Primer combination for obtaining whole genome sequence of American strain of porcine reproductive and respiratory syndrome virus, kit and application thereof

Abstract

The invention discloses a primer combination and a kit for obtaining a whole genome sequence of a porcine reproductive and respiratory syndrome virus American type strain and application of the primer combination and the kit. The primer combination can realize full coverage of a genome of the porcine reproductive and respiratory syndrome virus American type strain; in addition, the coverage rate of the primer combination on porcine reproductive and respiratory syndrome virus sequences in Asian areas in recent 10 years is far higher than that of an existing primer combination, and the reliability and the universality of complete acquisition of the genome of the American type strains of the porcine reproductive and respiratory syndrome are greatly improved. The invention provides a method for quickly acquiring the whole genome sequence of the porcine reproductive and respiratory syndrome virus, and provides a solid technical basis for advanced applications such as precise typing, traceability, virulence / immune escape analysis, vaccine evaluation, variation monitoring and the like based on the whole genome. The method can realize rapid tracing of blue ear pathogens, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a primer combination, a kit, and its application for obtaining the complete genome sequence of the American strain of porcine reproductive and respiratory syndrome virus, belonging to the field of viral genome detection. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most impactful diseases affecting the global swine industry. PPRSV can infect pigs of all ages, causing reproductive disorders in sows (abortion, mummified fetuses, stillbirths, and premature births) and respiratory symptoms in piglets and growing pigs, leading to high mortality rates and causing enormous losses to the global swine industry. First discovered in the United States in 1987, PPRSV has become one of the major infectious diseases seriously threatening the global swine industry. In early studies, experts classified PPRSV into two genotypes based on its genetic variation: the American type and the European type. In my country, PPRSV is mainly genotype II, the American type. PPRSV is highly prevalent and highly variable. Rapid genetic identification to obtain complete or near-complete PPRSV genome sequences will enhance our understanding of PPRSV epidemiology and provide a basis for clinical veterinary guidance and the implementation of control measures.

[0003] Nanopore sequencing technology enables direct sequencing of nucleic acids by immobilizing nanopore proteins on a resistive membrane and using dynamometer proteins to pull nucleic acid chains through the nanopores. Compared with traditional first-generation sequencing (Sanger sequencing) and second-generation sequencing (Roche 454, SOLiD, and Illumina), it offers significant advantages such as long read lengths, portability, and real-time sequencing. The emergence of nanopore long-read sequencing technology has provided a new solution for viral genome sequencing; however, currently, there is a lack of an efficient method specifically for constructing the full-length genome of PRRSV (Pleurotus ostreatus) based on nanopore long-read sequencing. Summary of the Invention

[0004] Objective of the Invention: The first objective of this invention is to provide a primer combination with high coverage for obtaining the complete genome sequence of the American strain of porcine reproductive and respiratory syndrome virus. The second objective of this invention is to provide a kit and detection method based on the above primer combination.

[0005] Technical solution: The primer combination for obtaining the whole genome sequence of the American strain of porcine reproductive and respiratory syndrome virus according to the present invention, wherein the primer sequences in the primer combination are shown in SEQ ID NO.1 to 33.

[0006] The kit for obtaining the whole genome sequence of the American strain of porcine reproductive and respiratory syndrome virus according to the present invention comprises the primer combination described above.

[0007] Furthermore, the kit also includes 5×VAHTS RT Multi-PCR Mix, Nuclease & Nucleic Acid-free ddH2O.

[0008] The application of the primer combinations and kits described in this invention in obtaining the whole genome sequence of the American strain of porcine reproductive and respiratory syndrome virus.

[0009] The method for obtaining the complete genome sequence of the American strain of porcine reproductive and respiratory syndrome virus according to the present invention includes the following steps:

[0010] (1) Sample collection and viral nucleic acid extraction;

[0011] (2) The extracted nucleic acid is reverse transcribed and amplified to obtain an amplified fragment; the amplification uses the primer combination of claim 1;

[0012] (3) Mix the amplified fragments to construct a sequencing library;

[0013] (4) Sequencing of the amplified fragments in the sequencing library using a nanopore sequencer;

[0014] (5) Bioinformatics analysis of sequencing information to obtain whole genome sequence.

[0015] Furthermore, the samples include saliva samples, nasal and oral swab samples, blood samples, fecal samples, tissue samples, environmental samples, or virus cultures.

[0016] Further, the amplification program in step (2) is: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 95℃ for 30 s, extension at 72℃ for 3 min, 35 cycles; extension at 72℃ for 5 min.

[0017] Furthermore, bioinformatics analysis includes sequencing data filtering, sequencing depth calculation, sequencing result assembly, viral sequence tracing, and finally obtaining the whole genome sequence.

[0018] Beneficial effects: Compared with existing technologies, this invention has the following significant advantages: The primer combination provided by this invention can amplify almost all American PRRSV strains from the past decade. The sequencing and analysis scheme provided by this invention can be used to rapidly obtain the full-length sequence of the highly variable American PRRSV, thereby enabling rapid typing and tracing, and can quickly provide a basis for clinical veterinary guidance and implementation of prevention and control measures. Attached Figure Description

[0019] Figure 1 This is a sequence identity curve of the American PRRSV type provided in an embodiment of this application.

[0020] Figure 2 This is a schematic diagram showing the coverage of the PRRSV sequence by the primer set amplification products in this invention.

[0021] Figure 3 This is a heatmap showing the coverage of PRRSV sequences in Asia over the past 10 years using the primer set in claim 1.

[0022] Figure 4 This is a heatmap showing the coverage of PRRSV sequences in Asia over the past 10 years by primer set amplification products from other patents (application number: 202510271967.1).

[0023] Figure 5 This is a schematic flowchart of a nanopore sequencing method provided in an embodiment of this application.

[0024] Figure 6 This is the whole genome sequencing depth in Example 2.

[0025] Figure 7 This is the result of the source analysis of the assembly sequence in Example 2. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0027] The biological materials used in the examples were obtained from the following sources: PRRSV01 to PRRSV09 were pure cultures of PRRSV isolated from pigs infected with PRRSV; the sample corresponding to PRRSV10 was a pure culture of strain XJ17-5, provided by the Preventive Veterinary Medicine Research Group of the College of Veterinary Medicine, Yangzhou University, with the reference genome being MK759853.1.

[0028] The reagents used in the examples were sourced from the following sources: QIAamp Viral RNA Mini Kit (52904) for virus extraction was purchased from Qiagen Biotechnology (Germany); PureScript II 1st StrandcDNA Synthesis Kit (Low Nucleic-acid Contamination, PR111), UltraClean Pathogen Multiplex PCR Mix for DNA & RNA (UNA312), and Equalbit 1×dsDNAHS Assay Kit (EQ121-01) for double-stranded DNA fluorescence quantitative detection were purchased from Nanjing Novizan Biotechnology Co., Ltd.; QNLK V1.1 direct barcode library preparation kit, QDS V1.5 sequencing kit, QCell-6k sequencing chip, and QPursue-6k gene sequencer were provided by Chengdu Qitan Technology Co., Ltd.; and AMPure XP nucleic acid purification kit was purchased from Beckman Coulter International Trading (Shanghai) Co., Ltd.

[0029] Example 1: Design of primer sequences for targeted amplification of American PRRSV and prediction of amplification effect.

[0030] Full-length PRRSV sequences from China over the past 5 years were collected from the NCBI virus database. These sequences were aligned using the bioinformatics software MAFFT, and a phylogenetic tree was constructed using the bioinformatics software FastTree, removing European-type sequences. A sequence consistency map was then plotted using the readDNAStringSet library from the Biostrings package. The results are shown below. Figure 1 As shown in the diagram. Highly conserved regions were selected for primer site design. Simultaneously, two forward primers and two reverse primers were designed for each primer site to enhance the coverage of the primer set for American PRRSV. Specifically, three forward primers were designed at the PRRSV1 IP2 site to improve coverage at this site. A schematic diagram illustrating the coverage of the final amplified products to the sequencing results is shown in the diagram. Figure 2 As shown in Table 1, the final primer sequences were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0031] Table 1 PRRSV primer sequences

[0032]

[0033]

[0034] PRRSV full-length sequences from Asia over the past 10 years were collected from the NCBI virus database. The bioinformatics software BLAST was used to calculate the identity between each primer and each PRRSV sequence. During the calculation, a maximum of two base mismatches or deletions were allowed to simulate the mismatches permitted during primer annealing. Based on the highest identity value of each primer site, a heatmap of primer pair coverage of PRRSV sequences from Asia over the past 10 years was plotted. The results are shown below. Figure 3 As shown, the horizontal axis represents different strains, and the vertical axis represents different primer sites. Using the same method as described above, a heatmap of primer pair coverage of PRRSV sequences in Asia over the past 10 years, as published in other patents (application number: 202510271967.1), was plotted, and the results are shown below. Figure 4 As shown. Compared to primer sets disclosed in other patents, the primers in this invention have extremely high coverage in different American PRRSV types.

[0035] Table 2 Coverage of different primer sites in this invention

[0036]

[0037] Table 3. Coverage of different primer sites in patent 202510271967.1

[0038]

[0039] Example 2: Obtaining the full-length genome of PRRSV American type.

[0040] The flowchart of the protocol for obtaining the complete genome of American porcine reproductive and respiratory syndrome virus (PRRSV) using the primer set constructed in Example 1 is as follows: Figure 5 As shown, the specific method is as follows:

[0041] First, nucleic acid was extracted from pure cultures of wild-type American porcine reproductive and respiratory syndrome virus (PRRSV) 01-10 using the QIAamp Viral RNA Mini Kit. The specific steps are as follows:

[0042] (1) Pipette 560 μL of Buffer ALV into a 1.5 mL centrifuge tube and add 140 μL of cell culture supernatant. Vortex for 15 seconds.

[0043] (2) Incubate at room temperature for 10 min, then centrifuge briefly to remove liquid droplets from the inner wall and inner cap.

[0044] (3) After adding 560 μL of ethanol, vortex for 15 seconds and then centrifuge briefly to remove liquid droplets from the inner wall and inner cap.

[0045] (4) Carefully aspirate 630 μL of the QIAamp Mini column obtained in the previous step, cap it, and centrifuge at 6000×g for 1 min. Transfer the QIAamp Mini column to a new 2 ml collection tube.

[0046] (5) Repeat the previous step.

[0047] (6) Carefully open the cap and add 500 μL BufferAW1. After capping, centrifuge at 6000×g for 1 min. Transfer the QIAamp Mini column to a new 2 ml collection tube and discard the old tube containing waste liquid.

[0048] (7) Carefully open the lid and add 500 μL BufferAW2. Close the lid and centrifuge at 12,000 × g for 3 min.

[0049] (8) Transfer the QIAamp Mini column to a new 2ml collection tube and centrifuge at 12,000×g for 1 min to remove residual ethanol.

[0050] (9) Transfer the QIAamp Mini column to a new 1.5ml centrifuge tube, carefully open the cap, and add 60μL BufferAVE onto the adsorption membrane. After capping, incubate at room temperature for 1 min. Centrifuge at 6,000×g for 1 min. Store the extracted viral RNA at -80℃.

[0051] Then, using the PureScript II 1st Strand cDNA Synthesis Kit, the RNA virus in the nucleic acid was reverse transcribed into cDNA for amplification and detection of the RNA. The specific steps are described below.

[0052] (1) RNA template denaturation. The reaction system consisted of 10 pg to 1 μg RNA, with enzyme-free water added to a final volume of 14 μL. The prepared system was heated at 65°C for 5 min, then rapidly cooled on ice and allowed to stand on ice for 2 min.

[0053] (2) cDNA synthesis reaction. The reaction system consisted of: 14 μL of the mixture from the previous step, 2 μL of 10×RTMix, 2 μL of PureScript Enzyme Mix, and 2 μL of Random Hexamers. The mixed sample was placed in a PCR instrument and incubated at 25℃ for 5 min, 37℃ for 45 min, and 85℃ for 5 s. The reverse transcription product was stored at -20℃.

[0054] The cDNA obtained in the previous step was amplified using two primer pools. The primer sequences used for amplification in primer pool 1 are SEQ ID NO: 1 to SEQ ID NO: 16, and the primer sequences used for amplification in primer pool 2 are SEQ ID NO: 17 to SEQ ID NO: 33.

[0055] The reaction mixture consisted of 10 μL Input cDNA, 5 μL Primer Mix, 5 μL 5×VAHTS RT Multi-PCR Mix, and 5 μL Nuclease & Nucleic acid-free ddH2O. The reaction program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 95℃ for 30 s, extension at 72℃ for 3 min, for 35 cycles; and extension at 72℃ for 5 min.

[0056] After the PCR reaction, primer pools Pool 1 and Pool 2 were mixed, and the amplification product from the previous step was purified using AMPure XP magnetic beads. The specific steps are as follows:

[0057] (1) Mix the AMPure XP magnetic beads by oscillation and bring them to room temperature.

[0058] (2) Use Nuclease & Nucleic acid-free ddH2O to bring the PCR product to 100 μL.

[0059] (3) After vortexing the AMPure XP magnetic beads, add 0.4 times the volume of magnetic beads (40 μL) to the above PCR reaction system, and gently pipette to ensure the system is homogeneous. Incubate at room temperature for 5 min to allow the library to bind to the magnetic beads.

[0060] (4) After briefly centrifuging the reaction tube, place it on a magnetic rack for 5 minutes to enrich the magnetic beads. Carefully discard the supernatant to avoid disturbing the magnetic beads.

[0061] (5) Add 200 μL of freshly prepared 80% ethanol, taking care to avoid disturbing the magnetic beads. After incubating for 30 seconds, carefully aspirate the supernatant.

[0062] (6) Repeat the previous step.

[0063] (7) Briefly centrifuge the sample, place the reaction tube on a magnetic rack for 30 seconds, and use a pipette to remove all residual ethanol.

[0064] (8) Open the lid and dry for 3-5 minutes. Avoid over-drying the magnetic beads, which will reduce nucleic acid production.

[0065] (9) Remove the reaction tube from the magnetic rack, add 22 μL of Nuclease & Nucleic acid-free ddH2O, mix well by pipetting, and incubate at room temperature for 2 min.

[0066] (10) Place the reaction tube on the magnetic rack and separate the magnetic beads until the solution is clear.

[0067] (11) Pipette 20 μL of supernatant into a new EP tube and store at -20°C.

[0068] The purified PCR products were quantified using Qubit4 according to the instructions of the Double-Stranded DNA Quantitative Detection Kit (Equalbit 1×dsDNA HSAssay Kit, EQ121-01). The quantified purified PCR products were then used to construct a third-generation nanopore sequencing library using the QNLKV1.1 direct barcode library construction kit. The specific steps are as follows:

[0069] (1) First, end repair was performed on the amplified products. The concentration of the PCR product was determined using the Equalbit 1×dsDNAHS Assay Kit, and the molar concentration of the sample was calculated according to the following formula: Molar concentration (fmol / μL) = 1613 * Mass concentration (ng / μL) / Sample fragment length (bp). The reaction mixture was prepared in the PCR tube according to the following system: 200 fmol of sample DNA, 2.5 μL of DNA repair buffer, 1 μL of end repair enzyme, and Nuclease-free water to make up to 20 μL. The tube was gently tapped, briefly centrifuged, and then placed in the PCR instrument. The reaction was carried out at 30℃ for 10 min, 65℃ for 10 min, and finally stored at 4℃.

[0070] (2) Ligation and purification of the end-repair products using barcode adapters. Remove the reaction tubes from the PCR instrument and prepare the reaction mixture as follows: 20 μL of the end-repair product obtained in the previous step, 1 μL of barcodes (BC01-10), 7.5 μL of 4× ligation buffer, and 1.5 μL of DNA ligase. Gently tap the tube wall, briefly centrifuge, and incubate at room temperature for 10 min. After incubation, add 2 μL of termination buffer to the ligation mixture, gently tap the tube wall to mix, and briefly centrifuge. Mix the samples ligated with different barcodes in a 1.5 mL centrifuge tube. Purify the ligation products using AMPure XP magnetic beads. The purification steps are as follows:

[0071] a. Vibrate the AMPure XP magnetic beads to mix them thoroughly and equilibrate to room temperature.

[0072] b. Based on the total volume of the reaction product in the 1.5 mL centrifuge tube from the previous step, add 0.4 times the volume of AMPure XP magnetic beads, mix by blowing and stirring, and let stand at room temperature for 5 min.

[0073] c. Perform instantaneous centrifugation. Place the centrifuge tube on a magnetic rack and let it stand for 5 minutes. Once the solution is clear, discard the supernatant.

[0074] d. Add 400 μL of freshly prepared 80% ethanol, wait 30 seconds, and then discard the supernatant.

[0075] e. Repeat step d.

[0076] f. After instantaneous centrifugation, place the centrifuge tube on a magnetic rack. Once the magnetic beads are fully adsorbed, use a pipette to remove all residual ethanol.

[0077] g. Open the lid and dry for 3-5 minutes. Avoid over-drying the magnetic beads, which will reduce nucleic acid production.

[0078] h. Remove the centrifuge tube from the magnetic rack, add 50 μL of Nuclease-free water to resuspend the magnetic beads, and let stand at room temperature for 3 minutes.

[0079] i. Place the centrifuge tube on the magnetic rack and let it stand for 3 minutes until the magnetic beads are completely adsorbed. Then, transfer the supernatant to a new 1.5 mL centrifuge tube.

[0080] j. Add one cup volume of AMPure XP magnetic beads to a new centrifuge tube, gently tap to mix, and let stand at room temperature for 5 minutes.

[0081] k. After instantaneous centrifugation, place the centrifuge tube on a magnetic rack and wait for the solution to clarify before discarding the supernatant.

[0082] 1. Add 200 μL of freshly prepared 80% ethanol, wait 30 seconds, and then discard the supernatant.

[0083] m. Repeat step l.

[0084] n. After instantaneous centrifugation, place the centrifuge tube on a magnetic rack. Once the magnetic beads are completely adsorbed, use a pipette to remove all residual ethanol.

[0085] o. Open the lid and dry for 3-5 minutes. Avoid over-drying the magnetic beads, which will reduce nucleic acid production.

[0086] p. Remove the centrifuge tube from the magnetic rack, add 35 μL of Nuclease-free water to resuspend the magnetic beads, and let stand at room temperature for 3 minutes.

[0087] q. Place the centrifuge tube on the magnetic rack and wait for the magnetic beads to be completely attracted before transferring the supernatant into a new 1.5 mL centrifuge tube.

[0088] (3) Ligation of the purified product with sequencing adapters. The concentration of the purified product from the previous step was determined using an Equalbit 1×dsDNAHSAssay Kit. The molar concentration of the sample was calculated using the following formula: Molar concentration (fmol / μL) = 1613 * Mass concentration (ng / μL) / [Sample fragment length (bp) + 120bp]. 100–200 fmol of the purified product was transferred to a new 1.5 mL centrifuge tube. The reaction mixture was prepared according to the following system: 200 fmol of purified product, 3.5 μL of sequencing adapter, 12.5 μL of 4× ligation buffer, 2 μL of DNA ligase, and Nuclease-free water. The volume was brought to 50 μL. The tube was gently tapped, and the tube was briefly centrifuged. The mixture was then incubated at room temperature for 10 min. After incubation, the ligation product was purified using AMPure XP magnetic beads. The specific steps are as follows:

[0089] a. Take 20 μL of AMPure XP magnetic beads that have been equilibrated at room temperature, add them to the reaction system of the previous step, gently tap the tube wall and let stand at room temperature for 5 min.

[0090] b. After instantaneous centrifugation, place the centrifuge tube on a magnetic rack and wait for the solution to clarify before discarding the supernatant.

[0091] c. Add 200 μL of room temperature equilibrated short fragment washing buffer, gently tap to resuspend the magnetic beads, centrifuge briefly, place the centrifuge tube on a magnetic rack, and discard the supernatant after the magnetic beads have completely adhered.

[0092] d. Repeat step c.

[0093] e. Discard as much liquid as possible, immediately resuspend the magnetic beads in 30 μL of elution buffer, and let stand at room temperature for 5 min.

[0094] f. Place the centrifuge tube on the magnetic rack and wait for the magnetic beads to be completely attracted before transferring the supernatant into a new 1.5 mL centrifuge tube.

[0095] g. Use the Equalbit 1×dsDNAHSAssay Kit to determine the concentration of the purified product and calculate the library molar concentration according to the following formula: Molar concentration (fmol / μL) = 1613 * mass concentration (ng / μL) / [sample fragment length (bp) + 300bp].

[0096] After the sequencing library was constructed, nanopore long-read sequencing was performed using the QDS V1.1 direct barcoding library preparation kit and the QNome-3841 gene sequencer. The main steps are as follows:

[0097] (1) Place the bridging compound, sequencing reaction buffer and 1 tube of bridging buffer at room temperature to thaw and vortex to mix.

[0098] (2) Unseal the sequencing chip and turn the valve clockwise by about 90° until it touches the limit. Use a 200μL pipette to insert into the sample port of the sequencing chip to draw a small amount of liquid from the chip, preventing the introduction of air bubbles. Then use a 200μL pipette to draw 200μL of conduction buffer, rotate the pipette roller to remove air bubbles from the tip, insert the tip into the sample port, and slowly and evenly add the conduction buffer to the sequencing chip until there is a small amount of liquid remaining at the tip.

[0099] (3) Repeat Step 2 once.

[0100] (4) Take 6 μL of the conductive linker and add it to 294 μL of conductive buffer, vortex to mix, and then centrifuge briefly.

[0101] (5) Take 200 μL of the mixture obtained in the previous step and add it to the sequencing chip until there is a little liquid residue at the end of the pipette tip. Turn the valve counterclockwise 90° until it touches the limit. After reattaching the sealing film, let it stand at room temperature for 15 minutes.

[0102] (6) Prepare a mixed solution in a 1.5 mL centrifuge tube according to the following system: 35 μL sequencing reaction buffer, 15 fmol of DNA library with sequencing adapters ligated, and Nuclease-free water to bring the total to 70 μL.

[0103] (7) Unseal the sequencing chip again, turn the valve 90° clockwise until it touches the limit, slowly and evenly add 70μL of the mixed sample obtained in the previous step to the sequencing chip until a small amount of liquid remains at the tip of the pipette. Turn the valve counterclockwise about 90° until it touches the limit, and reseal the chip. Set the waiting time to 15 minutes before starting sequencing.

[0104] The third-generation sequencing data obtained in the previous step were filtered using the bioinformatics software FastP and Seqkit. The filtered sequencing data were then aligned with American PRRSV sequences in the database using Minimap2. The PRRSV sequence with the highest base coverage at a sequencing depth of 30× was selected as the reference sequence for each sample. Sequencing depth was calculated using Minimap2 and Samtools. Simultaneously, the filtered sequences were assembled using Canu software. After assembly, the assembly results were aligned with the PRRSV sequences in the database using Maft software, a phylogenetic tree was constructed using FastTree software, and visualization was performed using Itol software.

[0105] The results of sequencing depth are as follows Figure 6 As shown, in PRRSV01–10, the sequencing results achieved good coverage of the full sequence length. This indicates the primer coverage for different wild-type PRRSV strains.

[0106] The results of the phylogenetic tree are as follows Figure 7 As shown, PRRSV01–10 exhibit a relatively large genetic distance. This indicates that this primer set has broad applicability to American porcine reproductive and respiratory syndrome virus (PRRSV).

Claims

1. A primer combination for obtaining the complete genome sequence of a porcine reproductive and respiratory syndrome virus (PRRSV) American strain, characterized in that, The primer sequences in the primer combination are shown in SEQ ID NO.1 to 33.

2. A kit for obtaining the complete genome sequence of a porcine reproductive and respiratory syndrome virus (PRRSV) American strain, characterized in that, The kit comprises the primer combination of claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes 5×VAHTS RT Multi-PCR Mix, Nuclease & Nucleic acid-free ddH2O.

4. The application of the primer combination of claim 1 and the kit of claim 2 in obtaining the whole genome sequence of the American strain of porcine reproductive and respiratory syndrome virus.

5. A method for obtaining the complete genome sequence of a porcine reproductive and respiratory syndrome virus (PRRSV) American strain, characterized in that, Includes the following steps: (1) Sample collection and viral nucleic acid extraction; (2) The extracted nucleic acid is reverse transcribed and amplified to obtain an amplified fragment; the amplification uses the primer combination of claim 1; (3) Mix the amplified fragments to construct a sequencing library; (4) Sequencing of the amplified fragments in the sequencing library using a nanopore sequencer; (5) Bioinformatics analysis of sequencing information to obtain whole genome sequence.

6. The method according to claim 5, characterized in that, The samples include saliva samples, nasal and oral swab samples, blood samples, fecal samples, tissue samples, environmental samples, or virus cultures.

7. The method according to claim 5, characterized in that, The amplification program in step (2) is: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 95℃ for 30 s, extension at 72℃ for 3 min, 35 cycles; extension at 72℃ for 5 min.

8. The method according to claim 5, characterized in that, Bioinformatics analysis includes sequencing data filtering, sequencing depth calculation, sequencing result assembly, viral sequence tracing, and finally obtaining the whole genome sequence.

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