Primer group for amplifying serotype dengue type II virus genome, kit and application

Abstract

The invention discloses a primer group for amplifying a serotype dengue type II virus genome. The primer group comprises a primer pair 1 to a primer pair 16, and the nucleotide sequences of the 16 primer pairs are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 32. The invention also discloses a targeted amplification kit which contains the primer group and is used for rapidly amplifying the serotype dengue type II virus. The invention also discloses application of the primer group and the kit in preparation of a product for amplifying a serotype dengue type II virus genome. The invention also discloses a method for rapidly obtaining a serotype dengue type II virus whole genome based on targeted amplification. The targeted amplification primer group consists of 16 pairs of primers, can cover and amplify all current epidemic serotype dengue II virus strains, and is high in amplification efficiency and less in cross interference; meanwhile, genomes obtained by a downstream sequencing method and an analysis method constructed by the amplicon are high in sequencing precision and large in sequencing flux.

Description

Technical Field

[0001] The invention belongs to the field of genome sequencing and relates to a primer set, a kit and an application thereof for amplifying a serotype dengue II virus genome. Background Art

[0002] Dengue virus is an important mosquito-borne virus belonging to the Flaviviridae family. It is a single-stranded positive-sense RNA virus, divided into four serotypes: I, II, III and IV. Dengue virus can cause an acute infectious disease called dengue fever, with clinical symptoms such as fever, rash, and joint pain. In the past 30 years, dengue fever has shown a rapid increase and spread in the world. It has now spread to more than 140 countries around the world, seriously threatening people's health and posing severe challenges to the global public health system. In China, dengue fever has reappeared in Foshan, Guangdong Province since 1978. Especially after 1990, dengue fever has been prevalent every year, and it has shown a trend of spreading westward and northward.

[0003] Compared with other serotypes, dengue type II virus is more virulent and has a wider impact globally. At the same time, in recent years, the spread of dengue type II virus in my country has continued to expand, and its share in serotypes has increased from less than 5% to nearly 40% in 2018.

[0004] Rapid acquisition of dengue II genome is a key technology in the process of scientific and efficient monitoring, detection and tracing of dengue II virus. At present, commonly used viral genome sequencing technologies include PCR-Sanger sequencing and metagenomic sequencing technologies. However, the above schemes currently face technical problems such as low data utilization, high detection cost and large amount of data required for genome assembly, while genome acquisition based on targeted amplification is a new generation of technical solutions with low cost, high data utilization and low assembly difficulty. However, the genome sequencing and analysis technology based on targeted amplification, while ensuring that the multiple primer pairs covering the dengue II virus genome do not interfere with each other during targeted amplification, still needs to accurately identify the strain population currently prevalent in the world. To improve its detection efficiency, it is necessary to obtain a specific primer set with high matching rate and high recognition rate for the complex population with a large number of mutation sites in the dengue II virus genome, which is still a key and important technical difficulty to be solved urgently. Summary of the invention

[0005] The object of the present invention is to provide a primer set for amplifying the genome of serotype dengue II virus, in particular a primer set with a high matching rate for specifically amplifying the genome of serotype dengue II virus population.

[0006] The present invention also aims to provide a targeted amplification kit containing the above primer set for rapid and specific amplification of serotype dengue II virus.

[0007] The last object of the present invention is to provide the use of the above primer set or kit in preparing a product for amplifying the genome of serotype dengue II virus.

[0008] The above-mentioned first object of the present invention can be achieved by the following technical scheme: a primer set for amplifying the genome of serotype dengue II virus, the primer set comprising primer pair 1 to primer pair 16, each pair of primers consisting of a forward primer and a reverse primer, and the nucleotide sequences of primer pair 1 to primer pair 16 are shown in sequence as SEQ ID NO: 1-32.

[0009] As a preferred embodiment of the present invention, primer pair 1 to primer pair 16 in the primer set are divided into two primer libraries, namely a first primer library and a second primer library, wherein the first primer library consists of primer pair 1, primer pair 3, primer pair 5, primer pair 7, primer pair 9, primer pair 11, primer pair 13 and primer pair 15; the second primer library consists of primer pair 2, primer pair 4, primer pair 6, primer pair 8, primer pair 10, primer pair 12, primer pair 14 and primer pair 16.

[0010] Specifically, the above-mentioned second object of the present invention can be achieved by the following technical solution: a targeted amplification kit for rapid amplification of serotype dengue II virus, comprising the primer set.

[0011] Preferably, the primer set is divided into two primer pools.

[0012] As a preferred technical solution of the present invention, the kit of the present invention also includes a reverse transcription reagent component.

[0013] Preferably, the reverse transcription reagent components include reverse transcriptase and reverse transcriptase buffer.

[0014] Preferably, the reverse transcriptase is Super Script III reverse transcriptase.

[0015] As a preferred technical solution of the present invention, the kit of the present invention further comprises a targeted amplification reagent component, wherein the targeted amplification reagent component comprises a polymerase, dNTPs and a polymerase buffer.

[0016] As a further preferred technical solution of the present invention, the targeted amplification reagent components of the present invention include a high-fidelity polymerase, dNTPs and a buffer of the high-fidelity polymerase.

[0017] As a preferred technical solution of the present invention, the kit of the present invention also includes a sequencing library construction component.

[0018] Preferably, the sequencing library construction components include end repair enzyme, end repair buffer, T4 DNA ligase, and T4 DNA ligase buffer.

[0019] As a preferred technical solution of the present invention, the targeted amplification kit for rapid amplification of dengue type II virus of the present invention is stored at -20°C to minimize repeated freezing and thawing.

[0020] The last object of the present invention can be achieved by the following technical solution: use of the above primer set and kit in the preparation of a product for amplifying the genome of serotype dengue II virus.

[0021] The present invention also provides a method for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification, comprising the following steps:

[0022] (1) Sample RNA extraction;

[0023] (2) Reverse transcription to generate cDNA;

[0024] (3) targeted amplification using the above primer set;

[0025] (4) Build a sequencing library and analyze the sequencing data to obtain the complete genome of dengue II virus.

[0026] In the method for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification:

[0027] Preferably, the sample in step (1) is a serum sample.

[0028] Preferably, when the above primer set is used for targeted amplification in step (3), the primer set is divided into two primer pools as described above, and amplification is performed using the first primer pool and the second primer pool respectively, and then the amplification products of the two groups are collected.

[0029] Preferably, the sequencing sequence data is analyzed in step (4), including: (a) filtering and quality control of the sequencing data; (b) aligning the sequencing sequence to the dengue II virus reference genome; and (c) generating a whole genome of the consensus sequence.

[0030] Preferably, the sequencing data filtering quality control in step (a) includes: removing sequences with 28bp bases at both ends of the sequence; filtering sequences with a read length less than 500bp; filtering sequences with a base sequencing quality value less than 6 accounting for more than 50%.

[0031] As a preferred technical solution of the present invention, when analyzing the sequencing sequence data in step (4) of the present invention, the server is configured with a CPU of at least 16 cores and 32 threads and a memory of at least 32G. If the sequencing analysis operation is performed on a server with a configuration lower than this, the analysis time for finally obtaining the dengue II virus genome will be longer.

[0032] The present invention has the following advantages:

[0033] (1) The targeted amplification primer set of the present invention has the advantages of strong specificity, high sequencing accuracy and high throughput, and the cost is significantly lower than that of methods based on Sanger sequencing or metagenomic sequencing. The whole genome sequence can be obtained within 24 hours at the fastest, greatly shortening the experimental cycle;

[0034] (2) The targeted amplification primer set of the present invention consists of 16 pairs of primers, which can cover and amplify all currently popular dengue II virus strains, with high amplification efficiency and little cross-interference;

[0035] (3) The primers in the present invention are selected from the conserved regions of the DENV serotype II genome sequence published previously and are designed by adding degenerate bases. Preliminary analysis of primer matching shows that all subtypes of dengue II virus can be well amplified without worrying about the problem of poor primer binding efficiency;

[0036] (4) The downstream sequencing method and analysis method constructed by the amplicon of the present invention obtains genome sequencing with high accuracy and large sequencing throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the expected amplification products of each primer pool in Example 1 of the present invention;

[0038] Figure 2 The genome coverage depth result is obtained by sequencing in Example 2 of the present invention. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. The embodiments are only exemplary and do not constitute any limitation to the scope of the present invention.

[0040] Example 1

[0041] In the previous research, the research team of this application integrated the global spatiotemporal epidemiological background information related to dengue fever, and established a globally unified high-resolution genotyping framework for dengue virus based on phylogenetics, population genetics, phylogeography and phylogeographic dynamics. The four dengue virus serotypes contain a total of 21 genotypes, 156 genotypes and 969 gene branches. The population diversity at the three genotyping levels is mainly characterized by a decrease in the average genetic distance, which is 2-6%, 0.8-2% and ≦0.8%, respectively. Each serotype of dengue virus shows significant geographical restriction characteristics at the corresponding levels of intercontinental-genotype, regional-genotype and national-gene branch. Therefore, it is urgent and vital to accurately identify the epidemic strains of dengue virus and their epidemic transmission patterns, and to improve the global coordinated early warning monitoring and joint prevention and control capabilities. Based on the established global unified dengue virus high-resolution genotyping framework, the team of this application has built a dengue virus gene database (a total of 41,338 sequences). This application is based on this database and the population genetic structure of dengue II virus, targeting the dominant population of dengue II virus, and combined with the research team's more than 10 years of field application practice. In the primer design, the genetic diversity, geographical restrictions and spatiotemporal epidemic patterns of dengue II virus are fully considered, so that the primer pairs invented in this application widely cover the sequences of dengue II virus strains currently prevalent in the world, greatly improving the versatility of dengue II virus full genome sequence detection, that is, improving the detection efficiency of amplifying the sequences of dengue II virus strains currently prevalent in the world, and providing an advanced technical system for effectively curbing the rapid expansion of dengue fever worldwide.

[0042] Therefore, the design of the primer set in the present invention takes full account of the genetic diversity, geographical restriction and spatiotemporal epidemic law of dengue II virus, so as to ensure that it widely covers the current global dengue II virus strain sequence, incorporates 4515 dengue II virus genomes reported worldwide, selects its conservative regions through comprehensive analysis, and introduces merging bases to achieve its versatility, and combines the application practice on site to greatly improve the matching rate of global dengue II virus population full gene targeted amplification. The grouping of primer pairs comprehensively considers the length of the primer, annealing temperature, GC%, 3UTR end stability, secondary structure and the possibility of primer dimer formation, and maximizes the chance of successful PCR reaction.

[0043] The primer set provided in this embodiment for amplifying the genome of serotype dengue II virus includes primer pair 1 to primer pair 16, each primer pair consists of a forward primer and a reverse primer, and the nucleotide sequences of primer pair 1 to primer pair 16 are shown in SEQ ID NO: 1-32 in sequence.

[0044] Primer pair 1 to primer pair 16 in the primer set are divided into two primer libraries, namely a first primer library and a second primer library, wherein the first primer library consists of primer pair 1, primer pair 3, primer pair 5, primer pair 7, primer pair 9, primer pair 11, primer pair 13 and primer pair 15; the second primer library consists of primer pair 2, primer pair 4, primer pair 6, primer pair 8, primer pair 10, primer pair 12, primer pair 14 and primer pair 16.

[0045] That is to say, the 1st, 3rd, 5th, 7th, 9th, 11th, 13th and 15th pairs of the 16 pairs of primers shown in the above primer pairs SEQ ID NO: 1-32 are the first primer library, and the 2nd, 4th, 6th, 8th, 10th, 12th, 14th and 16th pairs of the 16 pairs of primers shown in the above primer pairs SEQ ID NO: 1-32 are the second primer library.

[0046] The use of primers needs to be explained. The above primers must be used in pairs, with "a pair" as a selection unit. For example, SEQ ID NO: 1 and SEQ ID NO: 2 constitute a pair of primers numbered 1 (mD2-64F and mD2-1002R, and the rest are similar.

[0047] The use of primers needs to be explained. The above primer pairs must be used as a set, with the "primer library" as a selection unit. The most preferred technical solution of the present invention is to select 16 primer pairs shown in SEQ ID NO: 1-32 to form 2 primer libraries, and use third-generation nanopore sequencing for targeted amplification sequencing to obtain the whole genome of dengue II virus ( Figure 1 ).

[0048] On the one hand, the grouping of primer pairs takes into account the length, annealing temperature, GC%, 3' stability, secondary structure and the possibility of primer dimer formation, maximizing the chance of a successful PCR reaction. The primers are divided into two pools with an annealing temperature of 64-68°C, which is a higher temperature and a narrower range, allowing the PCR reaction to be carried out in two steps and allowing multiple pairs of primers to be carried out in the same reaction.

[0049] On the other hand, from the perspective of site, the two sets of primers are cross-set and overlap each other ( Figure 1 ), which makes the PCR primer amplification region relatively independent, making the amplification system more stable, and at the same time avoiding the situation where a single primer potentially off-target causes a decrease in genome coverage.

[0050] The primer set of the present invention includes primers in the first primer library placed in primer pool 1, primers in the second primer library placed in primer pool 2, and the matching rate of each primer in the global dengue type 2 strain population (4515 sequences) is shown in Table 1 below:

[0051] Table 1 Primer set of this example

[0052]

[0053]

[0054] The primer set can be used in a targeted amplification kit for rapid amplification of serotype dengue II virus.

[0055] The kit also includes a reverse transcription reagent component.

[0056] The reverse transcriptase may be Super Script III reverse transcriptase.

[0057] The kit further comprises a targeted amplification reagent component, wherein the targeted amplification reagent component comprises a polymerase, dNTPs and a polymerase buffer.

[0058] Furthermore, the targeted amplification reagent components include a high-fidelity polymerase, dNTPs and a buffer of the high-fidelity polymerase.

[0059] The kit also includes a sequencing library construction component.

[0060] The sequencing library construction components include end repair enzyme, end repair buffer, T4 DNA ligase, and T4 DNA ligase buffer.

[0061] The targeted amplification kit for rapid amplification of dengue type II virus of the present invention is stored at -20°C to minimize repeated freezing and thawing.

[0062] Example 2

[0063] The method provided in this embodiment for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification comprises the following steps:

[0064] Sample source: Clinical case samples from a certain hospital, samples are serum.

[0065] Step 1:

[0066] Extraction of sample RNA:

[0067] The sample RNA can be extracted by using Trizol according to the instructions of the kit. After the RNA is dissolved in RNase-free water, the extracted RNA is quantified using Nanodrop and Qubit.

[0068] Step 2:

[0069] The extracted RNA was reverse transcribed to generate cDNA. The configuration system was as shown in Table 2 according to the instructions of Super Script III reverse transcriptase:

[0070] Table 2 Configuration system according to Super Script III reverse transcriptase instructions

[0071] Components volume Extraction of RNA 1.00μL (not less than 50ng) Random Primers <5.0μg Nucleic acid-free water Refill to 10.00 μL Total volume 10.00μL

[0072] Set up the PCR machine and run the following program in Table 3:

[0073] Table 3 PCR reaction program

[0074] PCR procedure temperature time Number of cycles 1 65℃ 5 minutes 1 2 4℃ pause /

[0075] The reaction system is configured as shown in Table 4:

[0076] Table 4 PCR reaction system

[0077]

[0078]

[0079] Run the following program on the PCR instrument, as shown in Table 5:

[0080] Table 5 PCR operation program

[0081] PCR procedure temperature time Number of cycles 1 30℃ 10min 1 2 42℃ 30min 1 3 95℃ 5min 1 4 4℃ pause /

[0082] The obtained cDNA product proceeds to step 3.

[0083] Step 3:

[0084] Multiplex PCR targeted amplification is as follows:

[0085] Dissolve the primers in TE buffer and dilute the primers of the same primer pool in the same tube.

[0086] Primer pool 1 contains primers from the first primer library, namely primer pair 1, primer pair 3, primer pair 5, primer pair 7, primer pair 9, primer pair 11, primer pair 13 and primer pair 15. Each primer pair consists of a forward primer and a reverse primer. The final concentration of each primer pair is 0.015 μM.

[0087] Primer pool 2 contains primers from the second primer library, namely primer pair 2, primer pair 4, primer pair 6, primer pair 8, primer pair 10, primer pair 12, primer pair 14 and primer pair 16. Each primer pair consists of a forward primer and a reverse primer, and the final concentration of each primer pair is 0.015 μM.

[0088] Prepare the following system in a 0.2 mL centrifuge tube, as shown in Table 6:

[0089] Table 6 Multiplex PCR reaction system

[0090] Components volume cDNA products 2.50μL Q5 reaction buffer (5x), high-fidelity polymerase buffer 5.00μL dNTPs(10mM) 0.50μL Q5 DNA polymerase, high-fidelity polymerase 0.25μL Primer Pool 1 or Primer Pool 2 0.60μL Nucleic acid-free water 16.15μL Total volume 25.00μL

[0091] Run the following program on the PCR instrument, as shown in Table 7:

[0092] Table 7 PCR operation program

[0093]

[0094]

[0095] Step 4:

[0096] Product purification and quantification

[0097] Mix the two sets of primer pool multiplex PCR targeted amplification products and add an equal volume of nucleic acid purification magnetic beads, vortex mix and stand at room temperature for 10 minutes, and mix every 2 to 3 minutes. After a brief centrifugation, place it on a magnetic stand until the liquid is clear and carefully aspirate the liquid. Add 200.00μL 80% (volume percentage) freshly prepared ethanol, place the centrifuge tube on a magnetic stand and turn it to 45-75℃ to wash the magnetic beads, turn it back and aspirate the liquid. After repeating the above washing steps, carefully absorb the remaining ethanol and blow dry at room temperature. Add 15.00μL of nucleic acid-free water and mix well. After mixing and incubation, place it on a magnetic stand and aspirate the liquid into a new 1.5mL centrifuge tube. Use Nanodrop and Qubit to determine the library concentration.

[0098] Step 5:

[0099] DNA sequencing library preparation

[0100] The purified multiplex PCR targeted amplification product DNA was subjected to sequencing library construction reaction 1, i.e., DNA end repair and dA tail addition, in a 0.2 mL centrifuge tube according to the following system configuration, as shown in Table 8:

[0101] Table 8 Sequencing library construction reaction 1 system

[0102] Components volume Purified multiplex PCR targeted amplification product DNA About 200 μg NEBNext FFPE DNA Repair Mix 1.75μL NEBNext FFPE DNA Repair Buffer 1.00μL NEBNext End Repair Enzyme Mix 1.00μL NEBNext End Repair Reaction Buffer(10×) 1.00μL Klenow Fragment(3'-5'exo-) 0.50μL NEBNext dA-Tailing Reaction Buffer(10×) 0.75μL Nucleic acid-free water Refill to 30.00 μL Total volume 30.00μL

[0103] Run the following program on the PCR instrument, as shown in Table 9:

[0104] Table 9 PCR reaction program

[0105] PCR procedure temperature time Number of cycles 1 20℃ 30 minutes 1 2 65℃ 5 minutes 1 3 16℃ still /

[0106] Transfer the product of sequencing library construction reaction 1 to a 1.5 mL centrifuge tube, add an equal volume of resuspended purified magnetic beads and purify the nucleic acid.

[0107] The purified product of sequencing library construction reaction 1 was subjected to sequencing library construction reaction 2, i.e., sequencing motor protein adapter connection, and the following system was configured in a 1.5 mL centrifuge tube, as shown in Table 10:

[0108] Table 10 Sequencing library construction reaction 2 system

[0109] Components volume DNA end repair and dA tailing products 30.00μL Adapter Mix II (AMII) 5.00μL NEBNext Quick Ligation Reaction Buffer(5×) 20.00μL <![CDATA[Quick T4 DNA Ligase]]> 10.00μL Nucleic acid-free water Refill to 100.00 μL Total volume 100.00μL

[0110] Run the following program on the PCR instrument, as shown in Table 11:

[0111] Table 11 PCR reaction program

[0112] PCR procedure temperature time Number of cycles 1 20℃ 10 minutes 1 2 16℃ still /

[0113] Transfer the product of sequencing library construction reaction 2 to a 1.5mL centrifuge tube, add an equal volume of resuspended purified magnetic beads and perform nucleic acid purification. Add 200μL SFB and then perform magnetic bead purification, blow dry at room temperature. Add 13.00μL Eliution Buffer and mix well. After mixing and incubation, place on a magnetic stand and aspirate the liquid into a new EP tube. The liquid is the DNA sequencing library. Use Nanodrop and Qubit to measure the concentration of the DNA sequencing library.

[0114] The above-mentioned targeted amplification kit for rapidly obtaining dengue type II virus is stored at -20°C to minimize repeated freezing and thawing.

[0115] Step 6:

[0116] Library chip loading and sequencing start

[0117] Prepare the sequencing library suspension as shown in Table 12:

[0118] Table 12 Sequencing library suspension preparation system

[0119] Components volume Sequence Buffer (SQB) 37.50μL Loading Beads(LB) 25.50μL DNA sequencing library 12.00μL Total volume 75.00μL

[0120] Add 30 mL of Flush Tether (FLT) to a whole tube of Flush Buffer (FB) and mix well to prepare the library loading mixture.

[0121] Open the SpotON sample of the sequencing chip to exhaust air bubbles, and draw 200.00μL of library loading mixture into the PrimingPort hole until liquid reflux appears in the SpotON sample hole. Wash and blow to mix the sequencing library suspension, and add it drop by drop into the SponON sample hole smoothly, and cover the SpotON sample hole and the Priming Port hole.

[0122] Install the chip into the sequencer card slot, connect the data cable to the computer and start the sequencing control software MinKNOW. Select the appropriate sequencing model, chip, reagent kit and other related information parameters from the menu and start sequencing.

[0123] The server for sequencing analysis must have at least 16 CPU cores and 32 threads and at least 32 GB of memory. If sequencing analysis is performed on a server with a configuration lower than this, the time required to obtain the dengue II virus genome will be longer.

[0124] The average sequencing read length was 1,407.9 bp, which was consistent with the design.

[0125] Step 7:

[0126] Sequencing data analysis and genome assembly

[0127] The fast5 file generated by sequencing is used for base recognition in the high-precision mode of Guppy software in MinKNOW to convert the current signal into the corresponding base and remove the adapter sequence. After base recognition, it is converted into FASTQ format.

[0128] Bioinformatics analysis was used to remove sequences with 28bp bases at both ends of the sequence; sequences with read lengths less than 500bp were filtered out; and sequences with base sequencing quality values ​​less than 6 accounting for more than 50% were filtered out.

[0129] The filtered sequences were aligned to the reference genome (such as DENV serotype II strain 16681, GenBank: NC_001474), and then the sequences aligned to the reference genome were extracted and the sequencing depth of each site was calculated.

[0130] Perform SNP calling and generate VCF format files, create file indexes, and generate consensus sequences based on the reference genome.

[0131] The genome coverage depth obtained by sequencing is as follows Figure 2 As shown in Figure 2 It can be seen that the sequencing covers the entire length of the DENV genome and the sequencing depth is relatively large. The length of the obtained genome is 10459 bp. The genome sequence was submitted to GenBank for BLAST analysis. The results showed that the obtained dengue virus genome sequence and the dengue serotype II virus genome sequence with GenBank accession number KX655788 reached 100% similarity.

[0132] The library construction sequencing method used in this embodiment is the third-generation nanopore sequencing. The second-generation sequencing read length is relatively short, about 200bp, and the subsequent assembly is relatively difficult, and the continuity of the assembly is often poor, and the processing capacity for long repetitive sequences is limited. In addition, the second-generation sequencing instrument is large and expensive. Most laboratories are not equipped with second-generation sequencing instruments. Samples need to be sent to the company for sequencing. The degradation of sample RNA during the transportation of samples and the long sequencing time caused by the company's sequencing are problems that need to be solved. In recent years, the emerging third-generation nanopore sequencing technology has the characteristics of long sequencing read length, high throughput, and compact and portable instruments, which solves the problems of the above-mentioned second-generation sequencing.

[0133] In addition, the nanopore sequencing method uses 24 or 96 tags to detect 24 or 96 samples at the same time, reducing the cost price of each sample. When 24 samples are detected simultaneously, the sequencing price of each sample is 350 yuan, and the complete genome information of more samples can be obtained in a short time.

[0134] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A primer set for amplifying the genome of serotype dengue II virus, characterized in that: The primer set includes primer pair 1 to primer pair 16, each primer pair consists of a forward primer and a reverse primer, and the nucleotide sequences of primer pair 1 to primer pair 16 are shown in SEQ ID NOs: 1-32 in sequence.

2. The primer set for amplifying the genome of serotype dengue II virus according to claim 1, characterized in that: Primer pair 1 to primer pair 16 in the primer set are divided into two primer libraries, namely a first primer library and a second primer library, wherein the first primer library consists of primer pair 1, primer pair 3, primer pair 5, primer pair 7, primer pair 9, primer pair 11, primer pair 13 and primer pair 15; the second primer library consists of primer pair 2, primer pair 4, primer pair 6, primer pair 8, primer pair 10, primer pair 12, primer pair 14 and primer pair 16.

3. A targeted amplification kit for rapid amplification of serotype dengue II virus, characterized in that: The method comprises the primer set described in claim 1 or claim 2.

4. Use of the primer set according to claim 1 or 2 and the kit according to claim 3 in preparing a product for amplifying the genome of serotype dengue II virus.

5. A method for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification, characterized in that: The following steps are involved: (1) Sample RNA extraction; (2) Reverse transcription to generate cDNA; (3) using the primer set of claim 1 for targeted amplification; (4) constructing a sequencing library and analyzing the sequencing data to obtain the complete genome of the dengue II serotype virus.

6. The method for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification according to claim 5, characterized in that: The sample in step (1) is a serum sample.

7. The method for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification according to claim 5, characterized in that: When the primer set in claim 1 is used for targeted amplification in step (3), the primer set is divided into two primer pools according to claim 2, and the first primer pool and the second primer pool are used for amplification respectively, and then the amplification products of the two groups are collected.

8. The method for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification according to claim 5, characterized in that: In step (4), the sequencing sequence data is analyzed, including: (a) filtering and quality control of the sequencing data; (b) aligning the sequencing sequence to the dengue II virus reference genome; and (c) generating a whole genome of the consensus sequence.

9. The method for rapidly obtaining the whole genome of serotype dengue II virus based on targeted amplification according to claim 8, characterized in that: The sequencing data filtering quality control in step (a) includes: removing sequences with 28bp bases at both ends of the sequence; filtering sequences with a read length of less than 500bp; filtering sequences with a base sequencing quality value less than 6 accounting for more than 50%.

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