Universal method for deep sequencing of the whole genome of avian infectious bronchitis virus and application thereof

Abstract

The application provides a general avian infectious bronchitis virus whole genome deep sequencing method and application. The method realizes efficient amplification of long fragments of whole genomes of different genotype IBV epidemic strains by designing and applying a set of combinations containing 24 pairs of specific primers. The specific sequences of the primer combinations are shown in SEQ NO:1 to SEQ NO:48. Compared with a traditional sequencing method, the application significantly reduces the sequencing cost. Only two PCR reactions are needed, and more than 27 kb of IBV full-length genome sequence can be successfully obtained, which provides a more economical and efficient solution for IBV related scientific research and molecular epidemiology monitoring, and helps to promote the development of related field scientific research.

Description

TECHNICAL FIELD

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

[0002] Infectious bronchitis virus (IBV) is a coronavirus that can cause disease in chickens of different ages, leading to the occurrence of infectious bronchitis (IB). Sick chickens show severe respiratory symptoms and urogenital system damage, causing serious 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 serovar variant strains, and there is a large difference in antigenicity between the conventional vaccine strains, which has caused great difficulties in clinical immunization and prevention and control. Therefore, the current molecular epidemiological monitoring and variation analysis of the IBV strains prevailing in China have important guiding significance and reference value for the clinical prevention and control and vaccine development of IBV.

[0004] IBV is a single-stranded RNA virus, and its genome is about 27.6 kb in length, which is composed of four main structural genes: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). Spike protein S is a key component for virus invasion of host cells and the production of neutralizing antibodies, and is also a high-frequency region of gene mutation. Therefore, the genotyping of IBV is mainly based on the S1 gene encoding the receptor binding domain of S protein. By analyzing the nucleotide sequence difference of S1 gene, the currently prevalent IBV strains can be divided into seven genotypes and 35 lineages (GI-1 to GI-29, GII to GVII).

[0005] Meanwhile, it should be noted that although the S1 gene is the main antigenic gene of IBV, which is closely related to the pathogenicity and immunogenicity of the virus, it does not represent the entire genetic information of the virus, and other non-structural proteins outside the S1 gene can also play a key role in the infection and pathogenesis of IBV. In addition, due to the frequent recombination events of IBV strains, the phylogenetic tree constructed based on the whole genome is not completely consistent with the phylogenetic tree constructed based on the S1 gene. Therefore, S1 gene sequencing has limitations in revealing the overall genetic characteristics of the virus, and whole genome sequencing is needed to fully characterize IBV and understand its epidemiological characteristics, including its antigenicity, tissue tropism and pathogenicity. The traditional whole genome sequencing method needs to determine the specific genotype according to the S1 gene sequencing result, and design segmented sequencing primers covering the whole genome based on the sequence of the reference strain similar to it for PCR amplification, use first-generation sequencing methods such as Sanger sequencing to obtain segmented sequences, and use molecular biology tools for splicing. Although this sequencing method can meet the needs of IBV genotype detection to some extent, it has obvious shortcomings 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 cope with the challenges of the increasingly complex IBV epidemic trend. SUMMARY

[0006] The technical problem to be solved by the present application is how to economically and efficiently sequence the whole genome of the infectious bronchitis virus (IBV) strain,

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

[0008] In a second aspect, the present application provides a method for preparing a sequencing fragment of infectious bronchitis virus, which can comprise the following steps: performing PCR on a template using the IBV-specific primer composition 1 and the IBV-specific primer composition 2 respectively to obtain a sequencing fragment of infectious bronchitis virus; wherein the template is cDNA reverse transcribed from total RNA of the infectious bronchitis virus to be sequenced, or cDNA reverse transcribed from total RNA of the sample to be tested.

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

[0010] In the above method, the upstream primers with the nucleotide sequences as shown in SEQ ID NO: 1-12 are paired with the downstream primers with the nucleotide sequences as shown in SEQ ID NO: 13-24 respectively.

[0011] The upstream primers with the nucleotide sequences as shown in SEQ ID NO: 25-36 are paired with the downstream primers with the nucleotide sequences as shown in SEQ ID NO: 37-48.

[0012] In the above method, the annealing condition of the primers used in the PCR is 55°C for 5s.

[0013] In the above method, the reaction temperature program of the PCR is as follows: pre-denaturation at 98°C for 2min; then 35 cycles of denaturation at 98°C for 10s, annealing at 55°C for 5s, and extension at 65°C for 5min; and then extension at 68°C for 10min.

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

[0015] The method can comprise the steps of performing deep sequencing on the sequencing fragment obtained by the above method to obtain sequencing data, filtering the sequencing data, splicing the full-length genome sequence of IBV, constructing a phylogenetic tree, identifying the genotype, analyzing gene recombination, and identifying variant sites.

[0016] The sequencing is performed by the Illumina Miseq platform.

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

[0018] Preferably, the phylogenetic tree construction software is Mega.

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

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

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

[0022] In a fourth aspect, the present application also provides a reagent for detecting infectious bronchitis virus, which contains the primer composition described above.

[0023] In a fifth aspect, the present application also provides a kit for detecting infectious bronchitis virus, which contains the primer composition or the reagent described above.

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

[0025] B1) detecting or assisting in detecting infectious bronchitis virus,

[0026] B2) preparing a product for detecting or assisting in detecting infectious bronchitis virus,

[0027] B3) detecting or assisting in detecting the whole genome nucleic acid of infectious bronchitis virus,

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

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

[0030] B6) preparing a product for whole genome deep sequencing of infectious bronchitis virus.

[0031] The reagent or the kit can further comprise Tris-HCl, KCl, MgCl2, glycerol, Tween 20, dNTPs, KOD DNA polymerase, ddH2O, etc.

[0032] The above-mentioned use or method can be a use or method that is not for disease diagnosis. The above-mentioned use or method can not be directly aimed at obtaining a disease diagnosis result or health condition of a living human or animal body. The sample to be detected can be a sample from a non-living human or animal body, such as an environmental sample (e.g. air), clothes, towels, or animal tissues and / or organs as food. The sample to be detected can also be at least one of chicken embryo allantoic fluid, throat swab, trachea, kidney, and bursa of Fabricius.

[0033] In a specific embodiment of the present application, the method can comprise the following specific steps:

[0034] S1) collecting samples, extracting RNA, reverse transcribing the cDNA of the samples to be tested using the reagent or kit or composition described above, and performing multiplex PCR amplification to obtain PCR products;

[0035] S2) agarose gel electrophoresis is performed to detect the PCR products to see if a band is obtained;

[0036] S3) if no band is obtained in S2), the sample to be tested is not or is not likely to be infectious bronchitis virus, or the sample to be tested does not contain or is not likely to contain infectious bronchitis virus;

[0037] S4) if a band is obtained in S2), Illumina library preparation is performed on the obtained PCR products, sequencing is performed, and the sequencing results are used to identify or assist in identifying whether the sample is infectious bronchitis virus, and further bioinformatics methods can be used to splice the full-length genome sequence of IBV, construct an evolutionary tree, identify the genotype, analyze gene recombination, and identify variation sites.

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

[0039] In an embodiment of the present application, taking a total reaction volume of 50 μL as an example, the reaction system comprises: KODOneTM PCR Master Mix 25 μl, primer group 1 / primer group 2 2.4 μl, template cDNA 4 μl, ddH2O: 18.6 μl, and the total reaction volume is 50 μL.

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

[0041] In the present application, the infectious bronchitis virus to be sequenced can be avian infectious bronchitis virus.

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

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

[0044] Further, the multiplex PCR reagent or kit described above is used in A1)-A3) as follows:

[0045] A1) detecting or assisting in detecting avian infectious bronchitis virus in a sample;

[0046] A2) preparing full genome DNA of avian infectious bronchitis virus;

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

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

[0049] The designed 24 pairs of primers are applied to segment amplification of the representative strains GD, SD and YN of GI-7, GI-19 and GI-22 genotypes, and the results show that the detection primer set can achieve 100% coverage of the full genome of different genotypes of IBV, and has good specificity, single amplification band and high nucleic acid concentration, which provides a good technical means for obtaining more uniform and effective IBV genome sequencing samples, and good results are achieved in Illumina Miseq high-throughput sequencing. The detection primer set is suitable for preparing a product for detecting IBV and is widely used. Specifically, it can be a reagent or kit. The present application simultaneously protects the reagent or kit, specifically using the above detection primer set as primers to amplify and prepare sequencing fragments of the full genome of IBV.

[0050] The present application provides a universal full genome deep sequencing method for avian infectious bronchitis virus strains and its application. The method realizes efficient amplification of long fragments of the full genome of different genotypes of IBV epidemic strains by designing and applying a set of 24 pairs of specific primers. The specific sequences of the primer set are shown in SEQ NO. 1 to SEQ NO. 48. The present application also covers multiplex polymerase chain reaction (PCR) amplification system, amplification program, high-throughput sequencing technology based on Illumina Miseq platform and subsequent bioinformatics analysis process. The core advantage of the method is to ensure that the full-length genome of different genotypes of IBV strains is sequenced with >99% coverage, thereby greatly promoting the molecular epidemiological monitoring of the strains, the analysis of recombination events and the identification of variation sites in the full genome. Through the method, researchers can obtain more detailed and comprehensive genetic information of the strains, which is of great significance for in-depth understanding of the genetic variation rules and epidemiological characteristics of IBV. In addition, compared with traditional sequencing methods, the present application significantly reduces the sequencing cost. Only two PCR reactions are needed to successfully obtain more than 27 kb of IBV full-length genome sequence, which provides a more economical and efficient solution for IBV-related scientific research and molecular epidemiological monitoring, and helps to promote the development of related field research. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1Schematic diagram of the positions of 24 pairs of specific primers designed in the application in the IBV genome.

[0052] Figure 2 Segment amplification effect of 24 pairs of specific primers designed in the application on IBV strains GD, SD and YN of genotypes GI-7, GI-19 and GI-22.

[0053] Figure 3 Illumina Miseq sequencing depth coverage of GI-1 M41 strain in the application.

[0054] Figure 4 Illumina Miseq sequencing depth coverage of GI-7 GD strain in the application.

[0055] Figure 5 Illumina Miseq sequencing depth coverage of GI-19 SD strain in the application.

[0056] Figure 6 Illumina Miseq sequencing depth coverage of GI-22 YN strain in the application.

[0057] Figure 7 Illumina Miseq sequencing depth coverage of clinical isolated strain ck / CH / LN / TA / 2023M0204 in the application.

[0058] Figure 8 Illumina Miseq sequencing depth coverage of clinical isolated strain ck / CH / LN / TA / 20230406 in the application.

[0059] Figure 9 Construction of phylogenetic tree of whole genome of IBV strains of different genotypes in the application.

[0060] Figure 10 Recombination analysis result of IBV clinical isolated strain ck / CH / LN / TA / 20230406 in the application. DETAILED DESCRIPTION

[0061] The application will be further described in detail below with specific embodiments, and the embodiments given are only for illustrating the application, but not for limiting the scope of the application. The embodiments provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application in any way.

[0062] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

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

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

[0065] According to the genomic 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), whole-genome multiple sequence alignment analysis was performed using molecular biology software such as Geneious Prime, and the conserved regions were screened to design and verify multiple PCR primers covering the whole genome. The specific primer sequence information is as follows:

[0066] Table 1. Sequences of detection primer sets (5'-3') involved in the present application

[0067]

[0068]

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

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

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

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

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

[0074] The above-mentioned representative strains of IBV can be obtained from China Agricultural University, and the biological material is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0075] The method provided by the present application is used for multiplex PCR amplification and high-throughput deep sequencing, and the specific process is as follows:

[0076] 1) Preparation of viral nucleic acid sample: inoculate 10-day-old SPF chicken embryos with IBV strains, each embryo is inoculated with 0.2 mL of inoculum, and is placed in a 37℃ incubator for 40h, and the allantoic fluid of the chicken embryo is harvested; 200ul of allantoic fluid is aspirated, and total RNA is extracted by Trizol method or adsorption column method; 2ul of RNA is taken in a 0.2ml PCR tube, 4ul of RT Mix enzyme (5x) is added, 14ul of ddH2O is added, and it is mixed by flicking, and the mixture is centrifuged to the bottom to avoid air bubbles. According to the reverse transcription program (37℃ for 15min, 85℃ for 5s) in the PCR instrument, the obtained cDNA is stored at 4℃.

[0077] 2) Multiplex PCR amplification: according to the primers in Table 1, the first primer group (primer group 1) and the second primer group (primer group 2) are constructed, the 24 amplification primers in primer group 1 are mixed by taking 1ul in a PCR tube (tube 1), vortex mixing, and centrifuging for standby; the 24 amplification primers in primer group 2 are mixed by taking 1ul in another PCR tube (tube 2), vortex mixing, and centrifuging for standby. According to tube 1 and tube 2, two multiplex PCR reactions are divided, and the reaction system is: KOD One TMPCR Master Mix 25μl, primer set 1 or primer set 2 2.4μl, template cDNA 4μl, ddH2O: 18.6μl, total reaction volume 50μL. Primer set 1 in tube 1 consists of 24 single-stranded DNAs (24 amplification primers) with nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 24 in the sequence listing, respectively, and the content of the 24 single-stranded DNAs of SEQ ID NO: 1 to SEQ ID NO: 24 in tube 1 is 0.01μM; primer set 2 in tube 2 consists of 24 single-stranded DNAs with nucleotide sequences of SEQ ID NO: 25 to SEQ ID NO: 48, respectively, and the content of the 24 single-stranded DNAs (24 amplification primers) of SEQ ID NO: 24 to SEQ ID NO: 48 in tube 2 is 0.01μM.

[0078] 3) The multiplex PCR amplification reaction conditions of tube 1 and tube 2 are both: 98℃ pre-denaturation for 2min; 98℃ denaturation for 10s, 55℃ annealing for 5s, 65℃ extension for 5min; 68℃ final extension for 10min; wherein the denaturation, annealing and extension steps are performed for 35 cycles. The PCR products are stored at 4℃.

[0079] 3) NGS sequencing: combine the PCR reaction products of the two tubes into a 1.5ml EP tube, remove the non-amplified fragments by magnetic bead purification method to improve the purity of the amplified PCR products; amplify the DNA library by a PCR amplification primer with a sequencing adapter to make the library concentration meet the sequencing requirement; sort and purify the amplified DNA library by a two-step magnetic bead purification method; after the purified DNA library passes the quality inspection and is quantified, it can be sequenced (Illumina Miseq sequencing platform) to obtain sequencing data;

[0080] 4) Sequencing data analysis: filter and analyze the sequencing data by bioinformatics analysis software, the specific steps including:

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

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

[0083] Alignment: align the sequencing sequences to the IBV reference genome;

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

[0085] Genetic evolution analysis: use molecular biology software to perform IBV full-length genome sequence assembly, phylogenetic tree construction, genotype identification, gene recombination analysis and variation site identification.

[0086] Quality control standards: raw data Q30 (percentage of bases with Phred values greater than 30 in 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 (percentage of sites in the reference genome covered by at least one base) > 99%, average sequencing depth (total number of bases aligned to the reference genome / genome size) > 300x.

[0087] The results show that the whole genome sequence amplification and capture analysis of 4 different genotypes (GI-1, GI-7, GI-19, GI-22) IBV strains using the method is good, the 30x coverage of the initial data of Illumina Miseq deep sequencing is greater than 90.0%, the QC value is greater than 99.0%, the sequence that can be aligned to the IBV reference genome accounts for more than 90%, the average sequencing depth is greater than 300x, and the coverage is greater than 99.0% (see Table 2 and Figures 3-6 for specific amplicon capture analysis results). The results show that the standard of the present application can meet the needs of subsequent IBV full-length genome sequence assembly, phylogenetic tree construction, genotype identification, gene recombination analysis and variation site identification.

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

[0089]

[0090] Example 2, Phylogenetic tree construction and gene recombination analysis of clinical isolates ck / CH / LN / TA / 20230406 and ck / CH / LN / TA / 2023M0204 full genome

[0091] In this embodiment, two IBV clinical isolates of unknown genotype are subjected to multiplex PCR amplification and high-throughput deep sequencing using the method provided by the present application, and subsequent bioinformatics analysis steps such as full genome phylogenetic tree construction and gene recombination analysis. The specific process is as follows:

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

[0093] 2) The sequencing data is filtered and analyzed by bioinformatics analysis software, and the specific results are shown in Table 3 and Figure 7 , Figure 8

[0094] ​Table 3.2 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 phylogenetic trees of whole genome and S gene

[0099] 57 reference sequences of different genotypes of IBV strains were downloaded from NCBI for phylogenetic tree analysis. The phylogenetic trees of whole genome and S gene of ck / CH / LN / TA / 2023M0204 and ck / CH / LN / TA / 20230406 strains were constructed using the maximum likelihood method (ML) of MEGA7.0 software, using Kimura 2 parameter nucleotide substitution model. The results were verified by 1000 times bootstrap. The results are shown in Figure 9 From the results, it can be seen that ck / CH / LN / TA / 2023M0204 is consistent in the position of whole genome phylogenetic tree (A) and S gene phylogenetic tree (B), and belongs to GVI-1 branch. While ck / CH / LN / TA / 20230406 has different genotypes in the whole genome phylogenetic tree and S gene phylogenetic tree, respectively belonging to GI-19 genotype and GI-1 genotype, suggesting that the strain has the possibility of genetic recombination.

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

[0101] The putative recombination events and parent strains 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. The results are shown in Figure 10As shown, ck / CH / LN / TA / 20230406 is a recombinant strain of GI-19 genotype strain I0916 / 16 and GI-1 genotype strain Mass 41, the recombination event occurs between the 20228th nucleotide and the 25276th amino acid, and the S gene is just located in the position, so there is a difference between the phylogenetic tree of the S gene and the phylogenetic tree of the whole genome, which further illustrates the importance of using the method of the patent for whole genome sequencing.

[0102] The application has been described in detail above. For those skilled in the art, the application can be implemented within a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the present application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application.

Claims

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

2. A method for preparing sequencing fragments of infectious bronchitis virus, characterized in that, The method includes the following steps: performing PCR on the template using IBV-specific primer composition 1 and IBV-specific primer composition 2 as described in claim 1, respectively, to obtain the sequencing fragment of infectious bronchitis virus; the template is cDNA obtained by reverse transcription of the total RNA of the infectious bronchitis virus to be sequenced, or 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 DNAs are the same; in the IBV-specific primer composition 2, the amounts of the 24 single-stranded DNAs are the same.

4. The method according to claim 1 or 2, characterized in that, The primer annealing conditions used in the PCR were 55°C for 5 seconds.

5. The method according to claim 1 or 2, characterized in that, The PCR reaction temperature program was as follows: first, pre-denaturation at 98℃ for 2 min; then, 35 cycles were performed as follows: denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 65℃ for 5 min; and then extension at 68℃ for 10 min.

6. The application of the method described in any one of claims 1-5 for non-disease diagnosis and treatment purposes in bioinformatics analysis.

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

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

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

10. The use of the reagent of claim 8 or the kit of claim 9 for any of the following non-disease diagnostic and therapeutic purposes: B1) Detection or auxiliary detection of infectious bronchitis virus, B2) Prepare products for the detection or auxiliary detection of infectious bronchitis virus. B3) Detection or auxiliary detection of the whole genome nucleic acid of infectious bronchitis virus. B4) Prepare 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) Prepare products for whole-genome deep sequencing analysis of infectious bronchitis viruses.

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