Amplicon sequencing technology-based whole genome sequencing primer for fiveleaf virus GII.3 and application of primer

Abstract

The invention discloses a fiveleaf virus GII.3 whole genome sequencing primer based on an amplicon sequencing technology and application thereof, and belongs to the technical field of virus gene sequencing. According to the whole genome sequencing primer and the sequencing method for the fiveleaf virus GII.3, interference of other microorganisms on sequencing can be avoided, the gene sequencing depth is high, the advantages of being wider in whole genome coverage, high in detection accuracy and high in specificity are achieved, the sequencing result can reflect mutation characteristics of main epidemic strains of the fiveleaf virus, and the sequencing result can be used for detecting the mutation characteristics of the main epidemic strains of the fiveleaf virus GII.3. And a powerful tool is provided for rapid diagnosis and full prevention of the fiveleaf akebia viruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of viral gene sequencing, and particularly relates to a primer for whole-genome sequencing of sapovirus GII.3 based on amplicon sequencing technology and its application. Background Art

[0002] Sapovirus is an enveloped single-stranded positive-sense RNA virus belonging to the family of human caliciviruses. It is an important cause of acute gastroenteritis (AGE) worldwide, capable of causing outbreaks and sporadic cases of acute gastroenteritis. It ranks second in the pathogen spectrum of viral acute gastroenteritis and has been detected in various places such as kindergartens, schools, long-term care institutions, hospitals, restaurants, hotels, and cruise ships. The transmission routes of sapovirus are diverse, it has strong environmental resistance, rapid virus mutation, and short immune protection time, with high infectivity and rapid transmission ability.

[0003] The prevalence of sapovirus not only poses a serious threat to population health but also causes huge social and economic losses. In high- and middle-income regions where rotavirus vaccination programs have been successfully implemented, sapovirus is also the most common cause of acute gastroenteritis in children. People of all age groups are susceptible to sapovirus. Among them, the incidence of sapovirus is the highest in children ≤5 years old, accounting for about 50% of the whole age group. Reports from Spain show that the incidence of sapovirus is the highest in children aged 0 - 2 years, decreasing in older children and young adults, and then increasing in adults over 60 years old. Currently, there are 17 virus strains of sapovirus that can infect humans, with the main genotypes being GII.3 and GI.2, accounting for about 70% of all sapovirus infections.

[0004] Genetic detection and accurate typing of virus epidemic strains are effective means to achieve precise treatment and effective control of the epidemic. However, the technical personnel of the present invention found during long-term work that the whole-genome research of sapovirus in China is almost blank, there are no relevant technical methods, the conventional detection of sapovirus and the articles on whole-genome analysis of sapovirus included in PUBMED are very few, and it is the 1st-generation sanger sequencing method. Currently, there are only 43 GII.3 whole-genome sequences globally included in NCBI. Those skilled in the art know that obtaining the whole genome by first-generation sequencing requires more than 10 experiments, which is complex and time-consuming. Moreover, since only one sequence is obtained, information such as virus mutations is easily masked. Not only that, metagenomic sequencing based on fecal samples or anal swab samples contains many intestinal microorganisms and human-derived samples, resulting in less effective data. Sufficient sequencing depth is required, the cost is extremely high, and it is very difficult to obtain the target sequence. Currently, there is a lack of effective second-generation / third-generation whole-genome sequencing methods for sapovirus. Developing a simple and low-cost second-generation / third-generation sequencing method for sapovirus has become an urgent technical problem to be solved.

[0005] Amplicon sequencing is a highly targeted method for analyzing genetic variations in specific genomic regions. Amplicon sequencing mainly includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing, and target region amplicon sequencing, etc. As a complementary technology to whole genome sequencing, amplicon capture sequencing can greatly simplify the experimental process and analysis target. It is a rapid and effective technology that plays a unique role in the new generation of high-throughput sequencing.

[0006] Patent document CN119913242A discloses a method for virus whole genome sequencing. The method includes extracting virus genes, performing reverse transcription, then using an amplicon sequencing primer set for targeted amplification, constructing a library, and performing bioinformatics analysis after sequencing. In a specific embodiment, this document discloses a primer set (10 groups) for sapovirus, but it does not explain the genotype of sapovirus to which this primer set is applicable, or whether it is applicable to the whole genome sequencing of all genotypes of sapovirus, and does not verify the number of reaction systems for amplification required for the provided primer set, the applicable sample concentration, as well as its feasibility and detection accuracy.

[0007] Based on this, the present invention provides a primer set for the whole genome sequencing of sapovirus GII.3 and a simplified amplicon-based enrichment sequencing method. Using the primer set for whole gene sequencing can avoid interference from other microorganisms during sequencing, ensure a higher sequencing depth of the GII.3 target gene, and has the advantages of a wider whole genome coverage, high detection accuracy, strong specificity. Moreover, the amplicon enrichment method is easy to operate, has low requirements for sample concentration, and the sequencing results can reflect the mutation characteristics of the main epidemic strains of sapovirus, providing a powerful tool for the rapid diagnosis and full prevention of sapovirus. Summary of the Invention

[0008] To achieve the whole genome sequencing of the sapovirus epidemic strain GII.3, one object of the present invention is to provide a primer set. The primer set can obtain the whole genome sequence by performing specific multiplex PCR amplification on sapovirus through 2 reaction systems. The amplicons generated by the primer set cover the whole genome sequence of sapovirus in a tiled manner, achieving deep sequencing of the sequence of the sapovirus epidemic strain GII.3. The second object of the present invention is to provide an application of the primer set in the preparation of a product for the whole genome sequencing of sapovirus GII.3. The third object of the present invention is to provide a kit including the primer set. Another object of the present invention is to provide a method for whole genome detection of sapovirus that is not for the purpose of disease diagnosis and treatment.

[0009] The objects of the present invention are achieved by the following technical solutions: In the first aspect of the present invention, the present invention provides a set of primer pairs for the whole genome sequencing of Sapovirus GII.3, characterized in that the set of primer pairs consists of primer pairs 1-21.

[0010] Specifically, the set of primer pairs consists of primer pair 1 shown in SEQ ID NO.1-2, primer pair 2 shown in SEQ ID NO.3-4, primer pair 3 shown in SEQ ID NO.5-6, primer pair 4 shown in SEQ ID NO.7-8, primer pair 5 shown in SEQ ID NO.9-10, primer pair 6 shown in SEQ ID NO.11-12, primer pair 7 shown in SEQ ID NO.13-14, primer pair 8 shown in SEQ ID NO.15-16, primer pair 9 shown in SEQ ID NO.17-18, primer pair 10 shown in SEQ ID NO.19-20, primer pair 11 shown in SEQ ID NO.21-22, primer pair 12 shown in SEQ ID NO.23-24, primer pair 13 shown in SEQ ID NO.25-26, primer pair 14 shown in SEQ ID NO.27-28, primer pair 15 shown in SEQ ID NO.29-30, primer pair 16 shown in SEQ ID NO.31-32, primer pair 17 shown in SEQ ID NO.33-34, primer pair 18 shown in SEQ ID NO.35-36, primer pair 19 shown in SEQ ID NO.37-38, primer pair 20 shown in SEQ ID NO.39-40, and primer pair 21 shown in SEQ ID NO.41-42.

[0011] In a specific embodiment of the present invention, the set of primer pairs is respectively in two primer pools. Among them, primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 are in primer pool 1, and primer pairs 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 are in primer pool 2.

[0012] In the second aspect of the present invention, the present invention provides an application of the primer pair set described in the first aspect of the present invention in the preparation of a product for the whole genome sequencing of Sapovirus GII.3.

[0013] The product includes but is not limited to reagents, reagent kits, chips, test strips, membrane strips or detection platforms.

[0014] In the third aspect of the present invention, the present invention provides a reagent kit, characterized in that the reagent kit includes the primer pair set described in the first aspect of the present invention, or the reagent kit includes a buffer solution containing the primer pair set described in the first aspect of the present invention.

[0015] Furthermore, the kit further includes reverse transcriptase, PCR reaction premix, and sequencing adapters.

[0016] The sequencing adapters are common sequencing adapters known to those skilled in the art and applicable to second-generation / third-generation sequencing platforms. The sequencing platforms include, but are not limited to, Illumina, Ion, or MGI. The sequencing adapters can be obtained by purchasing through commercially available kits.

[0017] The PCR reaction premix includes nuclease-free water, buffer, DNA polymerase, Mg 2+ , dNTPs, and the PCR reaction premix can be obtained by purchasing through commercially available channels.

[0018] Fourthly, the present invention provides a method for whole-genome sequencing of sapovirus GII.3 not for the purpose of disease diagnosis and treatment. The method includes the following steps: (1) Extract nucleic acid from the sample to be detected and reverse transcribe to obtain cDNA strands; (2) First-round PCR amplification Using the cDNA strands obtained in step (1) as a template, perform specific PCR amplification using the primer set described in the first aspect of the present invention, and collect the amplification products; (3) Purify and splice the PCR amplification products; (4) Construct a library; (5) Sequencing on a machine; (6) Bioinformatics analysis.

[0019] Preferably, the sample to be detected described in step (1) includes, but is not limited to, blood, throat swabs, saliva, and infected tissues.

[0020] In the specific embodiments of the present invention, the primer set is respectively in two primer pools. Among them, primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 are in primer pool 1, and primer pairs 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 are in primer pool 2.

[0021] Preferably, the primer concentrations in primer pool 1 and primer pool 2 are 10 - 50 µM.

[0022] More preferably, the primer concentrations in primer pool 1 and primer pool 2 are 10 µM.

[0023] In a specific embodiment of the present invention, after purification of the PCR product, a library is constructed using the Nextera® XT Library Prep Kit, and sequencing is performed using Miniseq. For the downloaded sequences, with the Genbank accession number LC790161.1 (GII.3) sequence as the reference sequence, the CLC Genomics Workbench 23.0 software is used for splicing.

[0024] The technical solution provided by the present invention has the following advantages: 1) According to the sequence characteristics of the sapovirus GII.3 gene, the present invention designs 21 pairs of primer pairs using the overlapping principle. In actual application, the primer pairs are divided into two primer pools for specific PCR amplification, which is compatible with 10 - 15 reaction systems in conventional detection into 2 reaction systems, effectively shortening the amplification time and difficulty.

[0025] 2) The primer set provided by the present invention can effectively improve the coverage of primer amplicons, ensuring coverage of 1.5 - 1.8 times the viral sequence. Compared with the coverage of 1 - 1.2 times in first-generation sequencing and the inability of metagenomic sequencing to effectively obtain all targeted sequences, the present invention better guarantees the acquisition of a whole-genome sequence with rapid full coverage.

[0026] 3) The high-fidelity primers provided by the present invention ensure the authenticity of sequence mutations, have a high accuracy rate for virus genotyping detection, and effectively improve the application and promotion of the GII.3 genotype in the prevention and control of sapovirus outbreaks and the exploration of epidemic trends. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a flowchart of the whole-genome sequencing of sapovirus GII.3 based on amplicon sequencing; Figure 2 It is the concentration after purification of the multiplex PCR products of two primer sets; Figure 3 It is the position of the primer set provided by the present invention relative to the target gene and the amplicon coverage area; Figure 4 It is the distribution map of the whole-genome sequence products of clinical samples of sapovirus GII.3. SPECIFIC EMBODIMENTS

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0029] Example 1: Design of Primers for the Whole Genome Sequencing of Sapovirus GII.3 The basic principle of amplicon sequencing is to use the PCR technology to amplify specific DNA regions, and then sequence these amplification products. First, specific primers are designed to amplify the genomic regions of interest; these regions are amplified by PCR technology to generate short, specific DNA fragments, namely amplicons, which cover the whole genome sequence of sapovirus in a tiled manner to achieve deep sequencing of the target sequence; then high-throughput sequencing technology is used to sequence these amplicons.

[0030] The process of obtaining the primers for the whole genome sequencing of sapovirus GII.3 in the present invention is as follows: referring to all the whole genome sequences of sapovirus GII.3 collected by NCBI, multiple pairs of primer sets covering different gene fragment regions of the whole gene are designed using multiple PCR primer design software, and then manual screening is carried out.

[0031] The manual screening process described in the present invention is as follows: a. First, primers containing simple repeat sequences and inverted repeat sequences are removed; b. Primers with a PCR primer length shorter than 15 bp or longer than 25 bp, or a GC content lower than 35% or higher than 65% are removed; c. The remaining primer sequences are re-annotated in the sapovirus genome sequence to identify the regions to which the primers belong, and primers covering the high mutation regions of the genome and primers spanning different gene regions are removed; d. The 5' and 3' end primers are corrected to cover the entire genome; The primer pairs obtained from different regions are combined to cover the whole genome and have a high amplification efficiency, and are suitable for amplification after mixing in 2 tubes: according to the principles that the amplification region is <800 bp, the length difference of the primer fragments amplified in each tube is <200 bp, the primers cover more than 1.5 times of the genome, the annealing temperature (the annealing temperature difference of the primers in the same group exceeds 10 °C), and it is not easy to generate internal dimers, the primers are evaluated and combined. Finally, 2 groups of optimal primer sets are obtained in the present invention. The optimal primer sets are shown in Table 1 and Table 2 respectively. Next, the present invention uses the 2 groups of optimal primer sets for subsequent experiments.

[0032] The 21-pair primer pool and the 18-pair primer pool shown in Table 1 and Table 2 are respectively used to perform PCR amplification on the test samples. The concentration statistics results of the purified PCR products are as Figure 2 shown. It can be seen that the concentration of the purified multiple PCR products of the 21-pair primer pool is significantly higher than that of the 18-pair primer pool, and the difference is statistically significant (p < 0.05). Therefore, the present invention preferably selects the 21-pair primer set shown in Table 1 as the best primer set screened in the present invention. The positions and sequence coverage regions of the 21 pairs of primers are as Figure 3 shown, where red represents the primers and pink-purple represents the obtained sequence regions.

[0033] Table 1 21 primer sets for amplicon sequencing of Sapporo virus GII.3 。

[0034] Table 2 18 primer sets for amplicon sequencing of Sapporo virus GII.3 。

[0035] Example 2: Method for whole genome sequencing of Sapporo virus GII.3 Step 1: Extract nucleic acid from the sample to be tested Use a commercially available kit to extract RNA from the sample to be tested.

[0036] Step 2: Reverse transcription Using the RNA obtained in Step 1 as a template, add reverse transcriptase to obtain cDNA. The reverse transcription system includes: 8 μl of RNA template, 2 μl of reverse transcriptase (5X RT SuperMix). The reverse transcription reaction conditions are: 25°C, 2 min; 55°C, 20 min; 95°C, 1 min; store at 4°C.

[0037] Step 3: Multiplex PCR amplification Primer pool 1: Includes primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 shown in Table 1.

[0038] Primer pool 2: Includes primer pairs 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 shown in Table 1.

[0039] Using the cDNA obtained in Step 2 as a template, perform specific PCR amplification in primer pool 1 and primer pool 2 respectively. The amplification system includes: 5 μl of cDNA product, 15 μl of 2X high-fidelity enzyme; 3 μl each of 10 μmol primer pool 1 / primer pool 2; 7 μl of ddH2O.

[0040] The PCR amplification program is as follows: 。

[0041] After multiplex PCR amplification, purify and splice the amplification products, then use the Nextera® XT Library PrepKit to construct a library for sequencing on Miniseq. Using the sequence with Genbank accession number LC790161.1 (GII.3) as the reference sequence for the sequences downloaded from the instrument, use the CLC Genomics Workbench 23.0 software for splicing.

[0042] Step 3: Optimize the primer pool concentration: After synthesizing the dry powder of primer pool 1 and primer pool 2, dilute them with ddH2O to 50 µmol, 20 µmol, 10 µmol, and 5 µmol respectively, and perform PCR amplification according to the matrix concentration combination method shown in Table 3. Statistically analyze the concentration of the purified PCR products, and the results are shown in Table 3.

[0043] Table 3 Concentrations of purified matrix paired multiplex PCR of primer pool 1 and primer pool 2 (ng / µl) 。

[0044] From the results in the above table, it can be seen that when the concentrations of primer pool 1 and primer pool 2 are between 10 - 50 µmol, the concentrations of the purified PCR products after amplification are all above 67 ng / µl. Considering the detection cost, the preferred primer pool concentration in the present invention is 10 µmol.

[0045] Example 3: Clinical application verification Take 7 fecal specimens of sapovirus GII.3 with Ct values of 15.68, 18.67, 22.40, 22.57, 24.91, 28.89, and 29.85 in fluorescence PCR detection for clinical verification, and perform whole genome sequencing according to the method provided in Example 2 of the present invention.

[0046] Results: After specific PCR amplification by the method provided in Example 2 of the present invention, the concentrations of the PCR products of GII.3 are 67.50 ng / µl, 39.80 ng / µl, 59.00 ng / µl, 34.75 ng / µl, 21.70 ng / µl, 48.80 ng / µl, and 15.95 ng / µl respectively. After library construction for second-generation sequencing, the concentrations are 8.23 ng / µl, 7.87 ng / µl, 4.61 ng / µl, 5.28 ng / µl, 4.66 ng / µl, 3.01 ng / µl, and 2.02 ng / µl respectively, meeting the requirements for loading the second-generation sequencing products. After performing whole genome sequencing on the test samples according to the method provided in the present invention, typing the virus based on the sequencing results, and then using the traditional method to detect the genome of the test samples, the virus typing results after detection are shown in Table 4.

[0047] Table 4 Specificity typing of GII.3 whole genome sequence typing based on amplicons 。

[0048] The 7 fecal specimens of sapovirus GII.3 cover different gene sub-component types in the Beijing area from 2019 to 2022. After PCR amplification, purification, library construction, and loading for sequencing by the method provided in the present invention, the whole genome sequences are obtained. The length of the whole genome sequence of GII.3 is 7330 - 7447 bp, and the sequence distribution is shown inFigure 4 , the sequence information and gene subgroup distribution are shown in Table 5.

[0049] Table 5 Sequence information and gene subgroup distribution map of GII.3 .

[0050] From the results of this example, it can be seen that the primer set for the whole genome sequencing of sapovirus GII.3 provided by the present invention and the sequence obtained by the sequencing method can be successfully used for virus genotyping, and the genotyping results are completely consistent with those of the traditional genotyping method. It can be further used for virus recombination analysis. The traditional gene typing of sapovirus is based on about 300-800 bp of partial gene fragments in the VP1 gene region. As an RNA virus prone to mutation and recombination, the whole genome sequence can identify whether the gene regions other than the genotyping region are recombined with other genomes. The genomic sequence results obtained by the present invention are used for recombination identification, and no recombinant genotypes are found. At the same time, the whole genome can also be further used for epidemic tracing and virus evolution analysis.

[0051] The homology between the whole genome sequences of GII.3 clade1 and clade 3 obtained by the present invention is 95.80% to 99.80% at the nucleic acid level and 99.31% to 99.88% at the amino acid level. There are at most 137 nucleotide mutations and 17 amino acid mutations, and these mutations are mainly located in open reading frame 1 region. The strain of GII.3 clade 1 obtained by the present invention has the highest sequence identity with the strain detected in Japan in 2022 (LC790161.1), with a nucleic acid sequence similarity of 97.10% to 99.60% and an amino acid sequence similarity of 99.71% to 99.80%. The strain of GII.3 clade3 obtained by the present invention has the highest sequence identity with the strain detected in Taiwan, China in 2014 (MN102405.1), with a nucleic acid sequence similarity of 97.14% to 98.60% and an amino acid sequence similarity of 99.67% to 99.88%. The strain of GII.3 clade 4 obtained by the present invention has the highest sequence identity with the strain detected in the United States in 2017 (MN461476.1), with a nucleic acid sequence similarity of 98.45% and an amino acid sequence similarity of 99.82%.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for the whole genome sequencing of Sapporo virus GII.3, characterized in that, The primer set comprises primer pair 1 shown in SEQ ID NOs. 1-2, primer pair 2 shown in SEQ ID NOs. 3-4, primer pair 3 shown in SEQ ID NOs. 5-6, primer pair 4 shown in SEQ ID NOs. 7-8, primer pair 5 shown in SEQ ID NOs. 9-10, primer pair 6 shown in SEQ ID NOs. 11-12, primer pair 7 shown in SEQ ID NOs. 13-14, primer pair 8 shown in SEQ ID NOs. 15-16, primer pair 9 shown in SEQ ID NOs. 17-18, primer pair 10 shown in SEQ ID NOs. 19-20, primer pair 11 shown in SEQ ID NOs. 21-22, primer pair 12 shown in SEQ ID NOs. 23-24, primer pair 13 shown in SEQ ID NOs. 25-26, primer pair 14 shown in SEQ ID NOs. 27-28, primer pair 15 shown in SEQ ID NOs. 29-30, primer pair 16 shown in SEQ ID NOs. 31-32, primer pair 17 shown in SEQ ID NOs. It consists of primer pair 17 shown in SEQ ID NOs.33-34, primer pair 18 shown in SEQ ID NOs.35-36, primer pair 19 shown in SEQ ID NOs.37-38, primer pair 20 shown in SEQ ID NOs.39-40, and primer pair 21 shown in SEQ ID NOs.41-42.

2. Use of the primer set described in claim 1 in preparing a product for whole genome sequencing of Saruvirus GII.

3.

3. The application according to claim 2, wherein The products include reagents, test kits, chips, test strips, membrane strips or detection platforms.

4. Kit, characterized in that, The kit comprises the primer set according to claim 1, or the kit comprises a buffer containing the primer set according to claim 1.

5. The kit according to claim 4, wherein The kit also includes reverse transcriptase, PCR reaction premix, and sequencing adapter.

6. The kit according to claim 5, characterized in that, The sequencing adapter is selected from general sequencing adapters applicable to second-generation / third-generation sequencing platforms; the PCR reaction premix includes nuclease-free water, buffer, DNA polymerase, Mg 2+ , dNTPs.

7. A method for whole genome sequencing of Saruvirus GII.3 not for the purpose of disease diagnosis and treatment, the method comprising the following steps: (1) Extract nucleic acid from the sample to be tested and reverse transcribe to obtain cDNA chain; (2) First round of PCR amplification Using the cDNA chain obtained in step (1) as a template, performing specific PCR amplification using the primer set of claim 1, and collecting the amplified product; (3) Purification and splicing of PCR amplification products; (4) Establishing a library; (5) Sequencing on a machine; (6) Bioinformatics analysis 8. The method according to claim 7, characterized in that The samples to be tested in step (1) include blood, throat swabs, saliva, and infected tissues.

9. The method according to claim 7, wherein The primer sets in step (2) are respectively in two primer pools, wherein primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 are in primer pool 1, and primer pairs 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 are in primer pool 2; the primer concentrations in primer pool 1 and primer pool 2 are 10-50 µM.

10. The method according to claim 7, wherein After purifying the PCR products, libraries were constructed using the Nextera® XT Library Prep Kit, and sequencing was performed using Miniseq. For the downloaded sequences, with the Genbank accession number LC790161.1 (GII.3) sequence as the reference sequence, the CLC Genomics Workbench 23.0 software was used for splicing.

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