Universal avian infectious bronchitis virus whole genome deep sequencing method and application

Abstract

The invention provides a general type avian infectious bronchitis virus whole genome deep sequencing method and application, and the method realizes efficient amplification of whole genome long fragments of IBV epidemic strains with different genotypes by designing and applying a set of combination containing 24 pairs of specific primers. The specific sequences of the primer combination are shown as SEQ NO: 1 to SEQ NO: 48. Compared with a traditional sequencing method, the method has the advantage that the sequencing cost is remarkably reduced. According to the method, the IBV full-length genome sequence exceeding 27 kb can be successfully obtained only through two PCR reactions, a more economical and efficient solution is provided for IBV related scientific research and molecular epidemiological monitoring, and scientific research and development in related fields are promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal virus detection, and specifically relates to a universal avian infectious bronchitis virus whole genome deep sequencing method and application. Background Art

[0002] Infectious bronchitis virus (IBV) is a coronavirus that can cause illness in chickens of varying ages, leading to infectious bronchitis (IB). Affected birds display severe respiratory symptoms and urogenital damage, causing significant economic losses to the global poultry industry.

[0003] Similar to other coronaviruses, IBV mutates rapidly and is prone to recombination, leading to the continuous emergence of new genotypes or serotypes. These variants differ significantly in antigenicity from conventional vaccine strains, creating significant challenges for clinical immune control. Therefore, ongoing molecular epidemiological monitoring and mutation analysis of currently prevalent IBV strains in my country are of great guiding significance and reference value for clinical control and vaccine development.

[0004] IBV is a single-stranded, positive-sense RNA virus with a genome length of approximately 27.6 kb. It is composed of four major structural genes: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). The spike protein S is a key component for viral invasion of host cells and the production of neutralizing antibodies, and it is also a region of high mutation frequency. Therefore, IBV genotyping is primarily based on the S1 gene, which encodes the receptor-binding domain of the S protein. Analysis of nucleotide sequence differences within the S1 gene has led to the classification of currently circulating IBV strains into seven genotypes and 35 lineages (GI-1 to GI-29, GII to GVII). Since the GI-19 genotype (QX type) was first isolated in my country in 1998, its isolation rate has shown a significant annual increase and has replaced the GI-1 genotype (Mass type) as the predominant circulating genotype in my country. In addition, GI-7 (TW type) and GI-22 (YN type) strains are also frequently isolated in my country, and in recent years, the isolation rate of GVI-1 genotype strains in my country has also increased year by year, and the homology with the GI genotype strains is only 60-70%, which brings new challenges to the epidemiological monitoring and prevention and control of IBV.

[0005] At the same time, it's important to note that while the S1 gene is the primary antigenic gene of IBV and is closely associated with its pathogenicity and immunogenicity, it does not represent the entire genetic makeup of the virus. Nonstructural proteins beyond the S1 gene may also play a key role in IBV infection and pathogenesis. Furthermore, due to the frequent recombination events among IBV strains, phylogenetic trees constructed based on the whole genome and those constructed based on the S1 gene are not completely consistent. Therefore, S1 gene sequencing has limitations in revealing the overall genetic characteristics of the virus. Whole-genome sequencing is required to fully characterize IBV and understand its epidemiological characteristics, including its antigenicity, tissue tropism, and pathogenicity. Traditional whole-genome sequencing methods require determining the specific genotype based on S1 gene sequencing results. PCR amplification is then performed using primers for segmented sequencing covering the entire genome, using sequences from similar reference strains. First-generation sequencing methods, such as Sanger sequencing, are used to obtain segmented sequences, which are then assembled using molecular biology tools. While this sequencing approach can meet the needs of IBV genotyping to a certain extent, it has significant limitations in terms of time consumption, cost, technical complexity, and flexibility. With the continuous development of sequencing technology, more efficient, accurate, economical and flexible sequencing methods should be explored to meet the challenges of the increasingly complex IBV epidemic trends. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to economically and efficiently perform deep sequencing of the whole genome of infectious bronchitis virus (IBV) strains.

[0007] In order to solve the above problems, in a first aspect, the present invention provides a method for deep sequencing of infectious bronchitis virus, which may include the following steps: performing PCR on a template using an IBV-specific primer composition 1 and an IBV-specific primer composition 2, respectively, to obtain two PCR products, and performing deep sequencing on the two PCR products to obtain the genome sequence of the infectious bronchitis virus to be sequenced; the IBV-specific primer composition 1 is 24 single-stranded DNAs whose nucleotide sequences are SEQ ID NO: 1 to SEQ ID NO: 24 in the sequence listing, and the IBV-specific primer composition 2 is 24 single-stranded DNAs whose nucleotide sequences are SEQ ID NO: 25 to SEQ ID NO: 48 in the sequence listing; the template is cDNA obtained by reverse transcription of the total RNA of the infectious bronchitis virus to be sequenced, or is cDNA obtained by reverse transcription of the total RNA of the sample to be tested.

[0008] In a second aspect, the present invention provides a method for preparing infectious bronchitis virus sequencing fragments, which may include the following steps: performing PCR on the template using the IBV-specific primer composition 1 and the IBV-specific primer composition 2 described above, respectively, to obtain infectious bronchitis virus sequencing fragments; the template is cDNA obtained by reverse transcription of the total RNA of the infectious bronchitis virus to be sequenced, or is cDNA obtained by reverse transcription of the total RNA of the sample to be tested.

[0009] In the above method, in the IBV-specific primer composition 1, the amounts of the 24 single-stranded DNA substances are the same; in the IBV-specific primer composition 2, the amounts of the 24 single-stranded DNA substances are the same.

[0010] In the above method, the upstream primers whose nucleotide sequences are shown in SEQ ID NOs: 1 to 12 are paired with the downstream primers whose nucleotide sequences are shown in SEQ ID NOs: 13 to 24, respectively;

[0011] The upstream primers whose nucleotide sequences are shown in SEQ ID NOs: 25 to 36 are paired with the downstream primers whose nucleotide sequences are shown in SEQ ID NOs: 37 to 48, respectively.

[0012] In the above method, the primer annealing condition used in the PCR is annealing at 55° C. for 5 seconds.

[0013] In the above method, the reaction temperature program used in the PCR was: pre-denaturation at 98°C for 2 minutes; then 35 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and extension at 65°C for 5 minutes; and then extension at 68°C for 10 minutes.

[0014] In a third aspect, the present invention also provides the application of the above method in bioinformatics analysis.

[0015] The method may include performing deep sequencing on the sequencing fragments obtained by the above method to obtain sequencing data, filtering the sequencing data, splicing the IBV full-length genome sequence, constructing an evolutionary tree, identifying genotypes, analyzing gene recombination, and identifying mutation sites.

[0016] The sequencing was performed using the Illumina Miseq platform.

[0017] Preferably, the sequence filtering, splicing, and variant site identification software is Geneious Prime;

[0018] Preferably, the evolutionary tree construction software is Mega;

[0019] Preferably, the gene recombination analysis software is RDP4.

[0020] In a fourth aspect, the present invention also provides a primer composition for preparing infectious bronchitis virus sequencing fragments, wherein the primer composition consists of the IBV-specific primer composition 1 and the IBV-specific primer composition 2 described in claim 1 or 3.

[0021] The IBV-specific primer composition 1 and the IBV-specific primer composition 2 can be packaged independently and used separately in PCR.

[0022] In a fourth aspect, the present invention also provides a reagent for detecting infectious bronchitis virus, wherein the reagent contains the above-mentioned primer composition.

[0023] In a fifth aspect, the present invention also provides a kit for detecting infectious bronchitis virus, wherein the kit contains the above-mentioned primer composition or reagent.

[0024] The present invention also provides the use of the above reagent or kit in any of the following:

[0025] B1) Detection or auxiliary detection of infectious bronchitis virus,

[0026] B2) preparing products for detecting or assisting in the detection of infectious bronchitis virus,

[0027] B3) Detection or auxiliary detection of infectious bronchitis virus full genome nucleic acid,

[0028] B4) preparing products for detecting or assisting in the detection of the whole genome nucleic acid of infectious bronchitis virus,

[0029] B5) Whole genome deep sequencing analysis of infectious bronchitis virus,

[0030] B6) Prepare products for whole genome deep sequencing analysis of infectious bronchitis virus.

[0031] The reagents or kit may further include Tris-HCl, KCl, MgCl2, glycerol, Tween 20, dNTPs, KOD DNA polymerase, ddH2O, and the like.

[0032] The above-mentioned application or method may be a non-disease diagnosis application or method. The above-mentioned application or method may not be directly intended to obtain disease diagnosis results or health status of a living human or animal body. The sample to be tested may be a sample from a non-living human or animal body, such as an environmental sample (e.g., air), clothing, towels, or animal tissue and / or organs used as food. The sample to be tested may also be at least one of chicken embryo allantoic fluid, a pharyngeal swab, trachea, kidney, or bursa of Fabricius.

[0033] In a specific embodiment of the present invention, the method may include the following specific steps:

[0034] S1) collecting samples, extracting RNA, and performing multiplex PCR amplification on cDNA obtained by reverse transcription of the test samples using the above reagents or kits or the above composition to obtain PCR products;

[0035] S2) Detect the PCR product by agarose gel electrophoresis to see if a band is obtained;

[0036] S3) If there is no band in S2), then the sample to be tested is not or the candidate is not infectious bronchitis virus, or the sample to be tested does not contain or the candidate does not contain infectious bronchitis virus;

[0037] S4) If a band is present in S2), the resulting PCR product is subjected to Illumina library preparation and sequencing. Based on the sequencing results, the sample is identified or assisted in identifying whether it is infectious bronchitis virus. Bioinformatics methods can be further used to assemble the full-length IBV genome sequence, construct a phylogenetic tree, identify genotypes, analyze gene recombination, and identify variant sites.

[0038] Specifically, the molar concentration of the upstream primer and the downstream primer in the PCR reagent or kit or primer set 1 and primer set 2 in the PCR amplification reaction system in S1) is 10 μM.

[0039] In one embodiment of the present invention, taking the total reaction volume of 50 μL as an example, the reaction system includes: KODOne™ PCR Master Mix 25 μL, Primer Set 1 / Primer Set 2 2.4 μL, template cDNA 4 μL, ddH2O: 18.6 μL, and the total reaction volume is 50 μL.

[0040] Preferably, the sample includes at least one of chicken embryo allantoic fluid, pharyngeal swab, trachea, kidney, and bursa of Fabricius.

[0041] In the present invention, the infectious bronchitis virus to be sequenced may be avian infectious bronchitis virus.

[0042] In a specific embodiment of the present invention, the infectious bronchitis virus to be sequenced includes at least one of GI-1 (Mass type), GI-19 (QX type), GI-22 (YN type), GI-7 (TW type), GVI-1 genotype

[0043] Preferably, the amount of each primer in the above composition in the multiplex PCR reagent or kit is the same.

[0044] Furthermore, the above-mentioned multiplex PCR reagent or kit is used in the following A1)-A3):

[0045] A1) Detecting or assisting in the detection of avian infectious bronchitis virus in samples;

[0046] A2) preparing the complete genomic DNA of avian infectious bronchitis virus;

[0047] A3) Whole genome deep sequencing analysis of avian infectious bronchitis virus.

[0048] The beneficial effects of the present invention are:

[0049] The 24 designed primer pairs were used to perform segmented amplification on representative strains GD, SD, and YN of the GI-7, GI-19, and GI-22 genotypes. The results showed that the detection primer set can achieve 100% coverage of the entire genome of IBV of different genotypes, with good specificity, single amplified bands, and high nucleic acid concentration. It provides a good technical means for obtaining more uniform and effective IBV genome sequencing samples, and has achieved good results in Illumina Miseq high-throughput sequencing. The detection primer set is suitable for preparing products for detecting IBV and is widely used. Specifically, it can be a reagent or a kit. The present invention also protects the reagent or kit, specifically using the above-mentioned detection primer set as primers to amplify and prepare sequencing fragments of the entire IBV genome.

[0050] The present invention provides a universal whole-genome deep sequencing method for avian infectious bronchitis virus (IBV) strains and its application. This method achieves efficient amplification of long fragments of the whole genome of prevalent IBV strains of different genotypes by designing and applying a set of 24 pairs of specific primers. The specific sequences of this primer combination are shown in SEQ NO. 1 to SEQ NO. 48. The present invention also covers a multiplex polymerase chain reaction (PCR) amplification system, amplification procedures, high-throughput sequencing technology based on the Illumina Miseq platform, and subsequent bioinformatics analysis processes. The core advantage of this method is that it can ensure greater than 99% coverage of the full-length genome of IBV strains of different genotypes, thereby greatly facilitating molecular epidemiological monitoring of strains, analysis of recombination events, and identification of variant sites across the entire genome. Through this method, researchers can obtain more detailed and comprehensive genetic information of the strains, which is of great significance for a deeper understanding of the genetic variation patterns and epidemiological characteristics of IBV. In addition, compared with traditional sequencing methods, the present invention significantly reduces sequencing costs. Only two PCR reactions are needed to successfully obtain the full-length IBV genome sequence of more than 27kb, providing a more economical and efficient solution for IBV-related scientific research and molecular epidemiological monitoring, and helping to promote scientific research development in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Schematic diagram of the locations of the 24 pairs of specific primers designed in the present invention in the IBV genome.

[0052] Figure 2 The 24 pairs of specific primers designed in the present invention have segmented amplification effects on GI-7, GI-19, and GI-22 genotype IBV strains GD, SD, and YN.

[0053] Figure 3 This is the depth coverage of Illumina Miseq sequencing of the GI-1M41 strain implemented in the present invention.

[0054] Figure 4 This is the depth coverage of Illumina Miseq sequencing of the GI-7GD strain implemented in the present invention.

[0055] Figure 5 This is the depth coverage of Illumina Miseq sequencing of the GI-19SD strain implemented in the present invention.

[0056] Figure 6 This is the depth coverage of Illumina Miseq sequencing of the GI-22YN strain implemented in the present invention.

[0057] Figure 7 This is the Illumina Miseq sequencing depth coverage of the clinically isolated strain ck / CH / LN / TA / 2023M0204 implemented in the present invention.

[0058] Figure 8 This is the Illumina Miseq sequencing depth coverage of the clinically isolated strain ck / CH / LN / TA / 20230406 implemented in the present invention.

[0059] Figure 9 The whole genome evolutionary tree of different genotype IBV strains was constructed for the implementation of the present invention.

[0060] Figure 10 This is the result of recombination analysis of the clinically isolated IBV strain ck / CH / LN / TA / 20230406 implemented in the present invention. DETAILED DESCRIPTION

[0061] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0062] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0063] The avian infectious bronchitis virus strains are representative strains of different genotypes isolated and preserved in this laboratory from clinical samples. The sequence numbers of their full genomes or S1 gene reference sequences in the GenBank database of the National Center for Biotechnology Information (NCBI) are M41 (MK937830), GD (OQ117368), SD (KY421673), YN (JF893452), ck / CH / LN / TA / 2023M0204 (OR467930), and ck / CH / LN / TA / 20230406 (OR467920).

[0064] Example 1: Establishment of a universal method for deep whole-genome sequencing of infectious bronchitis (IBV) virus strains

[0065] Based on the genome sequences of the reference strains M41 (MK937830), GD (OQ117368), SD (KY421673), YN (JF893452), ck / CH / LN / TA / 2023M0204 (OR467930), and ck / CH / LN / TA / 20230406 (OR467920), molecular biology software such as Geneious Prime was used for whole-genome multiple sequence alignment analysis, screening for conserved regions, and designing and validating multiplex PCR primers covering the entire genome. Specific primer sequence information is as follows:

[0066] Table 1. Detection primer set sequences involved in the present invention (5'-3')

[0067]

[0068]

[0069] Four IBV representative strains of different genotypes (GI-1, GI-7, GI-19, and GI-22) were used.

[0070] Among them, the IBV strain of the GI-1 genotype is the Infectious bronchitis virus strain M41 (GenBank: MK937830.1, 18-JUN-2019), referred to as the GI-1M41 strain (M41).

[0071] The IBV strain of the GI-7 genotype is the infectious bronchitis virus strain GD (GenBank: OQ117368.1, 28-JAN-2023), referred to as the GI-7GD strain (GD).

[0072] The IBV strain of the GI-19 genotype is the infectious bronchitis virus strain SD (GenBank: KY421673.1, 16-OCT-2017), referred to as the GI-19SD strain (SD).

[0073] The IBV strain of the GI-22 genotype is the infectious bronchitis virus strain YN (GenBank: JF893452.2, 11-OCT-2018), referred to as the GI-22YN strain (YN).

[0074] The public can obtain the above-mentioned representative IBV strains from China Agricultural University. The biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0075] The method provided by the present invention is used to perform multiplex PCR amplification and high-throughput deep sequencing. The specific process is as follows:

[0076] 1) Preparation of viral nucleic acid samples: Inoculate 10-day-old SPF chicken embryos with IBV at a dose of 0.2 mL per embryo. Incubate in a 37°C incubator for 40 h. Harvest allantoic fluid. Aspirate 200 μL of allantoic fluid and extract total RNA using Trizol or an adsorption column. Transfer 2 μL of RNA to a 0.2 mL PCR tube, add 4 μL of 5× RT Mix and 14 μL of ddH₂O, gently flick to mix, and centrifuge briefly to the bottom, avoiding bubbles. Reverse transcription is performed in a PCR instrument (37°C for 15 min, 85°C for 5 s). The resulting cDNA is stored at 4°C.

[0077] 2) Multiplex PCR amplification: Construct the first primer set (primer set 1) and the second primer set (primer set 2) according to the primers in Table 1. Take 1 μl of each of the 24 amplification primers included in primer set 1 and mix them in a PCR tube (tube 1), vortex mix, and centrifuge for use; take 1 μl of each of the 24 amplification primers included in primer set 2 and mix them in another PCR tube (tube 2), vortex mix, and centrifuge for use. According to tubes 1 and 2, two multiplex PCR reactions were performed, and the reaction system was: KOD One TMPCR Master Mix (25 μl), Primer Set 1 or Primer Set 2 (2.4 μl), Template cDNA (4 μl), ddH₂O (18.6 μl), Total reaction volume: 50 μL. Primer Set 1 in Tube 1 consists of 24 single-stranded DNAs (24 amplification primers) with nucleotide sequences of SEQ ID NOs: 1 to 24 (Sequence Listing). The concentration of each of these 24 single-stranded DNAs in Tube 1 is 0.01 μM. Primer Set 2 in Tube 2 consists of 24 single-stranded DNAs with nucleotide sequences of SEQ ID NOs: 25 to 48 (Sequence Listing). The concentration of each of these 24 single-stranded DNAs in Tube 2 is 0.01 μM.

[0078] 3) The multiplex PCR amplification reaction conditions for both tubes 1 and 2 were: initial denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension at 65°C for 5 min; and a final extension at 68°C for 10 min. The denaturation, annealing, and extension steps were repeated 35 times. PCR products were stored at 4°C.

[0079] 3) NGS sequencing: Combine the two PCR reaction products into a 1.5ml EP tube and use magnetic bead purification to remove non-amplified fragments and improve the purity of the amplified PCR product. Use PCR amplification primers with sequencing adapters to exponentially amplify the DNA library to a concentration that meets sequencing requirements. Use a two-step magnetic bead purification method to separate and purify the amplified DNA library. Once the purified DNA library passes quality inspection and quantification, it can be sequenced on an Illumina Miseq sequencing platform to generate sequencing data.

[0080] 4) Sequencing data analysis: Filter and analyze the sequencing data using bioinformatics analysis software. The specific steps include:

[0081] Raw data quality inspection: testing the quality of high-throughput sequencing data;

[0082] Data filtering: filter low-quality sequencing data and process adapter sequence data;

[0083] Alignment: Align the sequence to the IBV reference genome;

[0084] Capture analysis: Analyze data uniformity, specificity, coverage, sequencing depth, etc.;

[0085] Genetic evolution analysis: Molecular biology software was used to assemble the full-length IBV genome sequence, construct an evolutionary tree, identify genotypes, analyze gene recombination, and identify mutation sites.

[0086] Quality control standards: raw data Q30 (the percentage of bases with a Phred value greater than 30 in the total bases, where Phred = -10log10(e), e is the error rate) > 90%, QC rate (number of valid reads obtained after filtering / number of raw sequencing data reads) > 99%, specificity (number of reads aligned to the reference genome / number of valid sequencing data reads) > 90%, coverage (the percentage of sites in the reference genome covered by at least one base in the genome) > 99%, and average sequencing depth (total number of bases aligned to the reference genome / genome size) > 300×.

[0087] The results showed that the method was effective in whole-genome sequence amplification and capture analysis of four IBV strains with different genotypes (GI-1, GI-7, GI-19, and GI-22). The 30× coverage of the initial data from Illumina Miseq deep sequencing was greater than 90.0%, the QC value was greater than 99.0%, the proportion of sequences that could be aligned to the IBV reference genome was greater than 90%, the average sequencing depth was greater than 300×, and the coverage was greater than 99.0% (see Table 2 and Table 3 for specific amplicon capture analysis results). Figure 3-6 ). This result shows that the standard of this technical invention can meet the needs of subsequent IBV full-length genome sequence splicing, evolutionary tree construction, genotype identification, gene recombination analysis and mutation site identification.

[0088] Table 2. Amplicon capture analysis results of 4 different genotypes of IBV reference strains

[0089]

[0090] Example 2. Construction of the whole genome phylogenetic tree and gene recombination analysis of clinical isolates ck / CH / LN / TA / 20230406 and ck / CH / LN / TA / 2023M0204

[0091] This example uses two clinically isolated IBV strains of unknown genotype, performs multiplex PCR amplification and high-throughput deep sequencing using the methods provided by the present invention, and then performs subsequent bioinformatics analysis steps such as whole-genome evolutionary tree construction and gene recombination analysis. The specific process is as follows:

[0092] 1) The nucleic acid of the clinical isolate to be tested was extracted in the same manner as in Example 1, and whole-genome multiplex PCR amplification was performed to obtain sequencing results.

[0093] 2) The sequencing data were filtered and analyzed using bioinformatics analysis software. The specific results are shown in Table 3 and Figure 7 、 Figure 8 shown.

[0094] Table 3. Results of amplicon capture analysis of 2 IBV clinical isolates

[0095]

[0096] The IBV strain of ck / CH / LN / TA / 2023M0204 genotype is Infectious bronchitis virus strain ck / CH / LN / TA / 2023M0204 (GenBank: OR467930.1, 10-OCT-2023).

[0097] The IBV strain of ck / CH / LN / TA / 20230406 genotype is Infectious bronchitis virus strain ck / CH / LN / TA / 20230406 (GenBank: OR467920.1, 10-OCT-2023).

[0098] 3) Construction of whole genome and S gene phylogenetic tree

[0099] Fifty-seven reference sequences of IBV strains of different genotypes were downloaded from NCBI for phylogenetic tree analysis. The full genome and S gene phylogenetic trees of the ck / CH / LN / TA / 2023M0204 and ck / CH / LN / TA / 20230406 strains were constructed using the maximum likelihood (ML) method of MEGA7.0 software and the Kimura 2-parameter nucleotide substitution model. The results were validated by 1000 bootstrap replicates. Figure 9 As shown in the results, ck / CH / LN / TA / 2023M0204 has the same position in the whole-genome phylogenetic tree (A) and the S gene phylogenetic tree (B), both belonging to the GVI-1 branch. However, the genotype of ck / CH / LN / TA / 20230406 in the whole-genome phylogenetic tree and the S gene phylogenetic tree differs, belonging to the GI-19 genotype and the GI-1 genotype, respectively, suggesting the possibility of genetic recombination in this strain.

[0100] 4) Recombination analysis of ck / CH / LN / TA / 20230406

[0101] The putative recombination event and parental strain of ck / CH / LN / TA / 20230406 were identified using RDP 4.0 recombination analysis software. The data were analyzed using multiple methods and program default settings, including RDP, Bootscan, GeneConv, Maxch, Chimaera, SIScan, and Phylpro. Figure 10As shown, ck / CH / LN / TA / 20230406 is a recombinant strain of the GI-19 genotype strain I0916 / 16 and the GI-1 genotype strain Mass 41. The recombination event occurred between nucleotide position 20228 and amino acid position 25276, and the S gene is located exactly in this position. Therefore, there is a difference between the S gene phylogenetic evolutionary tree and the whole genome phylogenetic evolutionary tree, which further illustrates the importance of using this patented method for whole genome sequencing.

[0102] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for deep sequencing of infectious bronchitis virus, characterized in that: The method comprises the following steps: performing PCR on a template using an IBV-specific primer composition 1 and an IBV-specific primer composition 2, respectively, to obtain two PCR products, and performing deep sequencing on the two PCR products to obtain a genome sequence of the infectious bronchitis virus to be sequenced; the IBV-specific primer composition 1 is 24 single-stranded DNAs whose nucleotide sequences are SEQ ID NO: 1 to SEQ ID NO: 24 in the sequence listing, and the IBV-specific primer composition 2 is 24 single-stranded DNAs whose nucleotide sequences are SEQ ID NO: 25 to SEQ ID NO: 48 in the sequence listing; and the template is cDNA obtained by reverse transcription of the total RNA of the infectious bronchitis virus to be sequenced, or is cDNA obtained by reverse transcription of the total RNA of a sample to be tested.

2. A method for preparing infectious bronchitis virus sequencing fragments, characterized in that: The method comprises the following steps: performing PCR on a template using the IBV-specific primer composition 1 and the IBV-specific primer composition 2 described in claim 1, respectively, to obtain an infectious bronchitis virus sequencing fragment; the template is a cDNA obtained by reverse transcription of the total RNA of the infectious bronchitis virus to be sequenced, or a cDNA obtained by reverse transcription of the total RNA of the sample to be tested.

3. The method according to claim 1 or 2, characterized in that In the IBV-specific primer composition 1, the amounts of the 24 single-stranded DNA substances are the same; in the IBV-specific primer composition 2, the amounts of the 24 single-stranded DNA substances are the same.

4. The method according to any one of claims 1 to 3, characterized in that The primer annealing condition used in the PCR was annealing at 55° C. for 5 seconds.

5. The method according to any one of claims 1 to 4, characterized in that: The reaction temperature program used in the PCR was: pre-denaturation at 98°C for 2 min; then 35 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 65°C for 5 min; and extension at 68°C for 10 min.

6. Use of the method according to any one of claims 1 to 5 in bioinformatics analysis.

7. A primer composition for preparing infectious bronchitis virus sequencing fragments, characterized in that: The primer composition consists of the IBV-specific primer composition 1 and the IBV-specific primer composition 2 described in claim 1 or 3.

8. A reagent for detecting infectious bronchitis virus, characterized in that: The reagent contains the primer composition according to claim 7.

9. A kit for detecting infectious bronchitis virus, characterized in that: The kit contains the primer composition according to claim 7 or the reagent according to claim 8.

10. Use of the reagent according to claim 8 or the kit according to claim 9 in any of the following: B1) Detection or auxiliary detection of infectious bronchitis virus, B2) preparing products for detecting or assisting in the detection of infectious bronchitis virus, B3) Detection or auxiliary detection of infectious bronchitis virus full genome nucleic acid, B4) Preparation of products for detecting or assisting in the detection of the whole genome nucleic acid of infectious bronchitis virus, B5) Whole genome deep sequencing analysis of infectious bronchitis virus, B6) Preparation of products for whole genome deep sequencing analysis of infectious bronchitis virus.

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