Primers and methods for whole genome sequencing of african swine fever virus based on a next generation sequencing platform and targeted enrichment

Abstract

This invention relates to the field of biotechnology, and particularly to primers and methods for whole-genome sequencing of African swine fever virus (ASFV) based on next-generation sequencing platforms and targeted enrichment. This invention discloses a primer set and method for whole-genome sequencing of ASFV based on next-generation sequencing platforms and targeted enrichment, comprising 32 pairs of specific targeted amplification primers. These primers exhibit high specificity and sensitivity, fully leveraging the advantages of next-generation sequencing—high throughput, low cost, high accuracy, and mature technology—to accurately and completely obtain the entire ASFV genome sequence. This invention can obtain complete genetic information of ASFV, identify and diagnose infections of different ASFV genotypes, and, by analyzing the genetic differences among different strains, determine the geographical origin and transmission dynamics of circulating strains, tracing the evolution of recombinant strains. It provides resources for basic research on virus-host interactions and drug resistance, and offers a theoretical basis for developing long-term prevention and control strategies. It also promotes innovation in diagnostic methods.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to primers and methods for whole-genome sequencing of African swine fever virus based on next-generation sequencing platforms and targeted enrichment. Background Technology

[0002] African swine fever (ASF) is an acute, highly contagious, and deadly viral infectious disease caused by the African swine fever virus (ASFV). The natural hosts of ASFV are domestic pigs, wild boars, and ticks (such as the African swine fever tick), and it is primarily transmitted through the digestive tract, respiratory tract, and vector-borne transmission. It is highly resistant to environmental factors, has a wide range of incubation periods in pig herds, is highly transmissible, and has a high mortality rate.

[0004] With the reduction in sequencing costs and the optimization of bioinformatics tools (such as second-generation sequencing platforms and Nanopore third-generation sequencing platforms), its application has expanded from the laboratory to front-line production, providing scientific support for global ASF prevention and control. Although third-generation nanopore sequencing has the characteristics of real-time, portability, and long read length, it has a high error rate and high cost, and it is difficult to obtain the whole genome for clinical samples, especially those with low viral load. Second-generation sequencing has the advantages of high throughput, low cost, high accuracy, and mature technology. Therefore, this study provides a set of targeted amplification primers suitable for African swine fever virus whole genome sequencing, and establishes a method for targeted whole genome sequencing of African swine fever virus, providing a reliable new means for molecular epidemiological research of African swine fever virus and source tracing in emergency response to the epidemic.

[0005] In existing technologies, patent CN119753240A discloses a primer set of 183 pairs for whole-genome detection of African swine fever virus; patent CN118703709A discloses a PCR primer set of 106 pairs for whole-genome detection of African swine fever virus based on nanopore sequencing. Both patents use more than 100 primer pairs for amplifying the entire genome of African swine fever virus, making amplification cumbersome and labor-intensive. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide primers and methods for whole-genome sequencing of African swine fever virus based on a second-generation sequencing platform and targeted enrichment, so as to solve the technical problems of cumbersome amplification and large workload in the existing technology.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a primer set, which includes primer X;

[0009] The primer x has:

[0010] (1) The nucleotide sequence as shown in SEQ ID NO: x; or

[0011] (2) A nucleotide sequence obtained by substitution, deletion or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or

[0012] (3) A nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence shown in (1) or (2);

[0013] Where x is selected from any integer in the range 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64;

[0014] The plurality can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0015] In some specific embodiments of the present invention, the primer set includes primers 1 to 64, whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.64 respectively;

[0016] In some specific embodiments of the present invention, the primer set mentioned above includes primer set X;

[0017] The primer set X includes primer 2X-1 and primer 2X;

[0018] Where X is selected from any integer among 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0019] This invention also provides the application of the above primer set in any of the following:

[0020] (i) Whole genome sequencing of African swine fever virus;

[0021] (ii) Prepare products with complete genome sequencing of African swine fever virus.

[0022] In some specific embodiments of the present invention, the above applications are as follows:

[0023] The sequencing was based on a second-generation sequencing platform and targeted enrichment.

[0024] The products include reagents, reagent kits, or devices.

[0025] The present invention also provides sequencing products (which may be reagents, kits or devices) comprising the above-described primer set.

[0026] In some specific embodiments of the present invention, the sequencing product includes:

[0027] The reagents include at least one of TARAKA 5× PrimeSTAR GXL Buffer, TARAKA PrimeSTAR GXL DNAPolymerase, and TARAKA dNTP Mixture;

[0028] The kit includes the reagent;

[0029] The device includes the reagent.

[0030] This invention also provides a method for whole-genome sequencing of African swine fever virus, comprising sequencing based on any of the following:

[0031] (i) The primer set mentioned above;

[0032] (ii) The sequencing products mentioned above.

[0033] In some specific embodiments of the present invention, the above-mentioned method for whole-genome sequencing of African swine fever virus includes the following steps:

[0034] S1. Collect the sample to be tested and extract the nucleic acid to be tested;

[0035] S2. Using the nucleic acid to be tested as a template, amplify it with the primer set or sequencing product to obtain the amplification product;

[0036] S3. Construct a sequencing library based on the added products;

[0037] S4. Sequencing is performed on the sequencing library to obtain the sequence.

[0038] In some specific embodiments of the present invention, the amplification of the above-mentioned African swine fever virus whole genome sequencing method is carried out in 8 reaction systems, and the reaction system is reaction system Y and / or reaction system Z;

[0039] The reaction system Y includes primer set 8Y-7, primer set 8Y-5, primer set 8Y-3, and primer set 8Y-1;

[0040] The reaction system Z includes primer set 8Z-6, primer set 8Z-4, primer set 8Z-2, and primer set 8Z;

[0041] Where Y is selected from any integer among 1, 2, 3, and 4, and Z is selected from any integer among 1, 2, 3, and 4.

[0042] In some specific embodiments of the present invention, the above-mentioned method for whole-genome sequencing of African swine fever virus further includes a bioinformatics analysis step after obtaining the sequence.

[0043] In some specific embodiments of the present invention, the above-mentioned African swine fever virus whole genome sequencing method is as follows:

[0044] The construction of the sequencing library includes at least one of the following steps: enzyme digestion and fragmentation, end repair and addition of dA, adapter ligation, PCR amplification, POOLING and single-strand circularization, enzyme digestion and preparation of DNB.

[0045] The bioinformatics analysis includes at least one of the following steps: sequence alignment, differentiation between host and non-host sequences, sequence assembly, whole-genome sequence identification and whole-genome strain typing, and virulence identification.

[0046] This invention discloses a primer set and method for whole-genome sequencing of African swine fever virus (ASFV) based on a next-generation sequencing platform and targeted enrichment. It comprises 32 pairs of specific targeted amplification primers, exhibiting high specificity and sensitivity, and simultaneously detecting 32 fragments. This fully leverages the advantages of next-generation sequencing—high throughput, low cost, high accuracy, and mature technology—to accurately and completely obtain the entire ASFV genome sequence. This invention provides complete genetic information of ASFV, enabling the identification and diagnosis of infections with different ASFV genotypes. By analyzing the genetic differences among different strains, the geographical origin and transmission dynamics of circulating strains can be determined, and the evolution of recombinant strains can be traced. It provides resources for basic research on virus-host interactions and drug resistance, and offers a theoretical basis for developing long-term prevention and control strategies. It also promotes innovation in diagnostic methods. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0048] Figure 1 The results of the genome map are shown. Detailed Implementation

[0049] This invention discloses primers and methods for whole-genome sequencing of African swine fever virus based on a next-generation sequencing platform and targeted enrichment. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0050] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0051] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0052] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0053] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0054] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0055] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0056] The basic concepts involved in this invention are as follows: Targeted enrichment technology is a method to improve sequencing or detection efficiency by specifically capturing target nucleic acid sequences. Its core principle includes the following key steps: specific primer design, enrichment and purification, and high-throughput sequencing and analysis; Next-Generation Sequencing (NGS) is a high-throughput, high-efficiency DNA / RNA sequencing method. Its core principle is to sequence a large number of DNA fragments in parallel and splice the results.

[0057] This invention provides a primer set based on the whole genome sequencing of African swine fever virus. The primer set includes 32 pairs of primers. This primer set is designed for different genotype strains by identifying conserved regions. After several rounds of testing and optimization, the problems of different amplification efficiencies of multiple primers and complementary pairing between primers are overcome. The sequences of the upstream and downstream primers of the different 32 pairs of primers are shown in SEQ ID NO.1~SEQ ID NO.64, as shown in Table 1.

[0058] In one implementation, the above primer set is applied to a method for targeted amplification primer enrichment and African swine fever virus whole genome sequencing detection, including the following steps:

[0059] (1) Collect samples and extract sample DNA;

[0060] (2) Multiplex PCR amplification was performed using the 32 pairs of PCR primers described above;

[0061] (3) Construct sequencing libraries;

[0062] (4) Sequencing;

[0063] (5) Bioinformatics analysis.

[0064] In practice, the samples include various types such as pig nasal swabs, pharyngeal swabs, blood samples, tissue samples, oral fluid, and saliva.

[0065] In practice, the nucleic acid extraction method is a fully automated magnetic bead extraction method.

[0066] In practice, the multiplex PCR is performed in eight tubes. In one example, each tube contains 10 μL of 5× PrimeSTAR GXL Buffer (TARAKA), 2 μL of PrimeSTAR GXL DNA Polymerase (TARAKA), 4 μL of dNTP Mixture (2.5 mM each) (TARAKA), 21 μL of enzyme-free water, and 5 μL of extracted viral nucleic acid. In tube 1, 1 μL of a 10 μM mixture of specific primer pairs for ASFV-1, ASFV-3, ASFV-5, and ASFV-7 is added. In tube 2, 1 μL of a 10 μM mixture of specific primer pairs for ASFV-2, ASFV-4, ASFV-6, and ASFV-8 is added. In tube 3, 1 μL of a 10 μM mixture of specific primer pairs for ASFV-9, ASFV-11, ASFV-13, and ASFV-15 is added. In tube 4, 1 μL of... ASFV-10, ASFV-12, ASFV-14, ASFV-16 specific primer pair mixture (10 μM) was added to reaction tube 5; ASFV-17, ASFV-19, ASFV-21, ASFV-23 specific primer pair mixture (10 μM) was added to reaction tube 6; ASFV-18, ASFV-20, ASFV-22, ASFV-24 specific primer pair mixture (10 μM) was added to reaction tube 7; ASFV-25, ASFV-27, ASFV-29, ASFV-31 specific primer pair mixture (10 μM) was added to reaction tube 8; and ASFV-26, ASFV-28, ASFV-30, ASFV-32 specific primer pair mixture (10 μM) was added to reaction tube 8. Finally, the amplification product was purified using 1:1 AMPureXP Beads (Beckman Coulter), and the nucleic acid concentration of the product was quantified using Qubit.

[0067] In one example, the reaction procedure for the multiplex PCR amplification is as follows: pre-denaturation at 95°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 68°C for 10 min, with a reaction cycle count of 30-45; and final extension at 68°C for 5 min.

[0068] In practice, the construction of the sequencing library for the second-generation sequencing platform includes the following steps: enzyme digestion and fragmentation, end repair and dA addition, adapter ligation, PCR amplification, POOLING and single-strand circularization, enzyme digestion, and DNB preparation.

[0069] In practice, the bioinformatics analysis includes sequence alignment, distinguishing between host (pig) sequences and non-host (virus) sequences, sequence assembly, whole genome sequence identification and whole genome strain typing, and virulence identification.

[0070] The aforementioned long PCR amplification-specific primers capable of amplifying the entire African swine fever virus (ASFV) genome bind specifically to the ASFV genome in the sample, improving the sensitivity, specificity, and sequencing coverage of ASFV detection, especially in samples with low viral infection levels. This primer set comprises 32 primer pairs, employing the high-fidelity TAKARA PrimeSTAR GXL DNA Polymerase, capable of amplifying products ≥30 kb, ensuring amplification efficiency, reducing primer pair usage, and providing a rapid and convenient way to obtain segments of the ASFV genome.

[0071] Based on this invention, complete genetic information of African swine fever virus can be obtained, and infection of different genotypes of ASFV can be identified and diagnosed. By analyzing the genetic differences of different strains, the geographical origin and transmission dynamics of circulating strains can be determined, and the evolution process of recombinant strains can be traced. It provides resources for basic research on virus-host interaction and drug resistance, and provides a theoretical basis for formulating long-term prevention and control strategies. At the same time, it also promotes the innovation of diagnostic methods.

[0072] The present invention will be further illustrated below with reference to the embodiments.

[0073] Example 1: Design and synthesis of primers for targeted amplification of African swine fever virus

[0074] Based on the publicly available genome sequences of ASFV type I, II, and type I-II recombinant strains in the GenBank database of the Center for Biotechnology Information in the United States, conserved regions were obtained through alignment, and primers were designed and validated. The primer sequences are shown in Table 1.

[0075] Table 1: Targeted amplification primer sequences involved in this invention

[0076]

[0077]

[0078] Example 2: Establishment of a method for specific targeted amplification primer enrichment and ASFV whole genome sequencing

[0079] The experimental samples in this embodiment were whole blood samples sent from five pig farms in Henan.

[0080] 300 μL of each of the above whole blood samples were extracted using the NPA-32E automated nucleic acid extractor and the MagaBio plus viral DNA / RNA purification kit from Hangzhou Borui Technology Co., Ltd., via magnetic bead extraction, yielding 80 μL of nucleic acid solution.

[0081] 1. ASFV whole-genome targeted primer amplification

[0082] Multiplex PCR was performed in eight tubes. Each tube contained 10 μL of 5× PrimeSTAR GXL Buffer (TARAKA), 2 μL of PrimeSTAR GXL DNA Polymerase (TARAKA), 4 μL of dNTP Mixture (2.5 mM each, TARAKA), 21 μL of enzyme-free water, and 5 μL of extracted viral nucleic acid. In tube 1, 1 μL of a 10 μM mixture of specific primer pairs for ASFV-1, ASFV-3, ASFV-5, and ASFV-7 was added. In tube 2, 1 μL of a 10 μM mixture of specific primer pairs for ASFV-2, ASFV-4, ASFV-6, and ASFV-8 was added. In tube 3, 1 μL of a 10 μM mixture of specific primer pairs for ASFV-9, ASFV-11, ASFV-13, and ASFV-15 was added. In tube 4, 1 μL of… ASFV-10, ASFV-12, ASFV-14, and ASFV-16 specific primer pair mixture (10 μM) were added to reaction tube 5. ASFV-17, ASFV-19, ASFV-21, and ASFV-23 specific primer pair mixture (10 μM) were added to reaction tube 6. ASFV-18, ASFV-20, ASFV-22, and ASFV-24 specific primer pair mixture (10 μM) were added to reaction tube 7. ASFV-25, ASFV-27, ASFV-29, and ASFV-31 specific primer pair mixture (10 μM) were added to reaction tube 8. ASFV-26, ASFV-28, ASFV-30, and ASFV-32 specific primer pair mixture (10 μM) were added to reaction tube 8. The reaction procedure was as follows: pre-denaturation at 95℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 68℃ for 10 min, with a reaction cycle of 30-45 cycles; and final extension at 68℃ for 5 min.

[0083] 2. Purification of PCR products

[0084] After the PCR reaction program is completed, combine 25 μL of each of the above 8 tubes of amplification products into one tube. Add 50 μL of AMPure XP Beads (Beckman Coulter) to the tube, incubate at room temperature for 5 min, then transfer to a magnetic rack and let stand for 2-5 min until the magnetic beads are completely adsorbed. Then discard the supernatant. Next, add 200 μL of freshly prepared 80% ethanol and let stand for 30 s. Then discard the supernatant. Add another 200 μL of freshly prepared 80% ethanol to the reaction tube and let stand for 30 s. Then discard the supernatant. Try to dry the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, you can centrifuge the tube briefly. After separating on a magnetic rack, use a small-capacity pipette to dry the liquid at the bottom of the tube. Keep the centrifuge tubes on the magnetic rack, open the caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked. Add 50 μL of TE buffer to resuspend the magnetic beads, remove them from the magnetic rack, and incubate at room temperature for 5 min. Then transfer them back to the magnetic rack and let them stand for 5 min until the magnetic beads are completely adsorbed. After that, aspirate the supernatant and transfer it to a 1.5 mL centrifuge tube. Quantify the concentration of the product nucleic acid in the supernatant using Qubit and store it temporarily at -20°C for later use.

[0085] 3. Sequencing library construction

[0086] Using the MGIEAsy DNA digestion library preparation kit, 200 ng of the amplification product obtained from step 2 was added to the library for construction. The specific operation is as follows.

[0087] 3.1 Enzyme digestion and fragmentation

[0088] Transfer the genomic DNA to be fragmented into a new 0.2 mL PCR tube, with a volume ≤45 μL. Make up any remaining volume with buffer if less than 45 μL (see Table 2). Vortex the PCR tube three times for 3 seconds each time, centrifuge, and place on ice. Prepare the enzyme digestion reaction solution on ice (see Table 3). Vortex three times for 2 seconds each time, briefly centrifuge to collect the reaction solution at the bottom of the tube, and then centrifuge rapidly again before placing on ice. The reaction procedure is shown in Table 4.

[0089] Table 2: Preparation of DNA fragmentation by enzyme digestion

[0090]

[0091] Table 3: Preparation of Enzyme Digestion and Disruption Reaction Solution

[0092]

[0093] Table 4: Enzyme cleavage interruption reaction conditions

[0094]

[0095] 3.2 Screening / Purification of Magnetic Bead Fragments

[0096] Use a pipette to add 36 μL of DNA Clean Beads to the 60 μL fragmentation product from step 3.1, and mix thoroughly.

[0097] Incubate at room temperature for 5 min. Perform a brief centrifugation, place the centrifuge tube on a magnetic rack, and let it stand for 2-5 min until the liquid is clear. Carefully pipette the supernatant into a new 1.5 mL centrifuge tube. Retain the supernatant and discard the magnetic beads.

[0098] Transfer 12 μL of DNA Clean Beads to 96 μL of supernatant using a pipette and mix thoroughly. Incubate at room temperature for 5 min. Briefly centrifuge, place the centrifuge tube on a magnetic rack, and let stand for 2–5 min until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette. Keeping the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls. Let stand for 30 s, then carefully aspirate and discard the supernatant. Repeat rinsing, aspirating as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, briefly centrifuge the tube on a magnetic rack, and after separation, aspirate the liquid at the bottom of the tube with a small-capacity pipette.

[0099] Keep the centrifuge tubes on the magnetic rack, open the centrifuge tube caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked.

[0100] Remove the centrifuge tube from the magnetic rack, add 43 μL of TE buffer to elute DNA, and gently pipette at least 10 times until completely mixed. Incubate at room temperature for 5 min. Centrifuge briefly, place the centrifuge tube on the magnetic rack, and let stand for 2-5 min until the liquid is clear. Transfer 41 μL of supernatant to a new 0.2 mL PCR tube.

[0101] 3.3 End-of-line repair & addition of dA tail

[0102] Take ≤100 ng of DNA for end repair. Prepare the end repair reaction solution on ice (see Table 5). Use a pipette to add 10 μL of the prepared end repair reaction solution to 40 μL of sample after magnetic bead fragment screening or purification. Perform the reaction according to the conditions in Table 6.

[0103] Table 5: Preparation of end-of-pipe repair reaction & dA tailing reaction solution

[0104]

[0105] Table 6: Conditions for terminal repair reaction & addition of dA tail reaction

[0106]

[0107] 3.4 Connector Connection

[0108] Add 5 μL of the corresponding MGIEasy DNA Adapters to the PCR tube in step 3.3, prepare the adapter ligation reaction solution on ice (see Table 7), and carry out the reaction according to the conditions in Table 8.

[0109] Table 7: Preparation of the reaction solution for connector connection

[0110]

[0111] Table 8: Reaction Conditions for Connector Connection

[0112]

[0113] 3.5 Purification of Ligation Products

[0114] Use a pipette to add 50 μL of DNA Clean Beads to the adapter ligation product from step 3.4, mix thoroughly, and follow the same purification steps as for targeted amplification PCR products. Finally, add 21 μL of TE Buffer to elute the DNA.

[0115] 3.6 PCR Amplification

[0116] Prepare the PCR reaction solution on ice (see Table 9) and carry out the reaction according to the conditions in Table 10.

[0117] Table 9: Preparation of PCR amplification reaction solution

[0118]

[0119] Table 10: PCR amplification reaction conditions

[0120]

[0121] 3.7 Purification of PCR Products

[0122] Add 50 μL of DNA Clean Beads to the PCR product from step 3.6. Follow the same purification steps as for the targeted amplification PCR product purification. Finally, add 32 μL of TE Buffer to elute the DNA.

[0123] 3.8 POOLING & Denaturation & Cycloning

[0124] After quantifying the PCR products, samples from different adapters were mixed into new 0.2 mL PCR tubes, with a total volume of 1 pmol. TE Buffer was then added to bring the total volume to 48 μL. The PCR tubes were placed on a PCR instrument, and the denaturation reaction was performed according to the conditions in Table 11. After the reaction, the PCR tubes were immediately transferred to ice, allowed to stand for 2 min, and then briefly centrifuged. Single-strand cyclization reaction solutions were prepared on ice (see Table 12) and (see Table 13).

[0125] Table 11: Denaturation Reaction Conditions

[0126]

[0127] Table 12: Preparation of Single-Chain Cycling Reaction Solution

[0128]

[0129] Table 13: Single-chain cyclization reaction conditions

[0130]

[0131] 3.9 Enzymatic digestion

[0132] Prepare the enzyme digestion reaction solution on ice (see Table 14) and the enzyme digestion reaction solution on ice (see Table 15).

[0133] Table 14: Preparation of Enzyme Digestion Reaction Solution

[0134]

[0135] Table 15: Enzyme digestion reaction conditions

[0136]

[0137] 3.10 Purification of Enzymatic Digestion Products

[0138] Add 170 μL of DNA Clean Beads to the enzyme digestion product from step 3.9 and incubate at room temperature for 10 min. Centrifuge briefly, place the centrifuge tube on a magnetic rack, and let it stand for 2–5 min until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette.

[0139] Keep the centrifuge tube on the magnetic rack, add 500 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant. Repeat the rinsing steps, aspirating as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, briefly centrifuge the tube and separate it on the magnetic rack. Use a small-range pipette to aspirate the liquid from the bottom of the tube. Keep the centrifuge tube fixed on the magnetic rack, open the tube cap, and allow it to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked. Remove the centrifuge tube from the magnetic rack, add 32 μL of TE buffer for DNA elution, and gently pipette at least 10 times until completely mixed. Incubate at room temperature for 10 min. Briefly centrifuge, place the centrifuge tube on the magnetic rack, and let stand for 2–5 min until the liquid is clear. Transfer 30 μL of supernatant to a new 1.5 mL centrifuge tube. Use Qubit to quantify the nucleic acid concentration of the product and store temporarily at -20°C for later use.

[0140] 3.11 DNB Preparation

[0141] Based on the concentration of the ssDNA library measured in 3.10 and the required fmol of library, calculate the volume of ssDNA library required for each DNB preparation system. The total amount of DNB required is 40 fmol. Take 0.2 mL of eight-tube or PCR tubes and prepare the reaction mixture on ice according to the following system (see Table 16). Perform the reaction according to the conditions in Table 17.

[0142] Table 16: DNB Preparation Reaction System 1

[0143]

[0144] Table 17: Hybridization conditions for DNB preparation reaction primers

[0145]

[0146] Once the PCR instrument reaches 4°C, remove the PCR tubes and centrifuge for 5 seconds using a mini centrifuge. Add the following components (see Table 18) to ice and react according to the reaction conditions in Table 19. After DNB preparation is complete, take 2 μL of DNB and use a Qubit... ® ssDNA Assay Kit and Qubit ® The concentration is measured using a fluorometer. A concentration of 8 ng / μL or higher is considered acceptable.

[0147] Table 18: DNB Preparation Reaction Component 2

[0148]

[0149] Table 19: Conditions for DNB preparation and rolling circle amplification

[0150]

[0151] 4. Sequencing

[0152] Take out a 0.5 mL cryovial, add reagents as shown in Table 20, and perform sequencing experiments according to the instructions for use of the MGISEQ-200RS high-throughput (rapid) sequencing reagent kit.

[0153] Table 20: DNB Loading System

[0154]

[0155] 5. Bioinformatics Analysis

[0156] The contigs were assembled using the MGAP Assemble software from the BGI bioinformatics analysis platform, and each contig was mapped to the reference genome. (See attached image.) Figure 1 We will conduct comparative analysis.

[0157] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set, characterized in that, Including primer X; The primer x has: (1) The nucleotide sequence as shown in SEQ ID NO: x; or (2) A nucleotide sequence obtained by substitution, deletion or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or (3) A nucleotide sequence that is at least 80% homologous to the nucleotide sequence shown in (1) or (2); Where x is selected from any integer from 1 to 64; The number of items is 2 to 50.

2. The primer set as described in claim 1, characterized in that, This includes primers 1 to 64, whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.64, respectively.

3. The use of the primer set as described in claim 1 or 2 in any of the following: (i) Whole genome sequencing of African swine fever virus; (ii) Prepare products with complete genome sequencing of African swine fever virus.

4. The application as described in claim 3, characterized in that, include: The sequencing was based on a second-generation sequencing platform and targeted enrichment. The products include reagents, reagent kits, or devices.

5. Sequencing products, including reagents, kits, or devices, characterized in that, Includes the primer set as described in claim 1 or 2.

6. The sequencing product as described in claim 5, characterized in that, include: The reagents include at least one of TARAKA 5× PrimeSTAR GXL Buffer, TARAKA PrimeSTAR GXL DNAPolymerase, and TARAKA dNTP Mixture; The kit includes the reagent; The device includes the reagent.

7. A method for whole-genome sequencing of African swine fever virus, characterized in that, Including sequencing based on any of the following: (i) The primer set as described in claim 1 or 2; (ii) The sequencing product as described in claim 5 or 6.

8. The method for whole-genome sequencing of African swine fever virus as described in claim 7, characterized in that, Includes the following steps: S1. Collect the sample to be tested and extract the nucleic acid to be tested; S2. Using the nucleic acid to be tested as a template, amplify it with the primer set or sequencing product to obtain the amplification product; S3. Construct a sequencing library based on the added products; S4. Sequencing is performed on the sequencing library to obtain the sequence.

9. The method for whole-genome sequencing of African swine fever virus as described in claim 8, characterized in that, After obtaining the sequence, the process also includes bioinformatics analysis.

10. The method for whole-genome sequencing of African swine fever virus as described in claim 9, characterized in that, include: The construction of the sequencing library includes at least one of the following steps: enzyme digestion and fragmentation, end repair and addition of dA, adapter ligation, PCR amplification, POOLING and single-strand circularization, enzyme digestion and preparation of DNB. The bioinformatics analysis includes at least one of the following steps: sequence alignment, differentiation between host and non-host sequences, sequence assembly, whole-genome sequence identification and whole-genome strain typing, and virulence identification.

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