7, characterized in that The PCR amplification reaction is a real-time fluorescence quantitative PCR reaction, and the method comprises the following steps: A1) extracting RNA from the sample to be tested; A2) reverse transcribing the RNA into cDNA, using the cDNA as a template, performing real-time fluorescence quantitative PCR reaction detection with the composition of any one of claims 2 to 4, and determining whether the sample to be tested contains live influenza A virus according to the qRT-PCR result.

9. Use of the primer pair according to claim 1 and / or the composition according to any one of claims 2 to 4 in the preparation of a product for detecting influenza A H1 and H3 viruses in the active replication phase.

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