Primers for whole genome sequencing of Saruvirus GII.3 based on amplicon sequencing technology and their application

Abstract

The present invention discloses a whole-genome sequencing primer for Zarovirus GII.3 based on amplicon sequencing technology and its application, belonging to the technical field of viral gene sequencing. The whole-genome sequencing primer and sequencing method for Zarovirus GII.3 provided by the present invention can avoid interference with sequencing by other microorganisms, achieve high gene sequencing depth, and have the advantages of wider whole-genome coverage, high detection accuracy, and strong specificity. The sequencing results can reflect the mutation characteristics of the main epidemic strains of Zarovirus, providing a powerful tool for rapid diagnosis and comprehensive prevention of Zarovirus.

Description

Technical Field

[0001] The present invention belongs to the technical field of viral gene sequencing, and specifically relates to a primer for sequencing the whole genome of the Sarcovirus 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 human Caliciviridae family. It is a major cause of acute gastroenteritis (AGE) worldwide, causing both outbreaks and sporadic outbreaks. It ranks second on the list of viral AGE pathogens and has been detected in a variety of settings, including kindergartens, schools, long-term care facilities, hospitals, restaurants, hotels, and cruise ships. Sapoviruses are highly contagious and spread rapidly due to their diverse transmission pathways, strong environmental resilience, rapid mutation rates, and short-lived immune protection.

[0003] Zarovirus epidemics not only pose a serious threat to public health but also result in significant socioeconomic losses. In high- and middle-income regions where rotavirus vaccination programs have been successfully implemented, Zarovirus is also the most common cause of acute gastroenteritis in children. People of all ages are susceptible to Zarovirus, with the highest incidence in children ≤5 years, accounting for approximately 50% of all age groups. Reports from Spain indicate that Zarovirus incidence is highest in children aged 0-2 years, decreases in older children and young adults, and then increases in adults over 60 years of age. Currently, there are 17 outbreak and sporadic strains of Zarovirus that can infect humans, with the predominant genotypes being GII.3 and GI.2, which account for approximately 70% of all Zarovirus infections.

[0004] Genetic testing and accurate typing of viral strains are effective means to achieve precise treatment and effectively control the epidemic. However, the technical personnel of the present invention have found in the long-term work process that there is almost no research on the whole genome of Zaru virus in China, and there are no relevant technical methods. There are very few articles on conventional Zaru virus detection and Zaru virus whole genome analysis included in PUBMED, and they are first-generation Sanger sequencing methods. Currently, there are only 43 GII.3 whole genome sequences included in NCBI worldwide. Those skilled in the art know that it takes more than 10 experiments to obtain a whole genome by first-generation sequencing, which is complicated and time-consuming. Moreover, because only one sequence is obtained, it is easy to cover up information such as viral mutations. In addition, metagenomic sequencing based on fecal samples or anal swab samples contains many intestinal microorganisms and human samples, and the effective data obtained is relatively small. Sufficient sequencing depth is required, the cost is extremely high, and it is difficult to obtain the target sequence. At present, there is a lack of effective second-generation / third-generation whole genome sequencing methods for Zaru virus. The development of a simple and low-cost second-generation / third-generation sequencing method for Zaru virus has become a technical problem that urgently needs to be overcome.

[0005] Amplicon sequencing is a highly targeted method used to analyze genetic variation in specific genomic regions. Amplicon sequencing primarily includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing, and targeted region amplicon sequencing. Amplicon capture sequencing, as a complementary technology to whole-genome sequencing, can significantly simplify experimental workflows and analytical objectives. It is a rapid and effective technology that plays a unique role in next-generation high-throughput sequencing.

[0006] Patent document CN119913242A discloses a method for whole-genome sequencing of a virus, comprising extracting viral genes, performing targeted amplification using an amplicon sequencing primer set after reverse transcription, constructing a library, and performing bioinformatics analysis after sequencing. In a specific embodiment, the document discloses a set of 10 primer sets specific to Sasavirus, but does not specify whether the primer sets are suitable for Sasavirus genotypes or for whole-genome sequencing of all Sasavirus genotypes. Furthermore, the document does not verify the number of reaction systems required for amplification using the provided primer sets, the applicable sample concentration, or their feasibility and detection accuracy.

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

[0008] In order to achieve whole genome sequencing of the Zarovirus epidemic strain GII.3, one object of the present invention is to provide a set of primer sets, which can perform specific multiplex PCR amplification of the Zarovirus through two reaction systems to obtain the whole genome sequence. The amplicons generated by the amplification of the primer set cover the whole genome sequence of the Zarovirus in a shingled manner, thereby achieving deep sequencing of the sequence of the Zarovirus 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 whole genome sequencing of the Zarovirus GII.3; the third object of the present invention is to provide a kit comprising the primer set; and another object of the present invention is to provide a method for whole genome detection of the Zarovirus that is not intended for disease diagnosis and treatment.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] In a first aspect of the present invention, the present invention provides a primer set for whole genome sequencing of Saprovirus GII.3, characterized in that the primer set consists of primer pair 1 to primer pair 21.

[0011] Specifically, the primer set 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 20 shown in SEQ ID NO. It consists of primer pair 16 shown in SEQ ID NOs.31-32, 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.

[0012] In a specific embodiment of the present invention, the primer groups are in two primer pools respectively, 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.

[0013] In a second aspect of the present invention, the present invention provides a use of the primer set described in the first aspect of the present invention in preparing a product for whole genome sequencing of Saruvirus GII.3.

[0014] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0015] In a third aspect of the present invention, the present invention provides a kit, characterized in that the kit comprises the primer set described in the first aspect of the present invention, or the kit comprises a buffer solution containing the primer set described in the first aspect of the present invention.

[0016] Furthermore, the kit also includes reverse transcriptase, PCR reaction premix, and sequencing adapter.

[0017] The sequencing adapter is a universal sequencing adapter known to those skilled in the art and applicable to second-generation / third-generation sequencing platforms, including but not limited to Illumina, Ion or MGI, and the sequencing adapter can be purchased through commercially available kits.

[0018] The PCR reaction premix includes nuclease-free water, buffer, DNA polymerase, Mg 2+ , dNTPs, and the PCR reaction premix can be purchased from commercial sources.

[0019] In a fourth aspect, the present invention provides 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:

[0020] (1) Extract nucleic acid from the sample to be tested and reverse transcribe to obtain cDNA chain;

[0021] (2) First round of PCR amplification

[0022] Using the cDNA chain 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 amplified product;

[0023] (3) Purification and splicing of PCR amplification products;

[0024] (4) Establish a library;

[0025] (5) Sequencing on a machine;

[0026] (6) Bioinformatics analysis.

[0027] Preferably, the sample to be tested in step (1) includes but is not limited to blood, throat swab, saliva, and infected tissue.

[0028] In a specific embodiment of the present invention, the primer groups are in two primer pools respectively, 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.

[0029] Preferably, the primer concentrations in primer pool 1 and primer pool 2 are 10-50 μM.

[0030] More preferably, the primer concentration in the primer pool 1 and the primer pool 2 is 10 μM.

[0031] In a specific embodiment of the present invention, the PCR product was purified and library construction was performed using the Nextera® XT Library Prep Kit. Sequencing was performed using Miniseq, and the sequence was spliced using CLC Genomics Workbench 23.0 software using the Genbank accession number LC790161.1 (GII.3) as the reference sequence.

[0032] The technical solution provided by the present invention has the following advantages:

[0033] 1) Based on the sequence characteristics of the Saruvirus GII.3 gene, the present invention uses the shingled principle to design 21 pairs of primer pairs. In practical applications, these primer pairs are divided into two primer pools for specific PCR amplification. The 10-15 reaction systems used in conventional detection are compatible with two reaction systems, effectively shortening the amplification time and difficulty.

[0034] 2) The primer set provided by the present invention can effectively improve the coverage of primer amplicon, ensuring coverage of 1.5-1.8 times the viral sequence. Compared with the 1-1.2-fold coverage of first-generation sequencing and the inability of metagenomic sequencing to effectively obtain all targeted sequences, the present invention can more effectively ensure the rapid and full coverage of the whole genome sequence.

[0035] 3) The high-fidelity primers provided by this invention ensure the authenticity of sequence mutations and have a high accuracy rate for virus typing detection, effectively improving the application and promotion of the GII.3 genotype in the prevention and control of Zaru virus outbreaks and the exploration of epidemic trends. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a flowchart for sequencing the whole genome of the Saruvirus GII.3 based on amplicon sequencing;

[0037] Figure 2 is the concentration of the purified multiplex PCR products of the two primer sets;

[0038] Figure 3 The position of the primer set provided by the present invention relative to the target gene and the area covered by the amplicon;

[0039] Figure 4 This is the distribution map of the whole genome sequence products of Saruvirus GII.3 clinical samples. DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0041] Example 1: Design of primers for whole genome sequencing of Sativa virus GII.3

[0042] The basic principle of amplicon sequencing is to use PCR technology to amplify specific DNA regions and then sequence these amplified products. First, specific primers are designed to amplify the genomic region of interest. These regions are amplified using PCR technology to generate short, specific DNA fragments, or amplicons. These amplicons overlap the entire Saruvirus genome sequence in a shingled pattern, enabling deep sequencing of the target sequence. These amplicons are then sequenced using high-throughput sequencing technology.

[0043] The process of obtaining primers for whole-genome sequencing of Saruvirus GII.3 in the present invention is as follows: referring to all whole-genome sequences of Saruvirus GII.3 included in NCBI, using multiplex PCR primer design software, multiple pairs of primer sets covering different gene fragment regions of the whole gene are designed, and then manual screening is performed.

[0044] The manual screening process described in the present invention is as follows: a. First, remove primers containing simple repeats and inverted repeats; b. Remove PCR primers shorter than 15 bp or longer than 25 bp, or with a GC content lower than 35% or higher than 65%; c. Remaining primer sequences are re-annotated in the genome sequence to identify the regions to which the primers belong, and remove primers covering highly mutated regions of the genome and primers spanning different gene regions; d. Modify the 5' and 3' end primers to cover the entire genome;

[0045] The primer pairs obtained for different regions were combined to cover the entire genome and have high amplification efficiency, and were suitable for amplification after mixing two tubes: the primers were evaluated and combined based on the principles of amplification region <800bp, primer fragment length difference of <200bp in each tube, primer coverage of more than 1.5 times the genome, annealing temperature (annealing temperature difference of primers in the same set greater than 10°C), and low production of internal dimers. The present invention ultimately obtained two optimal primer sets. The optimal primer sets are shown in Tables 1 and 2, respectively. Next, the present invention used these two optimal primer sets for subsequent experiments.

[0046] The 21-pair primer pool and the 18-pair primer pool shown in Table 1 and Table 2 were used to perform PCR amplification on the samples to be tested. The concentration statistics of the purified PCR products were as follows: Figure 2As shown in Table 1, it can be seen that the concentration of the purified multiplex PCR product 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 uses the 21-pair primer set shown in Table 1 as the optimal primer set screened by the present invention. The positions and sequence coverage areas of the 21-pair primers are shown in Table 1. Figure 3 As shown, red represents primers and pink and purple represent the acquired sequence regions.

[0047] Table 1 21 primer pairs for sequencing the Zaru virus GII.3 amplicon

[0048] .

[0049] Table 2 18 primer pairs for sequencing of Zaru virus GII.3 amplicon

[0050] .

[0051] Example 2: Whole-genome sequencing of Sativa virus GII.3

[0052] Step 1: Extract nucleic acid from the sample to be tested

[0053] RNA was extracted from the samples using a commercially available kit.

[0054] Step 2: Reverse transcription

[0055] Using the RNA obtained in step 1 as a template, add reverse transcriptase to generate cDNA. The reverse transcription system includes: 8µl of RNA template and 2µl of reverse transcriptase (5X RT SuperMix). Reverse transcription reaction conditions are: 25°C for 2 minutes; 55°C for 20 minutes; 95°C for 1 minute; and storage at 4°C.

[0056] Step 3: Multiplex PCR amplification

[0057] Primer pool 1: includes primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 shown in Table 1.

[0058] Primer pool 2: includes primer pairs 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 shown in Table 1.

[0059] Using the cDNA obtained in step 2 as a template, specific PCR amplification was performed in primer pool 1 and primer pool 2, respectively. The amplification system included: 5 µl of cDNA product, 15 µl of 2X high-fidelity enzyme, 3 µl of 10 µmol primer pool 1 / primer pool 2, and 7 µl of ddH2O.

[0060] The PCR amplification procedure is as follows:

[0061] .

[0062] After multiplex PCR amplification, the amplified products were purified and assembled. Libraries were constructed using the Nextera® XT Library Prep Kit for Miniseq sequencing. Sequences were assembled using CLC Genomics Workbench 23.0 software, using Genbank accession number LC790161.1 (GII.3) as a reference sequence.

[0063] Step 3: Optimize the primer pool concentration: Primer pool 1 and primer pool 2 were synthesized into dry powder and diluted with ddH2O to 50µmol, 20µmol, 10µmol, and 5µmol, respectively. PCR amplification was performed according to the matrix concentration combination method shown in Table 3. The concentration of the purified PCR products was calculated. The results are shown in Table 3.

[0064] Table 3 Concentrations of primer pools 1 and 2 after purification (ng / µl) for matrix-paired multiplex PCR

[0065] .

[0066] As can be seen from the results in the table above, when the concentrations of primer pools 1 and 2 were between 10 and 50 µmol, the concentrations of the purified PCR products were both above 67 ng / µl. Considering the cost of testing, the preferred primer pool concentration for this invention is 10 µmol.

[0067] Example 3: Clinical application verification

[0068] Seven stool specimens of Saruvirus GII.3 with Ct values of 15.68, 18.67, 22.40, 22.57, 24.91, 28.89, and 29.85 detected by fluorescence PCR were collected for clinical validation, and whole genome sequencing was performed according to the method provided in Example 2 of the present invention.

[0069] 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 were 67.50ng / µl, 39.80ng / µl, 59.00ng / µl, 34.75ng / µl, 21.70ng / µl, 48.80ng / µl and 15.95, respectively. The concentrations after the second-generation sequencing library construction were 8.23ng / µl, 7.87ng / µl, 4.61ng / µl, 5.28ng / µl, 4.66ng / µl, 3.01ng / µl and 2.02ng / µl, respectively, which met the requirements for the second-generation sequencing product machine. After whole genome sequencing of the test sample according to the method provided by the present invention, the virus was typed according to the sequencing results, and then the genome of the test sample was tested using the traditional method. The virus typing results after detection are shown in Table 4.

[0070] Table 4 GII.3 Amplicon-based whole genome sequence typing specificity

[0071] .

[0072] Seven stool specimens of Saprovirus GII.3 covering different genotypes in Beijing from 2019 to 2022 were amplified by PCR, purified, constructed, and sequenced using the method provided by the present invention to obtain the whole genome sequence. The obtained whole genome sequence length of GII.3 was 7330-7447 bp, and the sequence distribution is shown in Figure 2. Figure 4 , the sequence information and gene subgroup distribution are shown in Table 5.

[0073] Table 5 GII.3 sequence information and gene subgroup distribution

[0074] .

[0075] From the results of this example, it can be seen that the primer set for the whole genome sequencing of the Saruvirus GII.3 provided by the present invention and the sequence obtained by the sequencing method can be successfully used for virus genotyping. The typing results are completely consistent with the typing results of the traditional typing method and can be further used for virus recombination analysis. The traditional genotyping of Saruvirus is based on a partial gene fragment of about 300-800bp in the VP1 gene region. As an RNA virus that is prone to mutation and recombination, the whole genome sequence can identify whether the gene region other than the genotyping region has recombined with other genomes. The genome 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.

[0076] The homology between the whole genome sequences of GII.3 clade 1 and clade 3 obtained in this study ranges from 95.80% to 99.80% at the nucleic acid level and from 99.31% to 99.88% at the amino acid level. A maximum of 137 nucleotide mutations and 17 amino acid mutations are present, primarily located in the open reading frame 1 region. The GII.3 clade 1 strain obtained in this study 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 GII.3 clade 3 strain obtained in this study 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 GII.3 clade 4 strain obtained in the present invention has the highest sequence identity with the strain (MN461476.1) detected in the United States in 2017, with a nucleic acid sequence similarity of 98.45% and an amino acid sequence similarity of 99.82%.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for whole genome sequencing of Sativa 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 according to claim 1 in preparing a product for whole genome sequencing of Saruvirus GII.

3.

3. The use according to claim 2, characterized in that The products include reagents, test kits, chips, test strips, membrane strips or detection platforms.

4. A 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, characterized in that 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 a universal sequencing adapter suitable for the second generation / third generation sequencing platform; the PCR reaction premix includes nuclease-free water, buffer, DNA polymerase, Mg 2+ , dNTPs.

7. A method for whole-genome sequencing of a Sativa virus GII.3 not for the purpose of disease diagnosis and treatment, 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, perform specific PCR amplification using the primer set described in claim 1, and collect the amplified product; (3) Purification and splicing of PCR amplification products; (4) Establish 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, characterized in that 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 10-50µM.

10. The method according to claim 7, characterized in that After purification of the PCR product, the library was constructed using the Nextera® XTLibrary Prep Kit and sequenced using Miniseq. The sequences were assembled using CLC Genomics Workbench 23.0 software, with Genbank accession number LC790161.1 (GII.3) as the reference sequence.

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