A probe set, a kit for detecting a porcine reproductive and respiratory syndrome virus (PRRSV) and a preparation method and application thereof

Abstract

The application relates to the field of gene detection, in particular to a probe set for detecting a porcine reproductive and respiratory syndrome virus (PRRSV), a kit, a preparation method and application, nucleotide sequences of the probe set are shown as SEQ ID NO. 1-53; the probe set is designed by analyzing target gene library sequences composed of PRRSV American strains and European strains, and is capable of completely covering the whole genome of the PRRSV American strains and European strains; the probe set can specifically capture PRRSV sequences in a sample to be detected, so that the PRRSV is enriched, high-sensitivity detection of the PRRSV on the basis of culture-free, genome-level variation monitoring and typing are realized.

Description

Technical Field

[0001] This application relates to the field of gene detection, and in particular to a probe set, kit, preparation method, and application for detecting the porcine reproductive and respiratory syndrome virus (PRRSV). Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV), also known as blue ear disease. It is characterized by reproductive disorders in pregnant sows (e.g., abortion, stillbirth, mummified fetuses) and respiratory diseases in pigs of all ages, especially piglets. PRRSV is a single-stranded positive-sense RNA virus. Because RNA is prone to point mutations, deletions, insertions, and substitutions, PRRSV has one of the highest mutation rates among known RNA viruses. PRRSV includes two types: the American type, represented by ATCC VR-2332, and the European type, represented by Lelystad virus. Significant antigenic differences exist between European and American isolates, resulting in very little cross-reactivity. Extensive genomic variation exists among American isolates, while European isolates show greater conservation.

[0003] Current methods for detecting the presence of PRRSV in samples are primarily based on qPCR. However, qPCR only detects one gene locus, which has limitations such as false negatives due to viral mutations or degradation in primer regions and the inability to obtain sequence variation information. Existing techniques for detecting sequence variations are based on PCR combined with first-generation sequencing to detect variations in one or two gene regions, which is inefficient and has a limited monitoring range. Therefore, to obtain more comprehensive sequence variations, a typical approach is to divide the entire genome into multiple segments, such as 14 or 15 segments, use overlapping primers for PCR amplification and first-generation sequencing of the products, and then assemble the genome based on the sequencing results. However, this method relies on viral isolation and culture, which is difficult; even if cultured successfully, due to the high variability of the virus, it is difficult to amplify all fragments using existing primers, resulting in low sensitivity, low efficiency, and time-consuming limitations. Existing culture-free methods mostly use metatranscriptomics, which includes a large amount of host data in the sequencing data, leading to significant data waste, high costs, and difficulty in detecting low-abundance viruses. Therefore, how to provide a culture-free, highly sensitive probe set for PRRSV detection and variant monitoring has become an urgent problem to be solved. Summary of the Invention

[0004] This application provides a probe set, kit, preparation method, and application for detecting porcine reproductive and respiratory syndrome virus (PRRSV), which solves the problem in the prior art of achieving high-sensitivity detection and variant monitoring of PRRSV without culture.

[0005] In a first aspect, this application provides a probe set for detecting the porcine reproductive and respiratory syndrome virus (PRRSV), the nucleotide sequence of which is shown in SEQ ID NO.1 to 53.

[0006] Optionally, the nucleotide sequence length of the probe set is 120bp±30bp.

[0007] Optionally, the probe set is designed based on the sequences of PRRSV American strain and PRRSV European strain.

[0008] Secondly, this application provides a kit for detecting the porcine reproductive and respiratory syndrome virus (PRRSV), the kit comprising the probe set described in the first aspect.

[0009] Optionally, the detection limit of the kit is ≥10. -4 The CT value of the sample or the sample identified by fluorescent PCR is ≤27.

[0010] Thirdly, this application provides a method for preparing the probe set described in the first aspect, the method comprising:

[0011] The genome sequences of representative strains of PRRSV American strains and PRRSV European strains from multiple public databases were selected as the target genome library;

[0012] The genome sequence of a representative PRRSV strain was selected as a reference sequence. All 120bp sequences in the reference sequence were obtained, and the sequences with GC content of 30% to 70% and the highest number of genomes compared with the target genome library were selected as the first candidate probe group.

[0013] The first candidate probe set is compared with the target sequence set in the target genome library to obtain a second candidate probe set with a regional base difference rate of no more than 10% from the target sequence set;

[0014] Using the standard that each probe captures a target gene length of ≥400bp, the minimum number of probes that can cover all the target sequence groups is selected from the second candidate probe group to form the probe group.

[0015] Optionally, the PRRSV representative strain is ATCC VR-2332.

[0016] Fourthly, this application provides an application of a probe set for detecting porcine reproductive and respiratory syndrome (PRRSV) pathogen, the application of which includes using the probe set described in the first aspect to detect PRRSV.

[0017] Optionally, the specific steps of the detection are as follows:

[0018] Nucleic acid was extracted from the sample to be tested, and a transcriptome library containing the linker oligonucleotide sequence of the sample tag was obtained.

[0019] The transcriptome library was mixed and PRRSV sequences were captured using the kit described in the second aspect, followed by elution and PCR amplification to obtain the probe capture library.

[0020] The probe capture library was subjected to high-throughput sequencing, and the high-throughput sequencing data was used for quality control and analysis to calculate the genome alignment rate.

[0021] Based on the genome alignment rate, the genome sequence and type of the PRRSV strain infecting the sample to be tested are determined.

[0022] Optionally, the extraction of nucleic acids from the sample to be tested, followed by obtaining a transcriptome library containing adapter oligonucleotide sequences of the sample tag, specifically includes:

[0023] Nucleic acid was extracted from the sample to be tested, and then RNA-seq was performed to obtain double-stranded cDNA;

[0024] The double-stranded cDNA was broken and ligated with a linker oligonucleotide sequence containing the sample tag. Then, PCR amplification was performed using amplification primers to obtain a transcriptome library containing the linker oligonucleotide sequence containing the sample tag.

[0025] The amplification primers are amplification primers for amplifying the oligonucleotide sequence of the adapter.

[0026] The technical solutions provided in this application have the following advantages compared with the prior art:

[0027] This application provides a probe set for detecting porcine reproductive and respiratory syndrome (PRRS). It utilizes a target genome library composed of multiple representative PRRSV strains from the Americas and Europe, with the genome sequence of a representative PRRSV strain serving as a reference. By comparing the variation information of the representative strains, the Americas strain, and the Europe strain, a probe set containing multiple probes is designed within conserved and variable regions. This probe set can completely cover the genomes of both the Americas and Europe strains of PRRSV. The designed probe set can capture PRRSV sequences in the sample to be tested, thereby enriching PRRSV. Detection of the enriched PRRSV allows for highly sensitive detection of low-abundance viruses without the need for culture. Attached Figure Description

[0028] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 A schematic flowchart illustrating the preparation method of the probe set provided in the embodiments of this application;

[0031] Figure 2 A schematic flowchart illustrating the probe group method for detecting PRRSV provided in this application embodiment;

[0032] Figure 3 This is a schematic diagram of the data analysis process for the probe group detection PRRSV method provided in the embodiments of this application. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0034] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0035] This application provides a probe set for detecting the porcine reproductive and respiratory syndrome (PRRSV) pathogen, the nucleotide sequences of which are shown in SEQ ID NO.1 to 53.

[0036] In some alternative embodiments, the nucleotide sequence length of the probe set is 120bp ± 30bp.

[0037] In this embodiment, limiting the length of the nucleic acid sequence of the probe group ensures that the probe can capture a sufficient number of PRRSV sequences in the test sample, thereby improving the PRRSV enrichment effect.

[0038] In some alternative implementations, the probe set is designed based on the sequences of PRRSV American strain and PRRSV European strain.

[0039] In this embodiment, the specific design source of the probe set is limited, so that the probe set can completely cover the genomes of American and European strains of PRRSV, thereby improving the enrichment effect of PRRSV.

[0040] Based on a general inventive concept, this application provides a kit for detecting porcine reproductive and respiratory syndrome (PRRSV) pathogen, the kit comprising the probe set.

[0041] This kit is based on the probe set described above. The specific composition and sequence information of the probe set can be found in the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0042] In some optional implementations, the detection limit of the kit is ≥10. -4 The CT value of the sample or the sample identified by fluorescent PCR is ≤27.

[0043] In this embodiment of the application, the detection limit of the kit is limited. Since the probe set can completely cover the genomes of PRRSV American strain and PRRSV European strain, and the probe set is designed based on the conserved and variant regions of PRRSV, the kit composed of the probe set can have a low detection limit.

[0044] Based on a general inventive concept, such as Figure 1 As shown, this application provides a method for preparing the probe set, the method comprising:

[0045] S1. Select the genome sequences of representative strains of PRRSV American strains and PRRSV European strains from multiple public databases as the target genome library;

[0046] S2. Select the genome sequence of a representative PRRSV strain as a reference sequence, obtain all 120bp sequences in the reference sequence, and screen the sequences with GC content of 30% to 70% in the obtained sequences, and select the sequence with the highest number of genomes in the target genome library as the first candidate probe group.

[0047] S3. Align the first candidate probe set with the target sequence set in the target genome library to obtain a second candidate probe set with a regional base difference rate of no more than 10% with the target sequence set;

[0048] S4: Using the standard that each probe captures a target gene length of ≥400bp, select the minimum number of probes from the second candidate probe group that can cover all the target sequence groups to form the probe group.

[0049] In this embodiment of the application, by comparing the genome sequences of PRRSV American strains and PRRSV European strains with the genome sequences of representative strains, and then using the principle of full genome coverage, a probe set with high sensitivity against PRRSV can be obtained.

[0050] This kit is based on the probe set described above. The specific composition and sequence information of the probe set can be found in the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0051] In some alternative implementations, the PRRSV representative strain is ATCC VR-2332.

[0052] In this application embodiment, the positive effect of limiting the PRRSV representative strain to ATCC VR-2332 is that it is the most widely studied strain, easily accessible to the general public, and has a complete genome sequence.

[0053] Based on a general inventive concept, this application provides an application of a probe set for detecting porcine reproductive and respiratory syndrome (PRRSV) pathogen, the application of which includes using the probe set to detect PRRSV.

[0054] This application is based on the probe set described above. The specific composition and sequence information of the probe set can be found in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0055] In some alternative implementations, such as Figure 2 As shown, the specific steps of the detection are as follows:

[0056] S1. Extract nucleic acids from the sample to be tested, and then obtain a transcriptome library containing the linker oligonucleotide sequence of the sample tag;

[0057] S2. Mix the transcriptome library and capture the PRRSV sequence using the kit, then elute and amplify by PCR to obtain the probe capture library;

[0058] S3. Perform high-throughput sequencing on the probe capture library, and perform quality control and analysis based on the high-throughput sequencing data to calculate the genome alignment rate;

[0059] S4. Based on the genome alignment rate, detect the genome sequence and type of the PRRSV strain infecting the sample to be tested.

[0060] In this embodiment, specific detection steps are defined, and a probe set is introduced to capture PRRSV sequences. By utilizing the specific binding characteristics of the probe set and the PRRSV sequence, PRRSV can be fully enriched, thereby accurately measuring and calculating the genome alignment rate in the subsequent high-throughput sequencing stage. The alignment rate can then be used to clarify the PRRSV infection status in the sample to be tested, achieving highly sensitive detection of PRRSV.

[0061] Genome alignment rate refers to the proportion of sequencing sequences that align to each genome in a reference genome library formed by the PRRSV American representative strain, out of the total number of quality control qualified sequences in that sample.

[0062] In some alternative implementations, such as Figure 3 As shown, the extraction of nucleic acids from the sample to be tested, followed by the preparation of a transcriptome library containing adapter oligonucleotide sequences with sample tags, specifically includes:

[0063] S101. Extract nucleic acid from the sample to be tested, and then perform RNA-seq to obtain double-stranded cDNA;

[0064] S102. The double-stranded cDNA is broken and ligated with a linker oligonucleotide sequence containing the sample tag. Then, PCR amplification is performed using amplification primers to obtain a transcriptome library containing a linker oligonucleotide sequence containing the sample tag.

[0065] The amplification primers are amplification primers for amplifying the oligonucleotide sequence of the adapter.

[0066] In this embodiment of the application, the specific construction method of the transcriptome library is specified. The use of adapter oligonucleotide sequences can facilitate the calculation of the genome alignment rate in the subsequent high-throughput sequencing stage, thereby improving the accuracy of detection.

[0067] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. If there is no corresponding national standard, then general international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0068] Example 1

[0069] Probe assembly design:

[0070] 1131 publicly available PRRSV American and European strain genome sequences were collected from public databases. Nine sequences were selected to form a target genome sequence set. The genome sequence of the American strain ATCC VR-2332 (GenBank accession no. EF536003) was used as a reference sequence to obtain variation information between each genome sequence and the reference sequence. A sequence library of the ATCCVR-2332 genome sequence was constructed with a step size of 120 bp. Sequences with GC content of 30%–70% and the highest number of sequences aligned with the target genome sequence set were selected as the first candidate probe set. The first candidate probe set was compared with the target sequence set in the target genome library to obtain a second candidate probe set with a regional base difference rate of no more than 10%. Each probe can capture a length of approximately 400 bp. The minimum number of probes that can cover all target sequence sets was selected from the second candidate probe set to form the probe set.

[0071] In this embodiment, a total of 53 PRRSV probes covering the two subtypes of American and European strains were finally obtained. Each probe is about 120 bp in length, and its nucleotide sequence is shown in Table 1 as SEQ ID NO.1 to 53.

[0072] Table 1. Probe sequence distribution

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] Example 2

[0079] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is as follows:

[0080] The method for detecting PRRSV is CFWG-Seq.

[0081] 1. Constructing a transcriptome library:

[0082] After obtaining the nucleic acid of the sample to be tested, a commercial RNA-Seq kit was used to obtain the transcriptome library of the sample. The main steps include: obtaining cDNA using a commercial RNA-Seq kit, and then synthesizing double-stranded cDNA; breaking the double-stranded cDNA and ligating it with adapter oligonucleotide sequences containing the sample tag; and performing PCR amplification using primers that can amplify the adapter oligonucleotide sequences at both ends to obtain the transcriptome library of the sample.

[0083] 2. Construct a probe capture library:

[0084] Since sample tags have already been added in the first step, transcriptome libraries of 1 to 10 samples to be tested can be mixed in this step to form a mixed library.

[0085] A capture kit containing a designed probe set was used to add the probe set and hybridization solution to the formed mixed library for PRRSV sequence capture.

[0086] The hybridization product is bound to magnetic beads, and the unbound DNA is eluted to obtain the eluted product, which is the captured viral sequence.

[0087] The captured viral sequences were amplified by PCR using primers that can amplify adapter sequences, and the captured viral sequences were further enriched to obtain a probe capture library, which was then sequenced on a high-throughput sequencing platform.

[0088] 3. Sequencing data analysis:

[0089] After obtaining the sequencing data, the data is first allocated to each sample according to the sample tags added in the first step. The sequencing data in each sample undergoes quality control, and the quality-controlled sequencing sequences are aligned to a reference genome library formed by the genome sequences of representative PRRSV strains (including 17 American strains: ATCC_VR-2332, CH-1a, CH-1R, GD, HB-1, HENAN-HEB, HENAN-XINX, HUN4, HuN, JXA1_P80, JXA1, MN184B, MN184C, MN6, NADC30, NADC31, R98, and TJ). The proportion of sequencing sequences aligned to each genome to the total number of quality-controlled sequences in that sample is calculated, i.e., the genome alignment rate.

[0090] 4. Result determination:

[0091] The genome with the highest alignment rate was identified as the dominant strain in the sample.

[0092] 5. Genome assembly:

[0093] Based on the genotype represented by the most sequences in the sequence covered at each location, the genome is assembled to obtain the assembled genome sequence.

[0094] 6. Mutation detection

[0095] The assembled genome sequence is compared with the reference genome sequence of the dominant strain to obtain the genetic variation of the viral genome in the sample to be tested.

[0096] Example 3

[0097] Comparing Example 3 and Example 2, the differences between Example 3 and Example 2 are as follows:

[0098] Detection of three PRRSV representative strains of virus culture:

[0099] The genome sequences of the American strain JXA1, the American strain ATCC VR-2332, and the European strain Lelystad virus were obtained using designed probe sets, kits, and methods, respectively.

[0100] Each strain was tested three times, with RNA at three different gradients of 10 ng, 1 ng, and 0.1 ng added respectively. Three sterile water templates were also set up as negative controls, for a total of 12 tests.

[0101] As shown in Table 2, among the samples of each strain in the three gradients, according to the judgment and assembly method, the strains marked in red are the strains with the highest genome alignment rate, indicating that the method can accurately identify the strains in the test samples; after sequence assembly, more than 99% of the genome sequences were assembled, while the negative control failed to assemble the sequence, indicating the high efficiency of the set probe set.

[0102] Table 2. Identification and genome alignment rates of each strain.

[0103]

[0104]

[0105] Further 10-fold serial dilutions were performed on the RNA of the JXA1 and Lelystad virus strains, respectively, at 10... -2 ng, 10 -3 ng,10 -4 ng, 10 -5 ng and 10 -6 ng of RNA was used as the starting amount, and two sterile water templates were set up as negative controls. A total of 12 tests were performed.

[0106] The results show that the template quantity is 10.-4 Both ng samples accurately identified the virus strain in the test samples and assembled over 99% of the genome sequence. -5 ng and 10 -6 The failure to obtain assembled sequences from ng and negative control samples indicates that the invented probe set, kit, and CFWG-Seq method have a detection limit of 10 for nucleic acid levels in viral culture samples. -4 ng.

[0107] Example 4

[0108] Comparing Example 4 and Example 3, the differences between Example 4 and Example 3 are as follows:

[0109] Test results of 11 diseased samples from pig farms:

[0110] The invented probe set, kit, and CFWG-Seq method were used to test eight porcine reproductive and respiratory syndrome (PRRS) samples from different pig farms, as shown in Table 3. The sampling sites included blood and lungs. All eight samples were simultaneously confirmed by quantitative real-time PCR, with Ct values ​​ranging from 18 to 34. The results are shown in Table 4.

[0111] Table 3. Collection of Pathogen Samples for Swine Ear Disease

[0112]

[0113]

[0114] Table 4. Results of Detection by Real-Time PCR in 8 Samples

[0115]

[0116] The results showed that PRRSV sequences were detectable in all 8 samples, with two samples (S2 and S3) having relatively low genome alignment rates. After assembly, the sequences in the six samples with high genome alignment rates yielded 99% viral genome sequences, indicating that the method can obtain PRRSV genome sequences directly from host cases without culture. The two samples with low genome alignment rates had assembled sequences shorter than 3000 bp, and were considered assembly failures. The CT values ​​for samples S2 and S3 were 29.01 and 34.31, respectively, indicating that the detection limit of the method for clinical samples is ≤27 CT values.

[0117] One or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0118] (1) The probe set for detecting porcine reproductive and respiratory syndrome (PRRS) provided in this application involves analyzing the genomic sequences of multiple PRRSV American and European strains, with the American strain as a representative strain. By comparing the variation information of the representative strain, the American strain, and the European strain, a probe set containing multiple probes is designed in conserved and variable regions. This probe set can completely cover the genomes of the American and European strains of PRRSV. Thus, the designed probe set can capture the PRRSV sequence in the sample to be tested, thereby enriching PRRSV. By detecting the enriched PRRSV, high-sensitivity detection of low-abundance viruses can be achieved without culture.

[0119] (2) The method for detecting porcine reproductive and respiratory syndrome (PRRS) provided in this application adds a step of capturing PRRSV sequences in the sample transcriptome library using a PRRSV-specific probe set after construction. This enriches PRRSV, and the enriched viral sequences are then assembled through high-throughput sequencing and analysis to obtain the complete genome sequence of PRRSV. The addition of the probe capture step allows CFWG-Seq to detect low-abundance viruses without requiring a large amount of data.

[0120] (3) The method for detecting porcine reproductive and respiratory syndrome (PRRS) provided in this application, through testing PRRSV nucleic acid standards with known copy numbers, has a sensitivity of CGWG-Seq as low as 0.1 pg of viral RNA, and the assembled genome coverage range is 98% to 100%.

[0121] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0122] In this application, unless otherwise stated, directional terms such as "upper" and "lower" specifically refer to the orientation shown in the accompanying drawings. Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to."

[0123] In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any actual relationship or order between these entities or operations. In this document, "and / or" describes the association between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c," or "at least one of a, b, and c," can both represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0124] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A probe set for detecting porcine reproductive and respiratory syndrome (PRRSV) pathogen, characterized in that, The nucleotide sequences of the probe set are shown in SEQ ID NO.1 to 53.

2. The probe assembly according to claim 1, characterized in that, The nucleotide sequence length of the probe set is 120bp±30bp.

3. The probe assembly according to claim 1, characterized in that, The probe set was designed based on the sequences of PRRSV American strain and PRRSV European strain.

4. A kit for detecting porcine reproductive and respiratory syndrome (PRRSV) pathogen, characterized in that, The kit includes the probe set as described in any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The detection limit of the kit is ≥10^6 mcg of pure viral culture RNA. -4 The CT value of the sample or the sample identified by fluorescent PCR is ≤27.

6. A method for preparing a probe set as described in any one of claims 1-3, characterized in that, The method includes: The genome sequences of representative strains of PRRSV American strains and PRRSV European strains from multiple public databases were selected as the target genome library; Using the genome sequence of the American strain ATCCVR-2332 as a reference sequence, all 120bp sequences in the reference sequence were obtained, and the sequences with GC content of 30% to 70% and the highest number of genomes compared with the target genome library were selected as the first candidate probe group. The first candidate probe set is compared with the target sequence set in the target genome library to obtain a second candidate probe set with a regional base difference rate of no more than 10% from the target sequence set; Using the standard that each probe captures a target gene length of ≥400bp, the minimum number of probes that can cover all the target sequence groups is selected from the second candidate probe group to form the probe group.

7. The application of a probe set for detecting porcine reproductive and respiratory syndrome (PRRSV) pathogen, characterized in that, The application includes using the probe set as described in any one of claims 1-3 in the preparation of a diagnostic reagent for detecting porcine reproductive and respiratory syndrome (PRRS).

8. The application according to claim 7, characterized in that, The specific steps of the detection are as follows: Nucleic acid was extracted from the sample to be tested, and a transcriptome library containing the linker oligonucleotide sequence of the sample tag was obtained. The transcriptome library was mixed and PRRSV sequences were captured using the kit described in claim 4 or 5, followed by elution and PCR amplification to obtain the probe capture library. The probe capture library was subjected to high-throughput sequencing, and the high-throughput sequencing data was used for quality control and analysis to calculate the genome alignment rate. Based on the genome alignment rate, the genome sequence and type of the PRRSV strain infecting the sample to be tested are determined.

9. The application according to claim 8, characterized in that, The process involves extracting nucleic acids from the sample to be tested, and then obtaining a transcriptome library containing adapter oligonucleotide sequences with sample tags, specifically including: Nucleic acid was extracted from the sample to be tested, and then RNA-seq was performed to obtain double-stranded cDNA; The double-stranded cDNA was broken and ligated with a linker oligonucleotide sequence containing the sample tag. Then, PCR amplification was performed using amplification primers to obtain a transcriptome library containing the linker oligonucleotide sequence containing the sample tag. The amplification primers are amplification primers for amplifying the oligonucleotide sequence of the adapter.

Images