A primer and probe combination, method, and application for detecting the levels of H7N9 influenza virus vRNA, cRNA, and mRNA in vivo.

Abstract

This invention relates to a primer and probe combination, method, and application for detecting the levels of H7N9 influenza virus vRNA, cRNA, and mRNA in vivo, belonging to the field of biotechnology. To address the technical problem that existing technologies can only detect influenza virus vRNA, cRNA, and mRNA in in vitro cell lines and cannot be adapted to in vivo infection experiments with complex template quantities in animals, this invention designs a primer and probe combination for detecting the vRNA, cRNA, and mRNA of the PB2 or NA genes of H7N9 influenza virus in vivo. This primer and probe combination includes reverse transcription primers and quantitative real-time PCR primers and probes. This invention utilizes the obtained primer and probe combination and quantitative real-time PCR technology to determine the levels of vRNA, cRNA, and mRNA of the PB2 and NA genes of H7N9 virus in mice, providing an experimental protocol for studying the actual replication of H7N9 influenza virus in vivo.

Description

[0001] A primer and probe combination, method, and application for detecting the levels of H7N9 influenza virus vRNA, cRNA, and mRNA in vivo. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a primer and probe combination, method, and application for detecting the content of H7N9 influenza virus vRNA, cRNA, and mRNA in vivo. Background Technology

[0003] Influenza virus is a segmented, single-stranded, negative-sense RNA virus. Its replication in host cells involves the synthesis of viral genomic RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA). Studying the in vivo replication kinetics of influenza virus is crucial for understanding viral transmission, disease course, treatment, and prevention. First, understanding the replication kinetics of influenza virus in the host helps reveal the transmission mechanism, including how the virus enters host cells and spreads throughout the host, which is essential for answering scientific questions about influenza virus replication. Second, studying the in vivo replication kinetics of influenza virus helps understand the course of infection, including the replication rate, transmission routes, and timeline of infection in different tissues and cells. This helps identify key stages and potential treatment windows in the disease course. For example, understanding the replication rate and susceptibility of the virus at different stages of the disease can help doctors better plan treatments and improve efficacy. Third, understanding the replication kinetics of influenza virus in vivo can help researchers design and optimize antiviral drugs. Researchers can develop drugs targeting key steps in viral replication to inhibit or slow down the replication process within the host. For example, researchers can choose known anti-influenza drugs, such as oseltamivir, and investigate their effects on the replication rate and quantity of influenza viruses within cells. This helps verify the effectiveness of the drugs and provides a reference for the development of new drugs. They can also study which stage some drugs primarily act on—whether they affect the replication from vRNA to cRNA or the transcription from vRNA to mRNA—revealing scientific questions. Fourth, a deeper understanding of the replication kinetics of influenza viruses in vivo helps guide vaccine development. By understanding how the virus replicates within the host, it is possible to better select vaccine strains, determine the optimal vaccination time, and optimize vaccine design. For example, H5 and H9 avian influenza are currently the most harmful and widespread influenza virus subtypes affecting the poultry industry. Based on the previous research, exploratory studies can be conducted to investigate whether there are differences in the replication kinetics of H5 and H9 avian influenza in vivo. Fifth, studying the replication kinetics of influenza viruses in vivo also helps to understand the mechanisms of antiviral drug resistance development. This is of great significance for timely adjustment of treatment regimens and slowing the formation of drug resistance. For example, quantitative fluorescence can detect some cRNA and mRNA sequences. If these sequences contain key sites related to pathogenicity, drug resistance, or transmissibility, they can be detected promptly through sequencing. This allows for the rapid detection of virulence, drug resistance, and transmissibility, providing valuable information for virus surveillance. In summary, studying the replication dynamics of influenza viruses not only deepens our understanding of influenza infection but also provides crucial information for prevention and treatment, helping to address the challenges of human infection with avian influenza.

[0004] To study the in vivo replication dynamics of influenza virus, it is necessary to detect the levels of the three types of influenza virus RNA (vRNA, cRNA, and mRNA). However, existing detection methods can only detect these three types of influenza virus RNA in in vitro cell lines and are not suitable for in vivo infection experiments in animals with complex template amounts. Therefore, there is an urgent need to develop a method for detecting the three types of influenza virus RNA that is applicable to in vivo infection experiments in animals with complex template amounts. Summary of the Invention

[0005] To address the technical limitations of existing technologies, which can only detect the three RNAs of influenza virus in vitro on cell lines and are not suitable for in vivo infection experiments in animals (e.g., mice) with complex template quantities, this invention provides a chain-specific reverse transcription real-time quantitative PCR (qRT-PCR) method for detecting the content of the three RNAs of influenza virus H7N9 in vivo. This method provides an important tool for studying the replication kinetics of influenza virus H7N9 in vivo.

[0006] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a primer and probe combination for detecting the content of three RNAs of influenza virus H7N9 in vivo. The primer and probe combination is either primer and probe combination A for detecting the content of three RNAs of the H7N9 PB2 gene or primer and probe combination B for detecting the content of three RNAs of the H7N9 NA gene. Primer and probe combination A consists of strand-specific reverse transcription primers for PB2 vRNA, cRNA, and mRNA with nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, upstream and downstream primers and probes for PB2 vRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, upstream and downstream primers and probes for PB2 cRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, and upstream and downstream primers and probes for PB2 mRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.9. Primer and probe combination B consists of primers and probes with nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, upstream and downstream primers and probes for PB2 cRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.9. The primers for strand-specific reverse transcription of NA vRNA, cRNA and mRNA shown in NO.12, SEQ ID NO.13 and SEQ ID NO.14; the upstream and downstream primers and probes for NA vRNA real-time PCR with nucleotide sequences shown in SEQ ID NO.15, SEQ ID NO.5 and SEQ ID NO.16; the upstream and downstream primers and probes for NA cRNA real-time PCR with nucleotide sequences shown in SEQ ID NO.17, SEQ ID NO.8 and SEQ ID NO.18; and the upstream and downstream primers and probes for NA mRNA real-time PCR with nucleotide sequences shown in SEQ ID NO.19, SEQ ID NO.11 and SEQ ID NO.18.

[0007] A second objective of this invention is to provide the application of the above-mentioned primer and probe combination in the study of the in vivo replication dynamics of influenza virus H7N9.

[0008] A third objective of this invention is to provide a kit containing the above-described primer and probe combination.

[0009] A fourth objective of this invention is to provide the application of the above-mentioned kit in the study of the in vivo replication dynamics of influenza virus H7N9.

[0010] The fifth objective of this invention is to provide a method for detecting the levels of three RNA molecules of influenza virus H7N9 in vivo using the above-described primer and probe combination or the above-described kit, the method comprising the following steps: S1. Constructing a standard curve: Calculate the copy number of the full-length vRNA, cRNA, and mRNA products of the H7N9 PB2 or NA gene, which were transcribed and recovered in vitro, based on their molecular weight. Reverse transcribe them into cDNA using the corresponding strand-specific reverse transcription primers as standards for the corresponding RNA. Amplify the cDNA using the corresponding real-time PCR primers and probes for the three RNAs and record the Ct values. Plot a standard curve equation using the Ct values ​​and corresponding copy numbers of standards covering 6-8 consecutive 10-fold dilutions of RNA. S2. Extract RNA from mouse lungs and reverse transcribe it into cDNA using strand-specific reverse transcription primers used to detect the content of H7N9 PB2 or NA gene vRNA, cRNA, and mRNA, respectively. Amplify the cDNA using the corresponding fluorescent quantitative PCR primers and probes for the three RNAs and record the Ct values. Substitute the measured Ct values ​​of the three RNAs into the standard curve equation obtained in S1 to calculate the copy number of the three RNAs. Except for the different templates, the reaction system and reaction conditions for fluorescent quantitative PCR described in S1 and S2 are the same.

[0011] In one embodiment of the present invention, the reverse transcription reaction system consists of the following components: a 5.5 μL mixture of 2 μg RNA and 5 pmol strand-specific reverse transcription primers for vRNA, cRNA, or mRNA; 2 μL of 10×RT Mix; 2 μL of HiScript II Enzyme Mix; for reverse transcription of vRNA, 10.5 μL of RNase-free sterile water is added; and for reverse transcription of cRNA and mRNA, 10.5 μL of saturated trehalose solution is added.

[0012] In one embodiment of the present invention, the reverse transcription reaction conditions for vRNA are: incubation at 65°C for 5 min; incubation at 55°C for 45 min; incubation at 85°C for 2 min, and maintenance at 4°C; the reverse transcription reaction conditions for cRNA and mRNA are: incubation at 65°C for 5 min; incubation at 60°C for 45 min; incubation at 85°C for 2 min, and maintenance at 4°C.

[0013] In one embodiment of the present invention, the real-time PCR reaction system consists of the following components: 10 μL of Premix ExTaq (Cat No. RR390A; Takara), 0.4 μL each of 10 μM upstream and downstream primers, 0.8 μL of 10 μM probe, 0.4 μL of ROX, 2 μL of template, and 6 μL of ddH2O.

[0014] In one embodiment of the present invention, the reaction conditions for real-time PCR are as follows: 50℃ for 2 min; 95℃ for 1 min; 95℃ for 5 s, 60℃ for 30 s, 40 cycles, with a heating rate of 1.6℃ / s; and the signal is acquired at 60℃.

[0015] The sixth objective of this invention is to provide the application of the above-described method in the study of the replication kinetics of influenza virus H7N9 in vivo.

[0016] In one embodiment of the present invention, studying the in vivo replication kinetics of influenza virus H7N9 refers to detecting the copy number of three RNAs in mice infected with influenza virus H7N9 at different time points after H7N9 infection.

[0017] The beneficial effects of this invention are: The quantitative real-time primer and probe combination and method provided by this invention for detecting the levels of three RNAs (vRNA, cRNA, and mRNA) of the PB2 and NA genes of H7N9 influenza virus in vivo can be used to determine the levels of these three RNAs in mice. This invention provides an experimental protocol for studying the actual replication of H7N9 influenza virus in vivo, enabling the study of the replication kinetics of H7N9 influenza virus at various time points after infection. This further deepens our understanding of H7N9 influenza virus replication in vivo and is of great significance for understanding the virus's transmission, course, treatment, and prevention. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the principle of using qRT-PCR to detect the vRNA, cRNA, and mRNA of influenza virus H7N9 PB2 and NA genes in Example 1. Figure 2 The image shows the results of cross-amplification of cDNA from influenza virus H7N9 vRNA, cRNA, and mRNA using three pairs of primers via qRT-PCR. Figure 3 Standard curves of three RNAs of the influenza virus H7N9 PB2 gene or NA gene, plotted based on Ct values ​​and RNA copy numbers; Figure 4 Amplification curves of the vRNA, cRNA, and mRNA of the H7N9 influenza virus PB2 gene or NA gene; Figure 5 This is a graph showing the changes in the levels of PB2 or NA gene vRNA, cRNA, and mRNA in the lung tissue of mice infected with influenza virus H7N9 over time. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments are merely illustrative and not intended to limit the scope of the invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention can be made without departing from the spirit and scope of the invention, but such modifications and substitutions are all within the protection scope of the present invention. The processes, conditions, experimental methods, and reagents used in implementing the present invention, except as specifically mentioned below, are all common knowledge in the art and conventional market products; the present invention does not have any particular limitations.

[0020] Example 1: A quantitative fluorescence method for detecting the levels of three RNA genes (PB2 or NA) of influenza virus H7N9 in mice. This embodiment utilizes a qRT-PCR method to detect the vRNA, cRNA, and mRNA of the H7N9 PB2 or NA gene, as illustrated in the diagram below. Figure 1 As shown.

[0021] (1) Viral infection Six-week-old C57BL / 6 mice were subjected to tail clipping to collect a small amount of blood, which was then treated with receptor-destroying enzyme (RDE) for hemagglutination inhibition testing to rule out influenza virus infection. When all hemagglutination inhibition results were negative, the mice were weighed, and mice that were out of weight were discarded. The remaining mice, meeting the weight standard (15.5–16.5 g), were allowed to acclimatize in their cages for 2–3 days before experimental preparation. Dry ice was placed in a 2 L beaker and lined with absorbent paper to prevent frostbite, ensuring sufficient dry ice to maintain a CO2-rich anesthesia environment. An influenza virus (H7N9) suspension was prepared by diluting the original virus stock solution on ice with phosphate-buffered saline (PBS) to a volume of 50 μL, containing a total viral load of 10... 6 Chicken embryo median infection level (EID) 50 ); Ensure the mice are in good health and check for any abnormal signs. If none are found, begin anesthesia by placing the mice in a CO2-filled beaker. This will induce anesthesia. Once the mice are fully anesthetized, i.e., their thoracic-abdominal breathing has switched to abdominal breathing and they have inhaled three times, carefully remove the anesthetized mice and place them on a prepared surgical table. Use a pipette to drip a pre-diluted H7N9 virus suspension into the mice's nostrils for nasal infection. After infection, return the mice to their cages to allow them to recover naturally.

[0022] (2) RNA extraction Mice were anesthetized with CO2 and then euthanized. The lungs of the mice were removed, cut into small pieces, and soaked in 3 mL of RNAprotectTissueReagent (Cat. No. 76106; QIAGEN) protective agent. After being placed at 4°C overnight, they were transferred to -20°C for long-term storage.

[0023] For RNA extraction from lungs, 1 mL of Buffer RLT was added to Lysing Matrix A (Cat. No. 116910500; MP Biomedicals). Mouse lung fragments were evenly divided into three tubes and homogenized using a FastPrep-24 5G system (MP Biomedicals) at 6 M / s for 30 s, twice. After homogenization, RNA was extracted according to the RNeasyMini Kit (Cat. No. 74106; QIAGEN) instructions. RNA concentration was measured spectrophotometrically, and RNA integrity was assessed by agarose gel electrophoresis. Finally, the extracted RNA was aliquoted and stored at -70°C.

[0024] (3) Primer design Tags were designed for reverse transcription primers targeting three RNAs of the H7N9 influenza virus PB2 or NA gene. Appropriate sequences were extracted from a genome unrelated to mice for primer design (vRNA-Tag named S6TagR, nucleotide sequence as shown in SEQ ID NO. 5; cRNA-Tag named S1TagR, nucleotide sequence as shown in SEQ ID NO. 8; mRNA-Tag named S9TagR, nucleotide sequence as shown in SEQ ID NO. 11). After selecting the appropriate tags, RT primers specifically binding to the vRNA, cRNA, and mRNA tails were ligated to design the final tag-containing RNA chain-specific RT primers (PB2 vRNA RT primer named PB2vRNAS6RT-TagR, nucleotide sequence as shown in SEQ ID NO. 1; PB2 cRNA RT primer named PB2cRNAS1RT-TagR, nucleotide sequence as shown in SEQ ID NO. 2; PB2 mRNA RT primer named PB2mRNAS9RT-TagR, nucleotide sequence as shown in SEQ ID NO. 3; NA vRNA...). The RT primer is named NAvRNAS6RT-TagR, and its nucleotide sequence is shown in SEQ ID NO.12; the NA cRNA RT primer is named NAcRNAS1RT-TagR, and its nucleotide sequence is shown in SEQ ID NO.13; and the NA mRNA RT primer is named NAmRNAS9RT-TagR, and its nucleotide sequence is shown in SEQ ID NO.14.Using the tag sequences as downstream primers for qPCR, upstream primers and probes matching the three tag primers were designed using Primer Premier 5.0 software. (The upstream primers for qPCR of PB2 vRNA, cRNA, and mRNA were named PB2vRNA-F, PB2cRNA-F, and PB2mRNA-F, respectively, with nucleotide sequences shown in SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10; the probe corresponding to vRNA was named Probe-PB2vRNA, with a nucleotide sequence shown in SEQ ID NO.6; the probes corresponding to cRNA and mRNA were named Probe-PB2cmRNA, with nucleotide sequences shown in SEQ ID NO.9; the upstream primers for qPCR of NA vRNA, cRNA, and mRNA were named NAvRNA-F, NAcRNA-F, and NAmRNA-F, respectively, with nucleotide sequences shown in SEQ ID NO.15, SEQ ID NO.17, and SEQ ID NO.19; the probe corresponding to NA vRNA was named Probe-NAvRNA, with a nucleotide sequence shown in SEQ ID NO.16.) The corresponding probes for cRNA and mRNA are named Probe-NAcmRNA (nucleotide sequences are shown in SEQ ID NO.18). All probes are labeled with a FAM fluorescent group at the 5' end and an MGB quencher group at the 3' end. Information on all primers and probes is shown in Table 1 (PB2) and Table 2 (NA).

[0025] Table 1. Primer and probe information for detecting three RNAs in the PB2 gene of influenza virus H7N9.

[0026] Table 2. Primer and probe information for detecting three RNAs of the H7N9 influenza virus NA gene.

[0027] In this embodiment, the PB2 vRNA content of influenza virus H7N9 was detected using strand-specific RT primer PB2vRNAS6RT-TagR, qPCR primers PB2vRNA-F, S6TagR, and probe Probe-PB2vRNA; the PB2 cRNA content of influenza virus H7N9 was detected using RT primer PB2 cRNAS1RT-TagR, qPCR primers PB2cRNA-F, S1TagR, and probe Probe-PB2cmRNA; and the PB2 mRNA content of influenza virus H7N9 was detected using RT primer PB2mRNAS9RT-TagR, qPCR primers PB2mRNA-F, S9TagR, and probe Probe-PB2cmRNA.

[0028] In this embodiment, the NA vRNA content of influenza virus H7N9 was detected using strand-specific RT primer NAvRNAS6RT-TagR, qPCR primers NAvRNA-F and S6TagR, and probe Probe-NAvRNA; the NA cRNA content of influenza virus H7N9 was detected using RT primer NAcRNAS1RT-TagR, qPCR primers NAcRNA-F and S1TagR, and probe Probe-NAcmRNA; and the NA mRNA content of influenza virus H7N9 was detected using RT primer NAmRNAS9RT-TagR, qPCR primers NAmRNA-F and S9TagR, and probe Probe-NAcmRNA.

[0029] (4) Preparation of standard products RNA samples containing known concentrations of full-length vRNA, cRNA, and mRNA of influenza virus H7N9 PB2 or NA transcribed in vitro were used. The required target sequence copy number was calculated based on their molecular weight, as shown in Formula (1). Strand-specific RT was then performed to obtain cDNA. The obtained cDNA was diluted according to the calculated RNA copy number to a concentration of 10⁻⁶. 9 copies / 2 μL, 10 8 copies / 2 μL, 10 7 copies / 2 μL, 10 6 copies / 2 μL, 10 5 copies / 2 μL, 10 4 copies / 2 μL, 10 3 copies / 2 μL and 10 2 2 μL of diluted cDNA was added to the qPCR system as a template, and a standard curve was plotted based on the Ct value and RNA copy number.

[0030] RNA (copy) (1) (5) High-temperature reverse transcription Trehalose can thermally stabilize various reverse transcriptases, including MMLV, enabling them to perform their functions without inactivation at 60°C and reducing the annealing of nonspecific primers. This invention uses saturated trehalose as a stabilizer to increase the reverse transcription temperature and reduce nonspecific reactions. Reverse transcription was performed using the HiScript II 1st Strand cDNA Synthesis Kit (Cat No. R212-02; Vazyme). Specifically, 2 μg of RNA and 5 pmol of tag-containing strand-specific RT primers (PB2vRNAS6RT-TagR, PB2cRNAS1RT-TagR, PB2mRNAS9RT-TagR, NAvRNAS6RT-TagR, NAcRNAS1RT-TagR, or NAmRNAS9RT-TagR) were mixed to a 5.5 μL mixture and incubated at 65°C for 5 min. Then, 2 μL of 10×RT Mix and 2 μL of HiScript II Enzyme Mix were added. 10.5 μL of RNase-free sterile water was added to the vRNA system, and 10.5 μL of saturated trehalose was added to the cRNA and mRNA systems. After mixing thoroughly by pipetting, the vRNA was incubated at 55°C for 45 min, 85°C for 2 min, and maintained at 4°C; the cRNA and mRNA were incubated at 60°C for 45 min, 85°C for 2 min, and maintained at 4°C. After reverse transcription, cDNA was obtained.

[0031] (6) qPCR amplification qPCR was performed using the Premix Ex Taq (Probe qPCR) kit (Cat No. RR390A; Takara). The qPCR reaction mixture consisted of 10 μL Premix Ex Taq, 0.4 μL each of 10 μM forward and reverse primers, 0.8 μL of 10 μM probe, 0.4 μL ROX, 2 μL template, and 6 μL ddH2O.

[0032] The qPCR reaction conditions were: 50℃ for 2 min; 95℃ for 1 min; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles, with a heating rate of 1.6℃ / s; the signal was acquired at 60℃.

[0033] (7) qPCR specificity identification Total RNA was extracted from lung tissue of mice infected with H7N9 influenza virus. Three tag-containing strand-specific RT primers for each of the PB2 or NA genes were used to RT-transmit cDNA of PB2 or NA vRNA, cRNA, and mRNA, respectively, as templates. The three reverse-transcribed cDNAs of the PB2 or NA genes were amplified using qPCR primers targeting the three RNAs and corresponding probes, and Ct values ​​were measured. The Ct values ​​were normalized to the average Ct values ​​for each targeted RNA to calculate the relative abundance. The error bar represents the standard deviation of three independent experiments. Values ​​below the dashed line indicate no fluorescence detection or no amplification curve. The results showed that the PB2 vRNA amplification system could only detect fluorescence signals when cDNA RT-triggered by vRNA-Tag primers was used as a template, and could not detect signals when cDNA RT-triggered by both cRNA-Tag and mRNA-Tag primers was used as a template. The same results were obtained for cRNA and mRNA detection. Figure 2 The above results demonstrate that the qPCR method provided in this embodiment can specifically detect the vRNA, cRNA, and mRNA of the influenza virus H7N9 PB2 or NA gene in vivo.

[0034] Example 2: Application of a quantitative real-time method for detecting the levels of vRNA, cRNA, and mRNA of influenza virus H7N9 PB2 or NA genes in mice in the study of influenza virus replication kinetics in vivo. This invention verifies the practicality of the method described in Example 1 by detecting the viral RNA content in the lungs of mice infected with influenza virus H7N9. Fifteen C57BL / 6 mice were divided into 5 groups of 3 mice each, at a ratio of 10... 6 EID 50Mice infected with H7N9 influenza virus had their lungs collected at 6 h, 12 h, 24 h, 36 h, and 48 h post-infection to extract total RNA. Two μg of total RNA was used for PB2 or NA gene chain-specific RT-PCR to obtain cDNA corresponding to the three RNAs, which served as templates. qPCR was performed to detect the vRNA, cRNA, and mRNA of the PB2 and NA genes, and standard curves were plotted. Results showed good reproducibility between samples, with the detection limits for each RNA being as follows: PB2 vRNA 46.254 copies, PB2 cRNA 46.081 copies, PB2 mRNA 46.103 copies, NA vRNA 45.748 copies, NA cRNA 46.782 copies, and NA mRNA 46.119 copies. The amplification efficiencies of all primer sets were greater than 82% (the amplification efficiencies of PB2 gene vRNA, cRNA, and mRNA were 83.126%, 84.718%, and 88.037%, respectively, and the amplification efficiencies of NA gene vRNA, cRNA, and mRNA were 89.281%, 82.281%, and 85.006%, respectively), and the standard curves showed good linear correlation. Figure 3 and Figure 4 This indicates that a standard curve was successfully plotted, and the established qRT-PCR method is suitable for the quantitative detection of viral RNA in mouse lung tissue.

[0035] RNA detection results from mouse lungs at different time points post-infection showed that copy numbers of all three RNAs could be effectively detected in samples from 6 to 48 hours post-infection. At 6 hours post-infection, there were no significant differences in vRNA, cRNA, or mRNA levels between the PB2 and NA genes. However, starting from 12 hours post-infection for cRNA, and from 24 hours post-infection for vRNA and mRNA, differences in the average viral RNA copy number levels gradually appeared between the PB2 and NA genes, indicating different transcription or replication efficiencies of different viral gene fragments during viral infection. Within 24 hours post-infection, intra-group variation in viral RNA levels was small, but became increasingly pronounced after 24 hours, indicating increasing inter-host variability in viral replication. Furthermore, the overall trend for all three viral RNA levels was a rapid increase before 24 or 36 hours post-infection, followed by a certain degree of decline. Figure 5 ).

[0036] In summary, these results demonstrate that the qRT-PCR detection method provided in Example 1 can quantitatively analyze the types of influenza virus H7N9 PB2 and NA gene RNA in vivo and effectively capture the replication dynamics of the virus in mammalian hosts.

[0037] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A primer and probe combination for detecting the content of three RNAs of influenza virus H7N9 in vivo, characterized in that, The primer and probe combination is either primer and probe combination A for detecting the content of three RNAs in the H7N9 PB2 gene or primer and probe combination B for detecting the content of three RNAs in the H7N9 NA gene. Primer and probe combination A consists of strand-specific reverse transcription primers for PB2 vRNA, cRNA, and mRNA with nucleotide sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively; upstream and downstream primers and probes for PB2 vRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively; upstream and downstream primers and probes for PB2 cRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, respectively; and upstream and downstream primers and probes for PB2 mRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 9, respectively. Primer and probe combination B consists of primers and probes for NA with nucleotide sequences as shown in SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively. The primers and probes for chain-specific reverse transcription of vRNA, cRNA, and mRNA, with nucleotide sequences as shown in SEQ ID NO.15, SEQ ID NO.5, and SEQ ID NO.16, respectively; the primers and probes for NA vRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO.17, SEQ ID NO.8, and SEQ ID NO.18, respectively; and the primers and probes for NA mRNA real-time PCR with nucleotide sequences as shown in SEQ ID NO.19, SEQ ID NO.11, and SEQ ID NO.18, respectively.

2. A kit containing the primer and probe combination of claim 1.

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