Method for detecting seasonal influenza b virus live virus, primer probe and kit

Abstract

The application discloses a detection method, a primer probe and a kit for seasonal influenza B virus live virus. The application specifically discloses primer and probe sequences for detecting influenza B virus live virus. The primer, the probe, the composition, the kit and the detection method for detecting influenza B virus of the application can detect influenza B virus live virus replicated in cells, including Victoria and Yamagata, can effectively distinguish influenza virus live virus and inactivated virus, and has the characteristics of rapidness, accuracy, sensitivity, reliability and simple operation, good amplification curve, better slope and CT value, and the lowest detection sensitivity can reach 10 ‑6 copies / μL. The detection method of the application can better observe the virus multiplication in cells, and has great significance for early detection and early treatment of virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for detecting live seasonal influenza B virus, primers, probes, and reagent kits. Background Technology

[0002] Influenza, caused by the influenza virus (IV), is a zoonotic disease that seriously endangers human and animal health. Mammals susceptible to influenza virus include pigs, horses, dogs, raccoons, mink, ferrets, whales, etc. The main site of lesions is the respiratory system, and the clinical manifestations are mainly respiratory symptoms (To J, Torres J. Viroporins in the influenza virus[J]. Cells, 2019, 8(7)). Influenza IV is a highly infectious virus belonging to the Orthomyxoviridae family. Its genome is a single-stranded, negative-sense, segmented RNA. Currently, based on differences in their nucleoprotein (NP) and matrix protein (M), it is classified into four types: Influenza A virus (IAV), Influenza B virus (IBV), Influenza C virus (ICV), and Influenza D virus (IDV) (To J, Torres J. Viroporins in the influenza virus[J]. Cells, 2019, 8(7)). Among the four types of influenza viruses, IAV has the widest host range and is most prone to mutation. Infected hosts include birds, mammals (domestic and wild), and humans (Bi Y, Li J, Li S, et al. Dominant subtype switch in avian influenza viruses during 2016-2019 in China[J]. Nat Commun, 2020, 11(1): 5909.). Based on the antigenic characteristics of hemagglutinin (HA) and neuraminidase (NA), IAV is further divided into different subtypes (H1-H18, N1-N11), most of which are found in waterfowl, while subtypes H17, H18, and N10, N11 are found in bats (Tong S, Zhu X, Li Y, et al. New world batsharbor diverse influenza A viruses[J]. Plos Pathog, 2013, 9(10): E1003657.).In 1940, Velislava N Petrova first isolated IBV from a sick child and named it B / Lee / 40 influenza (Petrova V N, Russell CA. The evolution of seasonal influenza viruses[J].Nat RevMicrobiol,2018,16(1):47-60.). The virus continues to mutate, and based on differences in the HA gene, it is divided into two different lineages: the B / Victoria / 2 / 87 lineage and the B / Yamagata / 16 / 88 lineage (Petrova VN, Russell CA. The evolution of seasonal influenza viruses[J].Nat RevMicrobiol,2018,16(1):47-60.). These two lineages were divided between 1987 and 1989 and spread globally in the 1990s, with the Victoria lineage being dominant in the population (Horthongkham N, Athipanyasilp N, Pattama A, et al. Epidemiological, clinical and virological characteristics of influenza B virus from patients at the hospital Tertiary Care Units in Bangkok during 2011-2014[J]. Plos One, 2016, 11(7): E0158244.).

[0003] Currently, H1N1 and H3N2 subtypes of influenza A, as well as IBV, are mainly circulating in the human population, while the H2N2 virus gradually disappeared from the population after the pandemic. Other subtypes of IAV (H5, H7, H9, etc.) have also caused human infections, but due to their limited transmissibility, they have not caused global outbreaks or epidemics (Kawaoka Y, Neumann G. Influenza viruses: an introduction. [M]. Methods And Protocols. Methods Mol Biol. 2012; 865: 1-9.). Prior to 2017, seasonal influenza vaccines did not contain B / Yamagata strains, which was one of the reasons for the B / Yamagata outbreak in 2017 (Suntronwong N, Klinfueng S, Korkong S, et al. Characterizing genetic and antigenic divergence from vaccine strain of influenza B and D viruses circulating in Thailand, 2017-2020[J]. Sci Rep, 2021, 11(1):735.). According to the latest WHO report, influenza A virus was prevalent in Beijing in 2016-2017, while the H3N2 subtype was the main type in 2019-2020. Since December 2020, the B / Victoria lineage virus has been the main type circulating (https: / / apps.who.int / flumart / Default?ReportNo=1), causing local outbreaks of influenza and endangering public health.

[0004] Each gene segment of the influenza virus encodes a different protein, determining the genetic characteristics of the influenza virus. The genomes of IAV and IBV are divided into eight segments (e.g., Figure 1), while the genomes of ICV and IDV are divided into 7 segments (Cardenas-Garcia S, Caceres CJ, Rajao D, et al. Reverse genetics for influenza B viruses and recent advances in vaccine development[J]. Current Opinion In Virology,2020,44:191-202.). In IAV, gene segments 1-6 encode basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA), nucleoprotein (NP), and neuraminidase (NA), respectively; gene segment 7 encodes membrane proteins, which, due to splicing, can arbitrarily encode two proteins, M1 and M2, of the inner membrane matrix; segment 8 encodes the non-structural protein NS, which, through RNA splicing, can be further divided into two proteins, NS1 and NS2 (also known as NEP) (Li X, Liu B, Ma S, et al. High frequency of reassortment after co-infection of chickens with the H4N6 and H9N2 influenza A viruses and the biological characteristics of thereassortants[J].Vet Microbiol,2018,222:11-7.). In recent years, other proteins encoded by the IAV gene have been discovered, such as the accessory protein PB1-F2, which is translated from another open reading frame of PB1. Subsequently, six new proteins were discovered: PB1-N40, PA-X, PA-N155, PA-N182, M42, and NS3. The corresponding proteins have not yet been found in IBV (Vasin AV, Temkina OA, Egorov VV, et al. Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins[J]. Virus Res, 2014, 185: 53-63.).

[0005] Unlike IAV, IBV's sixth gene segment encodes not only the NA protein but also an NB ion channel protein (NB). The seventh gene segment encodes a BM2 ion channel protein (BM2), whose function is similar to that of the M2 protein in IAV, primarily involved in regulating the uncoating process of viral particles and promoting the release of mature viral particles. Influenza virus particles are known to exhibit many shapes, the most common being roughly spherical. The viral envelope consists of a lipid bilayer containing three viral transmembrane proteins: HA, NA, and M2 (e.g., NB, NA, NB ... Figure 1This lipid bilayer originates from the host's plasma membrane and is rich in cholesterol-rich lipid rafts and non-raft lipids (Scheiffele P, Rietveld A, Wilk T, et al. Influenza viruses select ordered lipid domains during budding from the plasma membrane[J]. J Biol Chem, 1999, 274(4): 2038-44.). HA is the most abundant envelope protein, accounting for about 80%, followed by NA, which accounts for about 17% of the viral envelope proteins; M2 is a very small component of the envelope, with only 16 to 20 molecules per virion (Nayak DP, Balogun RA, Yamada H, et al. Influenza virus morphogenesis and budding[J]. Virus Res, 2009, 143(2): 147-61.). Located below the viral lipid membrane is M1, which forms a matrix that accommodates vRNPs. These vRNPs are the core of the virus, composed of viral negative-strand RNAs. One end of each vRNP is connected to three polymerase proteins (PB1, PB2, and PA) that constitute the viral RNA polymerase complex (Nayak DP, Balogun RA, Yamada H, et al. Influenza virus morphogenesis and budding[J]. VirusRes, 2009, 143(2): 147-61.). Viral infection begins with the binding of viral particles to cell surface receptors containing sialic acid, followed by endocytosis. After fusion of the virus with the endosomal membrane, vRNPs are released into the cytoplasm and then transported to the nucleus. In the nucleus, viral RNA polymerase transcribes the vRNA fragments into mRNA (Cardenas-Garcia S, Caceres CJ, Rajao D, et al. Reverse genetics for influenza B viruses and recent advances in vaccine development[J]. Current Opinion In Virology, 2020, 44: 191-202.). Viral mRNA is translated via cellular mechanisms. Newly synthesized viral polymerases and nucleoproteins are introduced into the cell nucleus and bind to cRNA and vRNA, assembling vRNPs and cRNPs, respectively. After nuclear export, progeny vRNPs are transported to the cell membrane via the cytoplasm on the endosome, where the assembly of progeny viral particles takes place.Mature viral particles contain a large number of host proteins and are released through budding (Samji T. Influenza A: understanding the viral life cycle[J]. Yale J Biol Med, 2009, 82(4): 153-9.). Therefore, the nucleus exit of influenza virus vRNPs is of great significance to the viral replication cycle. Previously, it was thought that NS2 only existed in infected cells, until it was proven in 1991 that it existed in viral particles. Therefore, NS2 is not a non-structural protein in the strict sense. The gene encoding NS produces two mRNAs: mRNA 1 is an unspliced ​​transcript that encodes the NS1 protein. mRNA 2 is spliced ​​and encodes the NS2 protein, also known as the nuclear export protein (NEP), which is translated from the spliced ​​mRNA transcript (Richardson JC, Akkina R K. NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells[J]. Arch Virol, 1991, 116(1-4): 69-80.). NEP stimulates the synthesis of viral cRNP replication intermediates, which are believed to increase the production of vRNPs in the later stages of infection for packaging into progeny viral particles. Simultaneously, it mediates the export of viral ribonucleoproteins from the nucleus to the cytoplasm through nuclear export signals and independent interactions with the human chromosomal maintenance protein Crm1 (Subbarao K, Klimov A, Katz J, et al. Characterization of an avianinfluenza A(H5N1) virus isolated from a child with a fatal respiratory illness[J]. Science (New York, Ny), 1998, 279(5349):393-6.). Currently, it is generally believed that NEP and matrix protein M1 of influenza virus are crucial for the nuclear export of vRNPs, meaning that the nuclear export of vRNPs is mediated by the vRNP-M1-NS2 complex.Furthermore, as an adaptor protein, NEP can directly mediate the export of vRNP from the cell nucleus to be packaged into progeny viral particles in the cell periphery; therefore, NEP can regulate the transcription and replication of the viral genome in the absence of M1 (Gao R, Sheng Z, Sreenivasan CC, et al. Influenza A Virus Antibodies with Antibody-Dependent Cellular Cytotoxicity Function[J].Viruses,2020,12(3)).

[0006] Therefore, rapid and accurate diagnosis of influenza viruses is crucial. In recent years, PCR technology has been widely used in pathogen diagnosis, and there has been considerable research on PCR detection systems for influenza viruses. However, current influenza virus nucleic acid PCR detection methods cannot distinguish between live viruses and residual viral nucleic acids, and differentiating between the two is key to determining whether a virus is infectious. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to detect live influenza B virus (including the Victoria lineage and / or Yamagata lineage). The technical problem to be solved is not limited to the technical subject matter described herein, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0008] To address the aforementioned technical problems, this invention first provides primer pairs for detecting live seasonal influenza B virus, wherein the primer pairs may be any of the following:

[0009] A1) A primer pair consisting of primer FluB-Vic-F and primer FluB-Vic-R; wherein primer FluB-Vic-F is the single-stranded DNA molecule shown in SEQ ID No. 3; and primer FluB-Vic-R is the single-stranded DNA molecule shown in SEQ ID No. 4;

[0010] A2) A primer pair consisting of primer FluB-Yama-F and primer FluB-Yama-R; wherein primer FluB-Yama-F is the single-stranded DNA molecule shown in SEQ ID No. 6; and primer FluB-Yama-R is the single-stranded DNA molecule shown in SEQ ID No. 7.

[0011] The primers FluB-Vic-F and FluB-Yama-F are forward primers; the primers FluB-Vic-R and FluB-Yama-R are reverse primers.

[0012] The primers FluB-Vic-F and FluB-Vic-R are primer pairs that specifically amplify the NS gene of influenza virus B / Colorado / 06 / 2017 (Victoria-lineage), i.e., specific primer pairs used to detect live influenza B virus Victoria-lineage.

[0013] The primers FluB-Yama-F and FluB-Yama-R are primer pairs that specifically amplify the NS gene of influenza virus B / PHUKET / 3073 / 2013 (Yamagata-lineage), i.e., specific primer pairs used to detect live influenza B virus Victoria-lineage.

[0014] The nucleotide sequence of the NS gene of influenza virus B / Colorado / 06 / 2017 (Victoria-lineage) is SEQ ID No.1. The nucleotide sequence of the NS1 gene is from position 1 to 870 (counting from the coding region) in SEQ ID No.1, and the nucleotide sequence of the NS2 gene is from position 1 to 36 and position 713 to 1048 in SEQ ID No.1.

[0015] The nucleotide sequence of the NS gene of influenza virus B / PHUKET / 3073 / 2013 (Yamagata-lineage) is SEQ ID No.2. The nucleotide sequence of the NS1 gene is from position 1 to position 846 (counting from the coding region) in SEQ ID No.2, and the nucleotide sequence of the NS2 gene is from position 1 to position 33 and from position 689 to position 1024 in SEQ ID No.2.

[0016] The present invention also provides a composition for detecting live influenza B virus, said composition being any of the following:

[0017] B1) The composition comprises the primer pair described in A1) and the probe FluB-Vic-P, wherein the nucleotide sequence of the probe FluB-Vic-P is SEQ ID No. 5;

[0018] B2) The composition comprises the primer pair described in A2) and the probe FluB-Yama-P, wherein the nucleotide sequence of the probe FluB-Yama-P is SEQ ID No. 8.

[0019] In the above composition, the 5' end of the probe FluB-Vic-P and / or the probe FluB-Yama-P is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0020] 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 or Texas Red.

[0021] The quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1.

[0022] In the above composition, the 5' end of the probe FluB-Vic-P and / or the probe FluB-Yama-P is labeled 6-FAM, and the 3' end is labeled BHQ1.

[0023] The present invention also provides a kit for detecting live influenza B virus, the kit comprising the primer pairs described herein.

[0024] The above-mentioned kit also includes the probe FluB-Vic-P and / or the probe FluB-Yama-P.

[0025] In the above kit, the 5' end of the probe FluB-Vic-P and / or the probe FluB-Yama-P is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0026] 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 or Texas Red.

[0027] The quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1.

[0028] In the above kit, the 5' end of the probe FluB-Vic-P and / or the probe FluB-Yama-P is labeled with 6-FAM, and the 3' end is labeled with BHQ1.

[0029] Furthermore, the kit also includes Taq DNA polymerase, dNTPs, PCR buffer, and Mg2+ required for PCR amplification. 2+ One or more of them.

[0030] The various reagent components of the kit may be present in separate containers, or may be pre-assembled, in whole or in part, into a reagent mixture.

[0031] Furthermore, the kit may also include a readable carrier describing the method for detecting live influenza B virus as described herein. The readable carrier may be a kit instruction manual (e.g., a printed manual) or a computer-readable medium (e.g., a floppy disk, CD, etc.) on which information has been recorded.

[0032] The present invention also provides a method for detecting live influenza B virus, the method comprising performing a PCR amplification reaction using the primer pair or the composition, and determining whether the sample to be tested contains live influenza B virus based on the amplification product.

[0033] In the above method, the PCR amplification reaction is a real-time quantitative PCR reaction, and the method includes the following steps:

[0034] A1) Extract RNA from the sample to be tested;

[0035] A2) Further, the live influenza B virus B-Victoria-lineage was detected by real-time quantitative PCR (qRT-PCR) reaction using the primers FluB-Vic-F, FluB-Vic-R, and probe FluB-Vic-P;

[0036] Alternatively, the primers FluB-Yama-F, FluB-Yama-R, and probe FluB-Yama-P can be used to perform real-time quantitative PCR (qRT-PCR) to detect live influenza B virus B-Yamagata-lineage.

[0037] The qRT-PCR reaction system includes: RNase-free dH2O, 2×One Step RT-PCR Buffer III, TaKaRa Ex Taq HS (5U / μL), PrimeScript RT Enzyme Mix II, ROX Reference Dye II, upstream primer, downstream primer, probe, and RNA.

[0038] In one embodiment of the present invention, the 20 μL qRT-PCR reaction system comprises: 6.2 μL RNase-Free dH2O, 10 μL 2×One Step RT-PCR Buffer III, 0.4 μL TaKaRa Ex Taq HS (5 U / μL), 0.4 μL PrimeScriptRT Enzyme Mix II, 0.4 μL ROX Reference Dye II, 0.2 μL upstream primer (20 μM), 0.2 μL downstream primer (20 μM), 0.2 μL probe (20 μM), and 2 μL RNA;

[0039] The qRT-PCR reaction conditions were as follows: Step 1 (reverse transcription reaction): 42℃ for 5 min, 95℃ for 10 sec, 1 cycle; Step 2 (PCR reaction): 95℃ for 5 sec, 55℃ for 34 sec, 40 cycles.

[0040] The present invention also provides the primer pairs, and / or the use of the composition in the preparation of products for detecting live influenza B virus.

[0041] The sample to be tested may be a sample obtained from the subject's nasal swab, throat swab, pharyngeal swab, or nasal wash, or it may be a blood or tissue sample.

[0042] The purpose of the above applications and methods may be for disease diagnosis, disease prognosis and / or disease treatment, or they may be for non-disease diagnosis, non-disease prognosis and non-disease treatment purposes; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be for non-disease diagnosis, non-disease prognosis and / or non-disease treatment purposes.

[0043] In the above method, the method for determining whether the sample to be tested contains live influenza B virus based on the amplification product can be as follows: (1) If the amplification curve of real-time fluorescence quantitative PCR is an S-shaped curve, it is determined that the sample to be tested contains live influenza B virus; if the amplification curve of real-time fluorescence PCR is not an S-shaped curve, it is determined that the sample to be tested does not contain live influenza B virus.

[0044] (2) The negative / positive of the sample is determined according to the results of quantitative real-time PCR.

[0045] The influenza B virus mentioned in this article, also known as influenza B virus, includes, but is not limited to, the Victoria-lineage and / or Yamagata-lineage.

[0046] This invention designs primers and probes that can detect replicating viruses (i.e., live viruses), and establishes a rapid, accurate, and simple detection method, especially a method that can detect the presence of live viruses. This lays a good foundation for the study of live viruses, allows for better observation of viral proliferation within cells, and is of great significance for the early detection and treatment of viral infections.

[0047] Compared with the prior art, the technical effects of the present invention include:

[0048] (1) The primers, probes, compositions, kits and detection methods of the present invention for detecting influenza B virus can detect live influenza B virus after cell replication and can effectively distinguish between live influenza virus and viral nucleic acid residue.

[0049] (2) The primers, probes, compositions, kits, and detection methods of this invention for detecting influenza B virus are characterized by speed, accuracy, sensitivity, reliability, and simplicity of operation. They exhibit good amplification curves with better slope and CT values, and the detection sensitivity can reach as low as 10. -6 Copy / μL. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the structure and genome of the influenza B virus.

[0051] Figure 2 This is the experimental design diagram for Embodiment 2 of the present invention.

[0052] Figure 3 RT-PCR amplification curves for identifying the genome and subgene primers of influenza B virus B-Victoria-lineage. In the diagram, 1 represents the amplification curves of three samples using the genome primer (Genome primer) for identifying influenza B virus B-Victoria-lineage; 2 represents the amplification curves of three samples using the subgene primer (Subgene primer) for identifying influenza B virus B-Victoria-lineage.

[0053] Figure 4 RT-PCR amplification curves for identifying the genome and subgene primers of influenza B virus B-Yamagata-lineage. In the diagram, 3 represents the amplification curves of three samples using the genome primer (Genome primer) for identifying influenza B virus B-Yamagata-lineage; 4 represents the amplification curves of three samples using the subgene primer (Subgene primer) for identifying influenza B virus B-Yamagata-lineage.

[0054] Figure 5 RNA was directly extracted from the serially diluted supernatant of B-Victoria-lineage lung tissue and analyzed by qRT-PCR to determine CT-Value and viral copy number (copies / μL). Simultaneously, the HA titer of allantoic fluid from chicken embryos harvested after inoculation and the qRT-PCR results after RNA extraction were also analyzed. Allantoic fluid from chicken embryos with negative hemagglutination titers but positive CT values ​​was further inoculated into chicken embryos, and the results were used as the definitive test after three inoculations.

[0055] Figure 6RNA was directly extracted from the serially diluted supernatant of B-Yamagata-lineage lung homogenate for qRT-PCR detection of CT-Value and viral copy number (copies / μL). Simultaneously, the HA titer of allantoic fluid from chicken embryos harvested after inoculation with different dilutions of lung homogenate supernatant was compared with the qRT-PCR results after RNA extraction. Allantoic fluid from chicken embryos with negative hemagglutination titers but positive CT values ​​was further inoculated into chicken embryos, and the results were used as the definitive test after three inoculations. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] The qRT-PCR detection kit One Step PrimeScript described in the following examples TM The RT-PCR kit was from TAKARA, catalog number RR064A; the QIAamp Viral RNA Mini Kit was from QIAGEN, catalog number 52906. The qRT-PCR instrument was an ABI 7500fast real-time PCR system.

[0059] The B-Victoria-lineage influenza virus B / Colorado / 06 / 2017 (BV) and B-Yamagata-lineage influenza virus B / PHUKET / 3073 / 2013 (BY) used in the following examples were purchased from the Chinese Center for Disease Control and Prevention (China CDC).

[0060] The mice used in the following examples were SPF strain mice, purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0061] Example 1: Design of NS2 primers for influenza virus subgene

[0062] Specific primers and probes were designed based on the NS gene sequences of influenza viruses B / Colorado / 06 / 2017 (Victoria-lineage) and B / PHUKET / 3073 / 2013 (Yamagata-lineage), respectively.

[0063] The nucleotide sequence of the NS gene of influenza virus B / Colorado / 06 / 2017 (Victoria-lineage) is SEQ ID No.1. The nucleotide sequence of the NS1 gene is from position 1 to 870 (counting from the coding region) in SEQ ID No.1, and the nucleotide sequence of the NS2 gene is from position 1 to 36 and position 713 to 1048 in SEQ ID No.1.

[0064] The nucleotide sequence of the NS gene of influenza virus B / PHUKET / 3073 / 2013 (Yamagata-lineage) is SEQ ID No.2. The nucleotide sequence of the NS1 gene is from position 1 to position 846 (counting from the coding region) in SEQ ID No.2, and the nucleotide sequence of the NS2 gene is from position 1 to position 33 and from position 689 to position 1024 in SEQ ID No.2.

[0065] Table 1 shows the sequences and positions of the upstream and downstream primers and probes for the subgene (NS2). The primers and probes were synthesized by the company.

[0066] Table 1 Primers and probes for influenza virus B / Colorado / 06 / 2017 (Victoria-lineage) and influenza virus B / PHUKET / 3073 / 2013 (Yamagata-lineage).

[0067]

[0068] The modifying group 6-FAM is a fluorescent reporter group, and BHQ1 is a fluorescent quencher group.

[0069] The primer pair used to detect live influenza B virus (B-Victoria-lineage) consists of primer FluB-Vic-F and primer FluB-Vic-R; primer FluB-Vic-F is the single-stranded DNA molecule shown in SEQ ID No. 3; primer FluB-Vic-R is the single-stranded DNA molecule shown in SEQ ID No. 4.

[0070] The primer pair for detecting live influenza B virus (B-Yamagata-lineage) consists of primer FluB-Yama-F and primer FluB-Yama-R; primer FluB-Yama-F is the single-stranded DNA molecule shown in SEQ ID No. 6; primer FluB-Yama-R is the single-stranded DNA molecule shown in SEQ ID No. 7.

[0071] Example 2: Detection method for live influenza B virus and verification of the effectiveness of primer-probe combination.

[0072] The experimental design of this embodiment is as follows: Figure 2 As shown.

[0073] 1. Isolation and culture of viruses

[0074] (1) Egg testing

[0075] ① Use an egg-displaying lamp to examine the chicken embryo and mark the boundary between the air cell and allantois, as well as the location of the embryo.

[0076] ② If the chicken embryo is dead, unfertilized, cracked, underdeveloped, or has many water seepage holes on its surface, it should be discarded.

[0077] ③ How to determine the condition of the chicken embryo:

[0078] 1) Blood vessels: Blood vessels are clear in live embryos, but blurry in dead embryos, forming congested bands or clots.

[0079] 2) Fetal movement: Live embryos have obvious natural movements, while dead embryos have no fetal movement.

[0080] 3) Developmental boundary of the chorioallantoic membrane: The densely vascularized chorioallantoic membrane forms a clear boundary with the other side of the chicken embryo.

[0081] (2) Chicken embryo inoculation

[0082] ① Place the chicken embryos with the air cell facing up on the egg tray and label each chicken embryo.

[0083] ② Disinfect the chicken embryo with 75% alcohol, and drill a 1×1mm hole at the air cell end. About 5mm~15mm below the air cell, select an area with no blood vessels or few blood vessels, and drill a 1×1mm hole there as well.

[0084] ③ Use a 1mL syringe to draw 100μL of serially diluted mouse lung supernatant and attach a needle.

[0085] ④ Inject 100 μL of sample into the allantoic cavity of the chicken embryo through the hole under the air chamber.

[0086] ⑤ After repeatedly drawing 84 disinfectant solution 2-3 times with a syringe, place the needle in a sharps container and autoclave it at 121℃. Then, hand the sharps container over to a specialized medical waste disposal company. Dispose of the syringe barrel in a high-pressure bag, autoclave it at 121℃, and then discard it.

[0087] ⑥ Seal the opening with wax.

[0088] ⑦ Incubate chicken embryos at 37℃ (35℃ for mammalian influenza virus) 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 have died non-specifically and should be discarded.

[0089] (3) Determination of HA titer in chicken embryo allantoic fluid

[0090] ① Chicken embryos should be kept at 4°C overnight or at least for 4 hours before harvesting.

[0091] ② Disinfect the air cell end of the chicken embryo with 70%–75% alcohol. Tear open the eggshell of the air cell with sterile forceps, and puncture the chorioallantoic membrane where there are no large blood vessels.

[0092] ③ Determine the HA potency:

[0093] 1) Prepare a 1% chicken red blood cell suspension.

[0094] a. Mix the red blood cells stored in Alsever's solution thoroughly, and use a graduated pipette to transfer an appropriate amount of the red blood cell suspension to a centrifuge tube. Each tube should not exceed 1 / 3 of the total volume. Centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0095] b. Add at least twice the volume of sterile saline or PBS and wash three times, then mix well.

[0096] c. Centrifuge at 1500 rpm for 5 minutes twice, discarding the supernatant. Centrifuge at 1500 rpm for 10 minutes once more, discarding the supernatant. The deposited red blood cells are concentrated chicken red blood cells.

[0097] d. Aspirate 1 / 100 of the final required solution volume of chicken red blood cells, and repeatedly pipette with sterile physiological saline or PBS more than 10 times to prepare a 1% chicken red blood cell suspension. Label the suspension and store at 4℃ for later use.

[0098] e.1% chicken red blood cell suspension can be stored at 4°C for about 7 days. If hemolysis occurs, it should be discarded and prepared again. It is best to prepare it fresh and use it immediately.

[0099] 2) Determine HA titer:

[0100] a. Use a pipette tip with a filter to draw 25 μL of allantoic fluid into the first column (AH row) of a V-shaped microplate.

[0101] b. Use a 12-channel adjustable pipette to add 25 μL of PBS to all wells.

[0102] c. Load the 200μL pipette tip with filter cartridge into the 8th pipette, and blow and aspirate 5-10 times to mix the first column. Take 25μL of the mixture from each well of the first column into the second column, blow and aspirate 5-10 times to mix the second column, and continue diluting until the 11th column. Discard the 25μL liquid from the 11th column.

[0103] d. Add 25 μL of 1% red blood cell suspension to each well, and gently tap the V-shaped plate to mix the red blood cells and virus thoroughly.

[0104] e. Incubate at room temperature for 20–40 minutes, observe the red blood cell agglutination phenomenon and record the results.

[0105] f. Result Interpretation: The hemagglutination titer is determined by the highest dilution that produces complete agglutination. The reciprocal of this dilution is the viral hemagglutination titer. Complete agglutination is recorded as "+"; no agglutination or partial agglutination is recorded as "-".

[0106] (4) Harvesting chicken embryo allantoic fluid (toxin collection)

[0107] (1) Draw chicken embryo allantoic fluid into the appropriate collection tube using a 2mL or 5mL syringe. After harvesting the virus culture medium, place the chicken embryos in a sealed bag in a biosafety cabinet and then autoclave them.

[0108] (2) After centrifuging the chicken embryo harvest liquid at 3000 rpm for 5 min to remove blood and cells, virus samples 1 and 2, namely B / Colorado / 06 / 2017 (Victoria-lineage) and B / PHUKET / 3073 / 2013 (Yamagata-lineage), were obtained (virus stock solution). These can be used immediately for subsequent experiments or frozen at -80℃ for later use.

[0109] 2. Sample preparation:

[0110] (I) Influenza virus (BY and BV) infection in mice

[0111] (1) First, the experimental mice (SPF strain mice) were anesthetized by inhalation of isoflurane;

[0112] (2) After anesthetizing the mouse, hold it with its head facing upwards and use a 200μL pipette tip to drip the virus stock solution into the nasal cavity slowly along one side of the nasal cavity. 50μL of the virus stock solution is dripped into the nasal cavity, and the nasal cavity should be kept as clear as possible.

[0113] (3) After administering the nasal drops, the mouse's head should be held upright for 30-60 seconds to ensure that the virus does not leak out. BY-infected mice and BV-infected mice were prepared separately.

[0114] (II) Necropsy and Tissue Collection of Mice

[0115] After anesthetizing the mice (BY-infected and BV-infected mice), enucleate their eyes to collect blood, then euthanize them by dissecting their necks. Begin the dissection. Using the first set of scissors and forceps, cut the mouse's fur down to the mouth, using blunt force dissection. After completion, soak the scissors and forceps in a sterilizer or make a small incision in the middle of the mouse's abdomen, then use both hands to forcefully tear open the mouse's outer skin. Using the second set of scissors and forceps, cut open the mouse's abdominal and thoracic cavities, ensuring a clear and unobstructed view, avoiding touching the mouse's fur during the process. After completion, soak the scissors and forceps in a sterilizer. The third set of scissors and forceps was used to dissect the mouse's trachea from the throat, cut it off at the top of the larynx and hold it with the forceps. The scissors were bluntly dissected to peel the entire lung (connected to the heart) from the thoracic cavity along the trachea. The heart was then peeled off in a petri dish, the esophagus was removed, the entire lung was weighed, and it was placed in a tissue grinding tube. After grinding at 4000 rpm for 30 seconds three times, it was centrifuged at 7000 rpm for 10 minutes to obtain the test samples: lung grinding supernatant from B / Colorado / 06 / 2017 (Victoria-lineage) and lung grinding supernatant from B / PHUKET / 3073 / 2013 (Yamagata-lineage).

[0116] 3. Detection methods for live influenza B virus

[0117] 3-1. Extract RNA from the sample to be tested

[0118] RNA was extracted from the sample using the QIAamp Viral RNA Mini Kit. The specific steps are as follows:

[0119] (1) When using the kit for the first time, add 100% ethanol to the AW1 and AW2 buffers according to the volume indicated on the reagent bottle; dissolve the carrier RNA in 1550 μL AVE and add it all to the AVL, and aliquot it into 1.5 mL EP tubes, 560 μL per tube, and freeze at -80℃.

[0120] (2) Add 140 μL of the test sample and at least one corresponding negative control sample to the above liquid, vortex for 15 s to mix thoroughly, and incubate at room temperature (15-20℃) for 10 min. Briefly centrifuge to allow the liquid at the top of the centrifuge tube to reach the bottom.

[0121] (3) Add 560 μL of 100% ethanol to the centrifuged sample, vortex for 15 seconds to mix, and then briefly centrifuge to allow the liquid at the top of the centrifuge tube to fall to the bottom.

[0122] (4) Carefully add about 630 μL of liquid to the unwetted QIAamp filter column, cover it, centrifuge at 8000 rpm / 1 min, discard the collection tube, and place the column on a new 2 mL collection tube.

[0123] (5) Open the cap of the QIAamp filter column and repeat step (4) until all samples are centrifuged.

[0124] (6) Open the cap, add 500 μL of AW1 buffer to the filter column, close the cap, centrifuge at 8000 rpm / 1 min, discard the collection tube, and place the filter column on a new 2 mL collection tube.

[0125] (7) Open the cap, add 500 μL of AW2 buffer to the filter column, close the cap, and centrifuge at 14,000 rpm for 3 min. Place the filter column on a new 2 mL collection tube and centrifuge for 1 min (optional).

[0126] (8) Place the column on a new 1.5 mL centrifuge tube, add 40 mL of AVE elution buffer, and centrifuge at 8000 rpm for 1 min. Repeat this step and collect the centrifuged liquid, which is the extracted viral RNA. Perform subsequent detection immediately or store at -80 °C.

[0127] 3-2. qRT-PCR detection

[0128] The primers and probes designed in Example 1 were used to perform qRT-PCR detection on the test samples. The PCR system is shown in Table 2, and the PCR conditions are shown in Table 3.

[0129] Table 2 qRT-PCR reaction system

[0130] reagents Dosage <![CDATA[RNase Free dH2O]]> 6.2μL 2×One Step RT-PCR Buffer III 10μL TaKaRa Ex Taq HS (5U / μL) 0.4μL PrimeScript RT Enzyme Mix II 0.4μL ROX Reference DyeⅡ 0.4μL Upstream primer (20 μM) 0.2μL Downstream primer (20 μM) 0.2μL Probe (20 μM, 5'FAM-3'BHQ1) 0.2μL RNA 2μL Total volume 20μL

[0131] Table 3 qRT-PCR reaction conditions

[0132]

[0133] 3-3. Test Results

[0134] (1) Determine whether the sample contains live virus based on the PCR amplification products: If the amplification curve of real-time fluorescence quantitative PCR is an S-shaped curve, it is determined that the sample contains live influenza B virus; if the amplification curve of real-time fluorescence PCR is not an S-shaped curve, it is determined that the sample does not contain live influenza B virus.

[0135] (2) The negative / positive of the sample is determined according to the results of quantitative real-time PCR.

[0136] 4. Experiment to verify the effectiveness of specific primers for detecting live influenza B virus.

[0137] Samples 3, 4, and 5 were three mice infected with type B Victoria influenza virus via nasal drops. After 5 days post-mortem, the lungs were removed and ground up, following the sample preparation method described in step 2 above. RNA was extracted from the lungs and qRT-PCR was performed using genome primers and BV-Subgene primers and probes (specific primers and probes for detecting Victoria-lineage).

[0138] Samples 6, 7, and 8 were three mice infected with type B Yamagata influenza virus via nasal drops. After 5 days post-mortem, the lungs were removed and ground. The lungs were prepared as described in step 2 above. RNA was extracted from the lungs and detected by qRT-PCR using genomic primers and BY-Subgene primers and probes (specific primers and probes for detecting Yamagata-lineage).

[0139] The experiment included two treatments: subgene primer and probe treatment and genomic primer and probe treatment. For subgene primer and probe treatment, samples 1, 2, 3, 4, 5, 6, 7, and 8 were subjected to qRT-PCR detection using subgene primers and probes, following step 3. For genomic primer and probe treatment, samples 1, 2, 3, 4, 5, 6, 7, and 8 were subjected to qRT-PCR detection using genomic primers and probes, following step 3.

[0140] Among them, the subgene primers and probes are those listed in Table 1;

[0141] The genome primer and probe sequences used for comparison are as follows:

[0142] IBV-genome-F: 5'-GTCCATCAAGTCCAGTTTT-3',

[0143] IBV-genome-R: 5'-TCTTCTTACAGCTTGCTTGC-3',

[0144] IBV-genome-P: 5'-AAGYAGGTGGAGAYGGAGGGTCT-3'.

[0145] The qRT-PCR test was performed according to the method in step 3.

[0146] 4-1. Comparison of CT value results by qRT-PCR

[0147] The results are shown in Tables 4 and 5:

[0148] Table 4 Comparison of CT value results for subgenetic primers and genomic primers

[0149]

[0150] Note: U stands for Undetermined.

[0151] Samples 1 and 2 are the results obtained after extracting RNA from chicken embryo allantoic fluid (which was harvested after influenza virus propagation in chicken embryos, but not through cell propagation) and detecting it by qRT-PCR. Since the virus propagates through the allantoic fluid after inoculation into chicken embryos, without cellular amplification, it cannot be detected by qRT-PCR using subgenetic primers and probes. This indirectly demonstrates that the primers and probes designed in this invention can detect live viruses that have undergone cellular replication.

[0152] Samples 3, 4, and 5 were three mice infected with type B Victoria influenza virus via intranasal instillation. The mice were euthanized at 5 days post-mortem, and their lungs were harvested and ground. The lungs were prepared according to the sample preparation method described in step 2 above. RNA was extracted from the lungs for qRT-PCR detection. Samples 6, 7, and 8 were three mice infected with type B Yamagata influenza virus via intranasal instillation. The mice were euthanized at 5 days post-mortem, and their lungs were harvested and ground. The lungs were prepared according to the sample preparation method described in step 2 above. RNA was extracted from the lungs for qRT-PCR detection.

[0153] Although the sensitivity of subgene primers and probes is relatively weak, it demonstrates that they can detect live viruses, and the amplification curves are better than those using genomic primers and probes, with better slopes and CT values.

[0154] Table 5 Results of BY qRT-PCR and HA detection

[0155]

[0156] Note: U stands for Undetermined.

[0157] Table 5 shows the results of RNA extraction and qRT-PCR analysis of lungs from three mice (samples 9, 10, and 11, representing B-Yamagata-lineage influenza viruses) after passage in mice. The lungs were then ground to obtain the supernatant, and RNA was extracted and analyzed. Simultaneously, chicken embryo allantoic fluid from samples 9, 10, and 11 was inoculated with chicken embryos, and the hemagglutination titer (HA) and RNA extracted from these samples were analyzed using qRT-PCR.

[0158] When the B-Yamagata-lineage influenza virus was passaged in mice, after a certain generation, the mice were euthanized, and the lungs were collected. The lung supernatant was extracted, and RNA was extracted. qRT-PCR analysis revealed that no CT value could be detected using subgenetic primers and probes. Furthermore, hemagglutination titer (HA) and qRT-PCR results from the allantoic fluid of chicken embryos harvested after inoculation were both negative. This explains why, although nucleic acid residues were present in the mice, they were not live viruses and would not cause disease or death in the mice. Therefore, the subgenetic primers and probes of this invention could not detect live virus in mice.

[0159] 4-2 Comparison of qRT-PCR amplification curve results

[0160] The qRT-PCR results for samples 3, 4, 5, 6, 7, and 8 are as follows.

[0161] The experiment included two treatments: subgene primer and probe treatment and genomic primer and probe treatment. For subgene primer and probe treatment, samples 3, 4, 5, 6, 7, and 8 were subjected to qRT-PCR detection using subgene primers and probes, following the method described in step 3. For genomic primer and probe treatment, samples 3, 4, 5, 6, 7, and 8 were subjected to qRT-PCR detection using genomic primers and probes, following the method described in step 3.

[0162] The results are as follows Figure 3 and Figure 4 As shown in the qRT-PCR amplification curve results, although the sensitivity of using subgene primers and probes is relatively weak, it can still indicate that live viruses can be detected using subgene primers and probes. Furthermore, the amplification curves are better than those using genomic primers and probes, with better slopes and CT values.

[0163] 4-3. Viral load was detected by qRT-PCR after serial dilution of lung grinding supernatant.

[0164] (1)B / Colorado / 06 / 2017(Victoria-lineage)

[0165] On June 2017, 2017 (Victoria-lineage), RNA was directly extracted from the supernatant of lung homogenate after serial dilution and qRT-PCR was performed to detect CT-Value and viral copy number (copies / μL). Simultaneously, the HA titer of allantoic fluid from chicken embryos harvested and the qRT-PCR results after RNA extraction were compared. Allantoic fluid from chicken embryos with negative hemagglutination titers but positive CT values ​​was further inoculated into chicken embryos, and the results were used for three consecutive inoculations.

[0166] The results are as follows Figure 5As shown: Mice were inoculated on B / Colorado / 06 / 2017 (Victoria-lineage). Lung supernatant was obtained by necropsy after inoculation. Viral load was detected by qRT-PCR after serial dilution. The results showed that using Genome primers at 10... -7 It can still be detected during dilution gradients, but when using Subgene primers at 10... -7 It can no longer be detected; only 10 can be detected. -6 CT value.

[0167] The viral copy number results also showed this trend, so we serially diluted the lung grinding supernatant and inoculated it into chicken embryos to harvest chicken embryo allantoic fluid, detect HA titer and extract RNA, and perform qRT-PCR detection. Chicken embryo allantoic fluid with negative hemagglutination titer but CT value was inoculated into chicken embryos again, and the test results were used after three times. Figure 5 A negative sample is one that has been inoculated with chicken embryos three times and shows neither HA titer nor a negative result in qRT-PCR after RNA extraction. "+" indicates a positive HA result, "-" indicates a negative HA result, and "U" indicates Undetermined (negative qRT-PCR result).

[0168] Based on the HA titer results and the qRT-PCR detection results after RNA extraction, it can be seen that at 10 -5 The dilution gradient test was positive, 10 -6 The dilution gradient detected only two chicken embryos, 10 -7 and 10 -8 The results were undetectable in all five chicken embryos after a dilution gradient. The results obtained after three validations with chicken embryos were consistent with those obtained by directly extracting RNA from the lung supernatant of B-Victoria-lineage mice after gradient dilution and performing qRT-PCR.

[0169] Therefore, it is calibrated to 10. -5 The CT value and viral copy number detected by the dilution gradient are the positive criteria, 10 -6 The CT value and viral copy number detected by the dilution gradient are considered suspected. If this occurs, the test is repeated. If it is still suspected, it is judged as positive; if it is negative, it is judged as negative. 10 -7 The CT values ​​and viral copy numbers detected using the dilution gradient were negative criteria. These results correspond to the CT-Value and viral copy number detected using Subgene primers.

[0170] (2)B / PHUKET / 3073 / 2013(Yamagata-lineage)

[0171] Lung supernatant from B / PHUKET / 3073 / 2013(Yamagata-lineage)(B-Yamagata-lineage) was serially diluted and then qRT-PCR was performed to detect CT value and viral copy number (copy / μL). Simultaneously, the HA titer and qRT-PCR results of chicken embryo allantoic fluid harvested from chicken embryos were analyzed. Chicken embryo allantoic fluid with negative hemagglutination titer but a CT value was further inoculated into chicken embryos, and the results were used for three consecutive inoculations.

[0172] The results are as follows Figure 6 As shown:

[0173] Mice inoculated with B / PHUKET / 3073 / 2013 (Yamagata-lineage) were euthanized to obtain lung supernatant. Viral load was detected by qRT-PCR after serial dilution. The results showed that the viral load was significantly reduced using genomic primers and probes within 10... -7 It can still be detected at dilution gradients, but using subgene primers and probes at 10... -7 It can no longer be detected; only 10 can be detected. -5 CT value.

[0174] The viral copy number results also showed this trend, so we serially diluted the lung grinding supernatant and inoculated it into chicken embryos to harvest chicken embryo allantoic fluid, detect HA titer and extract RNA, and perform qRT-PCR detection. Chicken embryo allantoic fluid with negative hemagglutination titer but CT value was inoculated into chicken embryos again, and the test results were used after three times. Figure 6 A negative sample is one that has been inoculated with chicken embryos three times and shows neither HA titer nor a negative result in qRT-PCR after RNA extraction. "+" indicates a positive HA result, "-" indicates a negative HA result, and "U" indicates Undetermined (negative qRT-PCR result).

[0175] Based on the HA titer results and the qRT-PCR detection results after RNA extraction, it can be seen that at 10 -4 The dilution gradient test was positive, 10 -5 The dilution gradient detected only 1 chicken embryo, 10 -6 The results were undetectable in all five chicken embryos after a dilution gradient. The results obtained after three validations with chicken embryos were consistent with those obtained by directly extracting RNA from the lung supernatant of B-Victoria-lineage mice after gradient dilution and performing qRT-PCR.

[0176] Therefore, it is calibrated to 10. -4 The CT value and viral copy number detected by the dilution gradient are the positive criteria, 10 -5The CT value and viral copy number detected by the dilution gradient are considered suspected. If this occurs, the test is repeated. If it is still suspected, it is judged as positive; if it is negative, it is judged as negative. 10 -6 The CT values ​​and viral copy numbers detected using the dilution gradient were negative. This result corresponds to the CT values ​​and viral copy numbers detected using subgenetic primers and probes.

[0177] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> Methods, primers, probes, and kits for detecting live seasonal influenza B virus. <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 1048 <212> DNA <213> Influenza virus B / Colorado / 06 / 2017 (Victoria-lineage) <400> 1 atggcgaaca acaacatgac cacaacacaa attgaggtgg gtccgggagc aaccaatgcc 60 accataaact ttgaagcagg aattctggag tgctatgaaa ggctttcatg gcaaagagcc 120 cttgactacc ccggtcaaga ccgcctaaac agactaaaaa gaaaattaga gtcaagaata 180 aagactcaca acaaaagtga gcctgaaagt aaaaggatgt cccttgaaga gagaaaagca 240 attggagtaa aaatgatgaa agtactccta ttatgaatc cgtctgctgg aattgaaggg 300 tttgagccat actgtatgaa cagttcctca atagcaact gtacgaata caatggacc 360 gattaccctt cacaccaga gaggtgcctt gatgacatag aggagaacc agaggatgtt 420 gatggcccaa ctgaatagt attaagggac atgacaaca aagatgcaag gcaaagata 480 aaggagg taaacactca gaagaagggg aagttccgtt tgacataaa aagggatatg 540 cgtaatgtat tgtcctttga aggatgtggt tgaagtgtga gagtgttggt aaatggaaca 600 ttcctcaaac acccaatgg atacaagtcc ttcaactc tgcatagat gatgcatat 660 gaccagagtg gaaggcttgt tgctaaactt gttgccactg atgatcttac agtggaggt 720 gagaagatg gccatcggat cctcaactca ctctcgagc gtctcaatga aggacattca 780 aagccaattc gagcagctga aactgcggtg ggagtctt cccaatttgg tcaagagcac 840 cgatttacac cagaagagggg agacattag attggtcacg gagaacttt atcttttag 900 taaaagaattt gatgataca tactattcca caaacagtg atagctaca gctccataat 960 agctgacatg gttgtatcat tatcattatt agaaacattg tatgaatga aggatgtggt 1020 tgaagtgtac agcaggcagt gcttgtga 1048 <210> 2 <211> 1024 <212> DNA <213> PHUKET / 3073 / 2013(Yamagata-lineage) <400> 2 atggcggaca atatgaccac aacacaatt gaggtgggtc cgggagcaac caatgccacc 60 aaacttcg aagcaggaat tctggagtgc tatgaagac ttcatggca aagggccctt 120 gactaccctg gtcaagaccg tctaaacaga ctaagagga attagagtc aagaataag 180 actcacaaca aaagtgagcc tgaaagtaaa aggatgtctc ttgagagag aaagcaatt 240 ggagtaaaaa tgatgaaagt acttctattt atgaatccgt ctgctggaat tgaagggtttt 300 gagccatact gtatgaaaag ttcctcaag agcaactgtc cgaaatacag ttggattgat 360 taccctcaa ccccagggag gtgccttgat gatagaag aagaaccaga tgatgttgat 420 ggcccaactg aaatagtattt aagggacatg aaaaaaag atgcaaggca aagataag 480 gaggagtaa acactcagaa agaagggaag ttccgtttga cataaaaag ggatatgcgt 540 aatgtattgt ccctgagagt gttagtaaac ggaacattcc tcaaacaccc caatggatac 600 aagtccttat caactctgca tagattgaat gcatatgacc agagtggaag gcttgttgct 660 aaacttgttg ctactgatga tcttacagtg gaggatgaag aagatggcca tcggatcctc 720 aattcactct tcgagcgtct taatgaagga cattcaaagc caattcgagc agctgaaact 780 gcggtgggag tcttatccca atttggtcaa gagcaccgat tatcaccaga agagggagac 840 aattagactg gtcacggaag aactttatct tttaagtaaa agaattgatg ataacatatt 900 gttccacaaa acagtaatag ctaacagctc cataatagct gacatggttg tatcattatc 960 attattagaa acattgtatg aaatgaagga tgtggttgaa gtgtacagca ggcagtgctt 1020 gtga 1024 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 atggcgaaca acaacatgac 20 <210> 4 <211> 23 <212> DNA <213> Artificial sequence <400> 4 tctaattgtc tccctcttct ggt 23 <210> 5 <211> 26 <212> DNA <213> Artificial sequence <400> 5 acaaattgag tggaggatga agaaga 26 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <400> 6 atggcggaca atatgaccac a 21 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <400> 7 gtgaccagtc taattgtctc cct 23 <210> 8 <211> 25 <212> DNA <213> Artificial sequence <400> 8 acaaattgag tggaggatga agaag 25

Claims

1. The use of the composition in the preparation of products for detecting live influenza B virus, characterized in that, The composition is any one of the following: B1) Primers FluB-Vic-F, FluB-Vic-R, and probe FluB-Vic-P, wherein primer FluB-Vic-F is the single-stranded DNA molecule shown in SEQ ID No. 3; primer FluB-Vic-R is the single-stranded DNA molecule shown in SEQ ID No. 4; and the nucleotide sequence of probe FluB-Vic-P is SEQ ID No.

5. B2) Primers FluB-Yama-F, FluB-Yama-R, and probe FluB-Yama-P, wherein primer FluB-Yama-F is the single-stranded DNA molecule shown in SEQ ID No. 6; primer FluB-Yama-R is the single-stranded DNA molecule shown in SEQ ID No. 7; and the nucleotide sequence of probe FluB-Yama-P is SEQ ID No.

8. The application can distinguish between live influenza B virus and residual influenza B virus nucleic acid.

2. The application according to claim 1, characterized in that, The probes FluB-Vic-P and FluB-Yama-P are labeled with a fluorescent group at their 5' end and a quenching group at their 3' end.

3. The application according to claim 2, characterized in that, The 5' end of the probes FluB-Vic-P and FluB-Yama-P is labeled 6-FAM, and the 3' end is labeled BHQ1.

4. The application according to any one of claims 1-3, characterized in that, The application includes using the composition to perform PCR amplification reactions, and determining whether the sample to be tested contains live influenza B virus based on the amplification products.

5. The application according to claim 4, characterized in that, The PCR amplification reaction is a real-time quantitative PCR reaction, and the application includes the following steps: A1) Extract RNA from the sample to be tested; A2) The RNA is reverse transcribed into cDNA, and the cDNA is used as a template to perform real-time quantitative PCR reaction detection using any of the compositions described in claims 1-3. The presence of live influenza B virus in the sample is determined based on the amplification products.

6. The application according to claim 5, characterized in that, The method for determining whether a sample contains live influenza B virus based on the amplification products is as follows: if the amplification curve of real-time quantitative PCR is an S-shaped curve, it is determined that the sample contains live influenza B virus; if the amplification curve of real-time quantitative PCR is not an S-shaped curve, it is determined that the sample does not contain live influenza B virus.

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