Primer set, kit and method for whole genome sequencing of norovirus

Abstract

The present invention discloses a primer set, a kit and a method for whole genome sequencing of norovirus, and belongs to the technical field of viral gene sequencing. According to the gene sequence characteristics of norovirus GII.3[P12], the present invention adopts the shingled principle to design 22 pairs of primers, performs specific PCR amplification in two primer pools, shortens the amplification time and difficulty, improves the primer amplicon coverage and whole genome capture efficiency, and has a high accuracy rate for tracing the source of GII.3[P12] norovirus epidemic and identifying the spread trend, which is conducive to improving the application and promotion of GII.3P[12] in the prevention and control of norovirus outbreaks and the exploration of epidemic trends.

Description

Technical Field

[0001] The present invention belongs to the technical field of viral gene sequencing, and in particular relates to a method for sequencing the whole genome of a norovirus based on an amplicon technology, and a primer set and a kit used for sequencing. Background Art

[0002] Norovirus (NoV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the human Caliciviridae family. It is the leading cause of diarrhea in adults and the second leading cause of diarrhea in infants and young children under 5 years old. Norovirus has a variety of transmission routes, a low infection dose, a long detoxification time, strong environmental resistance, rapid viral mutation, and a short immune protection period. It is highly contagious and can spread rapidly. Globally, Norovirus is the main pathogen causing sporadic and outbreaks of acute gastroenteritis in humans.

[0003] The prevalence of norovirus not only poses a serious threat to human health, but also causes huge socioeconomic losses. In high- and middle-income areas where rotavirus vaccination programs have been successfully implemented, norovirus is also the most common cause of acute gastroenteritis in children. People of all ages are susceptible to norovirus. Among them, the incidence of norovirus is highest in children under 5 years old. 70% of norovirus cases in children aged 0 to 4 years occur between 6 and 23 months old. Patients infected with norovirus in low-income countries and medical institutions are younger. At present, the main strains causing norovirus outbreaks are GII.2[P16], GII.3[P12], GII.17[P17], GII.6[P7], and GII.4[P31]. Among them, GII.3[P12] has dominated the outbreaks caused by norovirus alone in the past two years.

[0004] Genetic testing and accurate typing of viral epidemic 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 conventional norovirus detection and articles involving norovirus whole genome analysis such as PUBMED and HowNet are all based on first-generation sequencing and metagenomic sequencing. Currently, there are only 11 GII.3[P12] and whole genome sequences included in NCBI. It is known to those skilled in the art that more than 10 experiments are required to obtain the whole genome by first-generation sequencing, which is complicated and time-consuming, and because only one sequence is obtained, it is easy to cover up information such as virus 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, requiring sufficient sequencing depth, 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 norovirus. Therefore, developing a simple and low-cost second-generation / third-generation sequencing method for norovirus has become a technical problem that urgently needs to be overcome.

[0005] Amplicon sequencing is a highly targeted method used to analyze gene variation in specific genomic regions. Amplicon sequencing mainly includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing, and target region amplicon sequencing. Amplicon capture sequencing, as a complementary technology to whole genome sequencing, can greatly simplify the experimental process and analysis targets. It is a fast and effective technology that plays a unique role in the new generation of high-throughput sequencing.

[0006] Patent document CN 117512213A discloses a whole genome capture kit for the GI-GIX subtype of norovirus, wherein the kit includes three primer pools, each primer in the primer pool is composed of a targeting primer, a UMI sequence and a barcdoe sequence arrangement, and the advantage is that the cost of connecting and adding the barcdoe sequence in the subsequent library construction process can be effectively reduced, thereby reducing the cost. However, the technical solution is used to detect the GI-GIX subtype of norovirus, and the detection range is wide, but the detection specificity for the epidemic strains of norovirus such as GII.3[P12] is not strong. Based on this, the present invention provides a whole genome sequencing method specifically for the epidemic strain of norovirus GII.3[P12], which can not only reflect the mutation characteristics of the main epidemic strains of norovirus, but also avoid the interference of other microorganisms on sequencing, ensure a higher sequencing depth of the GII.3[P12] target gene, obtain a wider coverage of the whole genome, and have a high detection accuracy and strong specificity. Summary of the invention

[0007] With respect to the whole genome sequencing of the epidemic strain of norovirus GII.3[P12], one object of the present invention is to provide a set of primer sets, which perform specific multiplex PCR amplification on norovirus, and the amplicons generated by the amplification of the primer sets cover the whole genome sequence of norovirus in a shingled manner; a second object of the present invention is to provide a use of the primer set in the preparation of a product for whole genome sequencing of norovirus; a third object of the present invention is to provide a kit comprising the primer set; and a further object of the present invention is to provide a method for whole genome detection of norovirus that is not intended for disease diagnosis and treatment.

[0008] The present invention includes the following technical solutions:

[0009] In a first aspect, the present invention provides a primer set, characterized in that the primer set consists of primer pair 1 to primer pair 22 in Table 1, and the primer set is used for whole genome sequencing of norovirus GII.3[P12].

[0010] Among them, primer pair 1 consists of the forward amplification primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2; primer pair n consists of the forward amplification primer shown in SEQ ID NO. (2n-1) and the reverse primer shown in SEQ ID NO.2n, and n is selected from an integer between 2-22.

[0011] 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, 20 and 22 are in primer pool 2.

[0012] In a second aspect, 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 norovirus GII.3[P12].

[0013] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0014] In a third aspect, 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 containing the primer set described in the first aspect of the present invention.

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

[0016] The sequencing adapter is a universal sequencing adapter known to those skilled in the art and applicable to second-generation / third-generation sequencing platforms, wherein the sequencing platforms include but are not limited to Illumina, Ion or MGI, and the sequencing adapter can be purchased through commercially available kits.

[0017] The PCR reaction premix includes nuclease-free water, buffer, DNA polymerase, Mg 2+ , dNTP, the PCR reaction premix can be purchased from commercial sources.

[0018] In a fourth aspect, the present invention provides a method for whole genome sequencing of norovirus GII.3 [P12] not for the purpose of disease diagnosis and treatment, the method comprising the following steps:

[0019] (1) Extract nucleic acid from the sample to be tested and reverse transcribe to obtain cDNA chain;

[0020] (2) First round of PCR amplification

[0021] Using the cDNA chain obtained in step (1) as a template, performing specific PCR amplification using the primer set described in the first aspect of the present invention, and collecting the amplified product;

[0022] (3) Purification and splicing of PCR amplification products;

[0023] (4) Establishing a library;

[0024] (5) Sequencing on a machine;

[0025] (6) Bioinformatics analysis

[0026] Preferably, the samples to be tested in step (1) include but are not limited to blood, throat swabs, saliva, and infected tissues.

[0027] 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, 20 and 22 are in primer pool 2.

[0028] Preferably, the primer concentration in the primer pool 1 and the primer pool 2 is 10-50 μM.

[0029] More preferably, the primer concentration in primer pool 1 and primer pool 2 is 10 μM.

[0030] In a specific embodiment of the present invention, after the PCR product is purified, a library is constructed using Nextera® XT Library Prep Kit, and sequencing is performed using Miniseq. The sequence of the machine is spliced ​​using the CLC Genomics Workbench 23.0 software with the Genbank accession number OP901693 as the reference sequence.

[0031] According to the gene sequence characteristics of norovirus GII.3[P12], the present invention adopts the shingled principle to design 22 pairs of primers, which are divided into two primer pools for specific PCR amplification in practical applications. 10-15 reaction systems of conventional detection are compatible in 2 reaction systems, effectively shortening the amplification time and difficulty.

[0032] The primer set provided by the present invention can effectively improve the coverage of primer amplicon, ensuring coverage of 1.5-1.8 times of the viral sequence. Compared with the 1-1.2 times 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 acquisition of rapid and full coverage of the whole genome sequence.

[0033] The high-fidelity primers provided by the present invention ensure the authenticity of sequence mutations and have a high accuracy rate in virus typing detection, effectively improving the application and promotion of GII.3P

[12] in the prevention and control of norovirus outbreaks and the exploration of epidemic trends. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Flow chart for norovirus whole genome sequencing;

[0035] Figure 2 is the concentration of the purified multiplex PCR products of the two primer pools;

[0036] Figure 3 The primer positions and sequence coverage areas provided by the present invention;

[0037] Figure 4 The concentration of clinical samples after amplicon amplification and library purification;

[0038] Figure 5 This is the distribution map of whole genome sequence products of 10 clinical samples. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1 Primer Pool

[0041] With reference to all GII.3P

[12] whole genome sequences collected in NCBI GenBank (as of January 2023), five sets of primers were designed using multiplex PCR primer design software, with amplification fragments of 400 bp, 500 bp, 600 bp, 700 bp, and 800 bp, respectively. First, the degree of internal dimerization after mixing multiple primer sets was evaluated, and the T value of primer design was improved while ensuring that the generation of primer dimers was reduced. m The consistency of the value and GC content.

[0042] The present invention finally selected two groups of optimal primers for clinical trials, the optimal primers are 22 pairs of primers shown in Table 1 and 17 pairs of primers shown in Table 2. The primers shown in Table 1 and Table 2 were used to perform specific PCR amplification on the samples to be tested, and the concentration statistical results of the purified PCR products are shown in the attached manual. Figure 2From the results shown in the figure, it can be seen that the concentrations of the 22 pairs of primers shown in Table 1 after purification are better in the high Ct value samples, and the primer preference is weakened, and the sequence distribution is more uniform. Therefore, the present invention prefers the 22 pairs of primers shown in Table 1. The positions and sequence coverage areas of the 22 pairs of primers are as shown in the attached manual. Figure 3 As shown, red represents primers and pink and purple represent the acquired sequence region.

[0043] Table 1 Primers used to specifically amplify the whole genome of GII.3P

[12]

[0044]

[0045] Table 2 Primers used for specific amplification of the whole genome of GII.3P

[12]

[0046]

[0047] Example 2 GII.3P

[12] Whole Genome Sequencing Method

[0048] Step 1: Extract nucleic acid from the sample to be tested

[0049] The RNA in the sample to be tested was extracted using a commercially available kit.

[0050] Step 2: Reverse transcription

[0051] The RNA obtained in step 1 was used as a template, and reverse transcriptase was added to obtain cDNA. The reverse transcription system includes: 8µl RNA template, 2µl reverse transcriptase (5X RT SuperMix). The reverse transcription reaction conditions are: 25℃, 2 min; 55℃, 20 min; 95℃, 1 min; 4℃, storage.

[0052] Step 3: Multiplex PCR amplification

[0053] Primer pool 1: includes primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 shown in Table 1.

[0054] Primer pair 2: includes primer pairs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 shown in Table 1.

[0055] 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: 5µl of cDNA product, 15µl of 2X high-fidelity enzyme; 3µl of 10µmol primer pool 1 / primer pool 2 respectively; and 7µl of ddH2O.

[0056] The PCR amplification procedure is as follows:

[0057]

[0058] After multiplex PCR amplification, the amplified products were purified and spliced, and then the library was constructed using Nextera® XT Library PrepKit for Miniseq sequencing. The sequence with Genbank accession number OP901693 was used as the reference sequence for the off-machine sequence, and CLC Genomics Workbench 23.0 software was used for splicing.

[0059] Step 3: Optimize the concentration of the primer pool: synthesize the dry powder of primer pool 1 and primer pool 2 and dilute them with ddH2O to 50µmol, 20µmol, 10µmol and 5µmol respectively, perform PCR amplification according to the matrix concentration combination method shown in Table 3, and calculate the concentration of the purified PCR product. The results are shown in Table 3.

[0060] Table 3 Concentrations of primer pools 1 and 2 after purification (ng / µl)

[0061]

[0062] 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 products after PCR amplification and purification are all above 49 ng / µl. Considering the detection cost, the preferred primer pool concentration of the present invention is 10 µmol.

[0063] Example 3 Clinical application verification

[0064] Ten stool specimens of norovirus GII.3P

[12] were collected for clinical verification and whole genome sequencing was performed according to the method provided in Example 2 of the present invention.

[0065] Results: The Ct values ​​of the fluorescent PCR detection of 10 stool specimens of Norovirus GII.3P

[12] were between 18 and 30. After specific PCR amplification by the method provided in Example 2 of the present invention, the median concentration of the PCR product was 32.88 ng / µl, the lowest concentration was 12.00 ng / µl, and the highest concentration was 66.50 ng / µl. After the second-generation sequencing library was built, the median concentration was 8.60 ng / µl, the lowest concentration was 5.90 ng / µl, and the highest concentration was 12.7 ng / µl, which all met the requirements for the second-generation sequencing product. The statistical results are detailed in the Appendix of the specification. Figure 4After the whole genome of the sample to be tested is sequenced according to the method provided by the present invention, the virus is typed according to the sequencing result, and then the genome of the sample to be tested is tested using the traditional method. The virus typing results after the test are shown in Table 4.

[0066] Table 4. GII.3P

[12] amplicon-based whole genome sequence typing specificity

[0067]

[0068] Ten stool specimens of norovirus GII.3P

[12] were amplified by PCR, purified, library constructed, and sequenced by the method provided by the present invention. Full genome sequences were obtained for 9 of them, one of which was partially missing. The length of the obtained full genome sequence was 7382-7450 bp. The sequence distribution is shown in the appendix of the specification. Figure 5 According to Table 4 and Figure 5 The results show that the sequence obtained by the whole genome sequencing method of Norovirus GII.3P

[12] provided by the present invention can be successfully used for virus genotyping, and the typing results are completely consistent with those of traditional typing methods.

[0069] From the above experimental results, it can be seen that the common norovirus GII.3P

[12] whole-genome sequencing method based on amplicon technology provided by the present invention can be successfully used for whole-genome sequencing of clinical samples. The method is simple, the captured sequence covers the whole genome, and has good sensitivity and specificity. The primers and method have good stability and have good application prospects.

[0070] Finally, it should be noted that 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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, characterized in that: The primer set consists of primer pair 1 to primer pair 22, and the primer set is used for whole genome sequencing of norovirus GII.3[P12]; primer pair 1 consists of the forward amplification primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2; primer pair n consists of the forward amplification primer shown in SEQ ID NO. (2n-1) and the reverse primer shown in SEQ ID NO.2n, and n is selected from an integer between 2-22.

2. The primer set according to claim 1, characterized in that The primer sets 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, 20 and 22 are in primer pool 2.

3. Use of the primer set described in any one of claims 1-2 in preparing a product for whole genome sequencing of norovirus GII.3[P12].

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

5. A kit, characterized in that The kit comprises the primer set according to any one of claims 1-2, or the kit comprises a buffer containing the primer set according to any one of claims 1-2.

6. The kit according to claim 5, characterized in that The kit also includes reverse transcriptase, PCR reaction premix, and sequencing adapter.

7. A method for whole genome sequencing of norovirus GII.3 [P12] 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 described in any one of claims 1 to 2, 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, characterized in that: After the PCR product was purified, the library was constructed using the Nextera® XT Library Prep Kit and sequenced using Miniseq. The sequence with Genbank accession number OP901693 was used as the reference sequence and spliced ​​using CLC Genomics Workbench 23.0 software.

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