A primer set and kit for amplifying serotype dengue type I virus genome and its application

Abstract

The present invention discloses a primer set for amplifying the genome of serotype I dengue virus (DENV‑1). The primer set includes primer pair 1 to primer pair 16, each pair of primers is composed of a forward primer and a reverse primer, and the nucleotide sequences of primer pair 1 to primer pair 16 are sequentially shown in SEQ ID NO: 1‑32. Also disclosed is a targeted amplification kit for rapid amplification of dengue type 1 virus containing the primer set. The targeted amplification primer set of the present invention is composed of 16 pairs of primers, which can cover and amplify all currently popular dengue type 1 virus strains, with high amplification efficiency and less cross-interference; at the same time, the downstream sequencing method and analysis method constructed by the amplicon of the present invention have high genome sequencing accuracy and large sequencing throughput.

Description

Technical Field

[0001] The invention belongs to the field of genome sequencing and relates to a primer set and a kit for amplifying the genome of serotype dengue type I virus and applications thereof. Background Art

[0002] Dengue virus is an important mosquito-borne virus belonging to the Flaviviridae family, genus Flavivirus. 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. Dengue fever is mainly prevalent in tropical and subtropical regions around the world, and has been rapidly increasing and spreading around the world in the past 30 years. Dengue fever poses a serious threat to public health.

[0003] Currently, commonly used viral genome sequencing technologies include PCR-Sanger sequencing and metagenomic sequencing technology.

[0004] (1) PCR-Sanger sequencing, which uses primers covering the entire genome fragment region for PCR amplification, and then performs Sanger sequencing (first-generation sequencing) on ​​the large number of amplified fragments to obtain fragment sequences for assembly. This technology is cumbersome, labor-intensive, and has a long operation cycle;

[0005] (2) In recent years, metagenomic second-generation sequencing technology has emerged, which can detect all viral sequences in a sample without the need for primers or probes, and can detect new viruses. However, it currently faces technical problems such as low data utilization, high detection costs, and a large amount of data required to assemble the genome. Summary of the invention

[0006] The object of the present invention is to provide a primer set for specifically amplifying the genome of serotype dengue type I virus.

[0007] The present invention also aims to provide a targeted amplification kit containing the above primer set for rapid and specific amplification of serotype dengue type I virus.

[0008] 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 type I virus.

[0009] 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 type I 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.

[0010] 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.

[0011] 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.

[0012] 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 form a pair of primers numbered 1 (mD1-36F and mD1-956R, and the rest are similar.

[0013] 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 for targeted amplification sequencing to obtain the full genome of dengue type I virus.

[0014] Specifically, the primer set of the present invention includes primers in a first primer library placed in primer pool 1 and primers in a second primer library placed in primer pool 2:

[0015]

[0016]

[0017] The above-mentioned second object of the present invention can be achieved by the following technical scheme: a targeted amplification kit for rapid amplification of serotype dengue type I virus, comprising the primer set.

[0018] Preferably, the primer set is divided into two primer pools.

[0019] As a preferred technical solution of the present invention, the kit of the present invention also includes a reverse transcription reagent component.

[0020] Preferably, the reverse transcription reagent components include reverse transcriptase and reverse transcriptase buffer.

[0021] Preferably, the reverse transcriptase is Super Script III reverse transcriptase.

[0022] 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.

[0023] 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.

[0024] As a preferred technical solution of the present invention, the kit of the present invention also includes a sequencing library construction component.

[0025] Preferably, the sequencing library construction components include end repair enzyme, end repair buffer, T4 DNA ligase, and T4 DNA ligase buffer.

[0026] As a preferred technical solution of the present invention, the targeted amplification kit for rapid amplification of dengue type I virus of the present invention is stored at -20°C to minimize repeated freezing and thawing.

[0027] 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 type I virus.

[0028] The present invention also provides a method for rapidly obtaining the whole genome of serotype dengue type I virus based on targeted amplification, comprising the following steps:

[0029] (1) Sample RNA extraction;

[0030] (2) Reverse transcription to generate cDNA;

[0031] (3) targeted amplification using the above primer set;

[0032] (4) Build a sequencing library and analyze the sequencing data to obtain the complete genome of dengue type I virus.

[0033] In the method for rapidly obtaining the whole genome of serotype dengue type I virus based on targeted amplification:

[0034] Preferably, the sample in step (1) is a serum sample or a mosquito sample.

[0035] Preferably, when the above primer set is used for targeted amplification in step (3), the primer set is divided into two groups as described above, and amplification is performed using the first primer library and the second primer library respectively, and then the amplification products of the two groups are collected.

[0036] 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 type I virus reference genome; and (c) generating a whole genome of consensus sequences.

[0037] 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%.

[0038] 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 type I virus genome will be longer.

[0039] The present invention has the following advantages:

[0040] (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;

[0041] (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 type I virus strains, with high amplification efficiency and little cross-interference;

[0042] (3) The primer pairs of the present invention can well amplify all subtypes of dengue type I virus without worrying about the problem of poor primer binding efficiency;

[0043] (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

[0044] Figure 1 This is a flow chart of the method for obtaining the genome of serotype dengue type I virus based on targeted amplification sequencing in Example 1 of the present invention;

[0045] Figure 2 This is the sequencing read length distribution result in Example 1 of the present invention;

[0046] Figure 3 The genome coverage depth result is obtained by sequencing a sample in Example 1 of the present invention. DETAILED DESCRIPTION

[0047] 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.

[0048] Example 1

[0049] like Figure 1 As shown, the method provided in this embodiment for rapidly obtaining the whole genome of serotype dengue type I virus based on targeted amplification comprises the following steps:

[0050] Sample source: Clinical case samples from a certain hospital, samples are serum.

[0051] Step 1:

[0052] Extraction of sample RNA:

[0053] 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.

[0054] Step 2:

[0055] The extracted RNA was reverse transcribed to generate cDNA. The configuration system was as shown in Table 1 according to the instructions of Super Script III reverse transcriptase:

[0056] Table 1 Configuration system according to Super Script III reverse transcriptase instructions

[0057] 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

[0058] Set up the PCR machine and run the following program in Table 2:

[0059] Table 2 PCR reaction procedure

[0060]

[0061]

[0062] The reaction system is configured as shown in Table 3:

[0063] Table 3 PCR reaction system

[0064] Components volume The product after adding the above primers and incubating at 65°C 10.00μL Reverse transcriptase buffer 4.00μL RNase inhibitors 0.50μL Super Script III reverse transcriptase (200U / μL) 1.00μL Nucleic acid-free water 4.50μL Total volume 20.00μL

[0065] Run the following program on the PCR instrument, as shown in Table 4:

[0066] Table 4 PCR operation program

[0067] PCR procedure temperature time Number of cycles 1 30℃ 10min 1 2 42℃ 30min 1 3 95℃ 5min 1 4 4℃ pause /

[0068] The obtained cDNA product proceeds to step 3.

[0069] Step 3:

[0070] Multiplex PCR targeted amplification is as follows:

[0071] Dissolve the primers in TE buffer and dilute the primers of the same primer pool in the same tube.

[0072] 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 consisting of a forward primer and a reverse primer. The final concentration of each primer pair is 1.25 μM, and the final concentrations of the forward primer and the reverse primer in each primer pair are both 1.25 μM.

[0073] 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 pair of primers consists of a forward primer and a reverse primer. The final concentration of each primer pair is 1.25 μM, and the final concentrations of the forward primer and reverse primer of each primer pair are respectively 1.25 μM.

[0074] Prepare the following system in a 0.2 mL centrifuge tube, as shown in Table 5:

[0075] Table 5 Multiplex PCR reaction system

[0076]

[0077]

[0078] Run the following program on the PCR instrument, as shown in Table 6:

[0079] Table 6 PCR operation program

[0080] PCR procedure temperature time Number of cycles 1 20℃ 30min 1 2 65℃ 5min 2-40 3 4℃ pause /

[0081] Step 4:

[0082] Product purification and quantification

[0083] 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.

[0084] Step 5:

[0085] DNA sequencing library preparation

[0086] 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 7:

[0087] Table 7 Sequencing library construction reaction 1 system

[0088] 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

[0089] Run the following program on the PCR instrument, as shown in Table 8:

[0090] Table 8 PCR reaction program

[0091] PCR procedure temperature time Number of cycles 1 20℃ 30 minutes 1 2 65℃ 5 minutes 1 3 16℃ still /

[0092] 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.

[0093] 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 9:

[0094] Table 9 Sequencing library construction reaction 2 system

[0095] 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 Quick T4 DNA Ligase 10.00μL Nucleic acid-free water Refill to 100.00 μL Total volume 100.00μL

[0096] Run the following program on the PCR instrument, as shown in Table 10:

[0097] Table 10 PCR reaction program

[0098] PCR procedure temperature time Number of cycles 1 20℃ 10 minutes 1 2 16℃ still /

[0099] 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.

[0100] The above-mentioned targeted amplification kit for rapidly obtaining dengue type I virus is stored at -20°C to minimize repeated freezing and thawing.

[0101] Step 6:

[0102] Library chip loading and sequencing start

[0103] Prepare the sequencing library suspension as shown in Table 11:

[0104] Table 11 Sequencing library suspension preparation system

[0105]

[0106]

[0107] Add 30 mL of Flush Tether (FLT) to a full tube of Flush Buffer (FB) and mix well to prepare the library loading mixture.

[0108] 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.

[0109] 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.

[0110] 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 I virus genome will be longer.

[0111] The sequencing read length distribution results are as follows Figure 2 As shown in Figure 2 It can be seen that the sequencing read length is concentrated around 1000bp.

[0112] Step 7:

[0113] Sequencing data analysis and genome assembly

[0114] 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.

[0115] 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.

[0116] The filtered sequences were aligned to the reference genome (such as DENV strain NV46, GenBank: KX458014), and then the sequences aligned to the reference genome were extracted, and the sequencing depth of each site was calculated.

[0117] Perform SNP calling and generate VCF format files, create file indexes, and generate consensus sequences based on the reference genome.

[0118] The genome coverage depth obtained by sequencing is as follows Figure 3 As shown in Figure 3 It can be seen that the sequencing covers the DENV genome and is deep. The length of the obtained genome is 10735bp. The genome sequence was submitted to GeneBank for BLAST analysis. The results showed that the obtained dengue virus genome sequence (its nucleotide sequence is shown in SEQ ID NO: 33) and the dengue virus genome sequence with Genbank accession number: OM281577 reached 99.62% similarity.

[0119] 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 virus type I, 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 type 1 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 type I virus, characterized by: The method comprises the primer set described in claim 1 or claim 2.

4. A method for rapidly obtaining the whole genome of serotype dengue type I 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) Build a sequencing library and analyze the sequencing data to obtain the complete genome of dengue type I virus.

5. The method for rapidly obtaining the whole genome of serotype dengue type I virus based on targeted amplification according to claim 4, characterized in that: The sample in step (1) is a serum sample or a mosquito sample.

6. The method for rapidly obtaining the whole genome of serotype dengue type I virus based on targeted amplification according to claim 4, characterized in that: When the primer set in claim 1 is used for targeted amplification in step (3), the primer sets are grouped as in claim 2, and amplification is performed using the first primer library and the second primer library respectively, and then the amplification products of the two groups are collected.

7. The method for rapidly obtaining the whole genome of serotype dengue type I virus based on targeted amplification according to claim 4, 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 type I virus reference genome; and (c) generating a whole genome of the consensus sequence.

8. The method for rapidly obtaining the whole genome of serotype dengue type I virus based on targeted amplification according to claim 7, 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%.

9. Use of the primer set according to claims 1-2 and the kit according to claim 3 in preparing a product for amplifying the genome of serotype dengue type I virus.

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