CN116287463A - Probe set and kit for detecting porcine reproductive and respiratory syndrome pathogen PRRSV, and preparation method and application thereof - Google Patents

Abstract

The application relates to the field of gene detection, in particular to a probe set for detecting porcine reproductive and respiratory syndrome pathogen PRRSV, a kit, a preparation method and application thereof; the nucleotide sequence of the probe set is shown as SEQ ID NO. 1-53; the probe set is designed in a conservation area and a variation area between the genomes of the strains by analyzing a target genome library sequence consisting of PRRSV American strains and European strain representative strains, and can completely cover the whole genomes 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, and the high-sensitivity detection of the PRRSV, the variation monitoring and the typing of genome level are realized on the basis of culture-free.

Description

A probe set, kit and preparation method for detecting PRRSV, a pathogen of porcine blue ear disease and application

Technical Field

The present application relates to the field of gene detection, and in particular to a probe set, a kit, a preparation method and an application thereof for detecting PRRSV, a pathogen of porcine blue ear disease.

Background Art

Porcine reproductive and respiratory syndrome is a highly contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV), also known as blue ear disease, characterized by reproductive disorders in pregnant sows (such as miscarriage, stillbirth, mummified fetuses) and respiratory diseases in pigs of all ages, especially piglets. The PRRSV genome is a single-stranded positive-strand RNA virus. Since RNA is prone to point mutations, deletions, insertions and substitutions, PRRSV is one of the viruses with a higher mutation rate among known RNA viruses. PRRSV includes two types: the American type represented by ATCC VR-2332 and the European type represented by Lelystad virus. There are significant antigenic differences between European and American isolates, so there is only a small amount of cross-reaction between the two. There is extensive genomic variation between American isolates, while European strains are more conservative.

The existing methods for detecting the presence of PRRSV in samples are mainly based on qPCR methods. However, the current qPCR method only detects one gene site, which has the limitations of false negative detection results due to mutation or degradation of the virus in the primer region and the inability to obtain sequence variation information. The current technology for detecting sequence variation is based on PCR combined with first-generation sequencing to detect variation information in 1 to 2 gene regions, which has the limitations of low efficiency and limited monitoring range. Therefore, in order to obtain more comprehensive sequence variation, the typical method 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 the isolation and cultivation of the virus, which is difficult to isolate and cultivate; even if the culture is successful, due to the high variability of the virus, it is difficult to successfully amplify all fragments using existing primers, which has the limitations of low sensitivity, low efficiency and time consumption. Existing culture-free methods mostly use macrotranscriptome methods. There is a large amount of host data in the sequencing data, which has the limitations of large data waste, high cost and difficulty in detecting low-abundance viruses. Therefore, how to provide a probe set for PRRSV detection and mutation monitoring without culture has become an urgent problem to be solved.

Summary of the invention

The present application provides a probe group, a kit, a preparation method and an application for detecting PRRSV, a pathogen of porcine blue ear disease, in order to solve the problem in the prior art that it is difficult to achieve high-sensitivity detection and variation monitoring of PRRSV on a culture-free basis.

In a first aspect, the present application provides a probe set for detecting PRRSV, a pathogen of porcine blue ear disease. The nucleotide sequence of the probe set is shown in SEQ ID NOs. 1 to 53.

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

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

In a second aspect, the present application provides a kit for detecting PRRSV, the pathogen of porcine blue ear disease, wherein the kit comprises the probe set described in the first aspect.

Optionally, the detection limit of the kit is ≥10 ^-4 ng or the CT value of the sample identified by fluorescent PCR is ≤27.

In a third aspect, the present application provides a method for preparing the probe group described in the first aspect, the method comprising:

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

A genome sequence of a representative strain of PRRSV is selected as a reference sequence, all sequences with a length of 120 bp in the reference sequence are obtained, and the sequences with a GC content of 30% to 70% and the highest number of genomes in the target genome library are selected from the obtained sequences as the first candidate probe group;

Comparing the first candidate probe set with the target sequence set in the target genome library to obtain a second candidate probe set whose base difference rate with the target sequence set region does not exceed 10%;

Taking the length of each probe capturing the target gene as ≥400 bp as the standard, the minimum number of probes that can cover all the target sequence groups is screened from the second candidate probe group to form the probe group.

Optionally, the representative strain of PRRSV is ATCC VR-2332.

In a fourth aspect, the present application provides an application of a probe group for detecting PRRSV, a pathogen of porcine reproductive and respiratory syndrome (PRRS). The application comprises using the probe group described in the first aspect for detecting PRRSV.

Optionally, the specific steps of the detection are:

Extracting nucleic acid from the sample to be tested, and then obtaining a transcriptome library containing the adapter oligonucleotide sequence of the sample tag;

The transcriptome library is mixed, and the PRRSV sequence is captured using the kit described in the second aspect, followed by elution and PCR amplification to obtain a probe capture library;

Performing high-throughput sequencing on the probe capture library, performing quality control and analysis based on the high-throughput sequencing data, and calculating the genome alignment rate;

According to the genome comparison rate, the genome sequence and type of the PRRSV strain infected by the sample to be tested are determined.

Optionally, the extraction of nucleic acid from the sample to be tested and then obtaining a transcriptome library containing the linker oligonucleotide sequence of the sample tag specifically includes:

Extract nucleic acid from the sample to be tested, then perform RNA-seq to obtain double-stranded cDNA;

The double-stranded cDNA is interrupted and connected to an adapter oligonucleotide sequence containing a sample tag, and then PCR amplification is performed with an amplification primer to obtain a transcriptome library of the adapter oligonucleotide sequence containing the sample tag;

Wherein, the amplification primer is an amplification primer for amplifying a linker oligonucleotide sequence.

The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

A probe group for detecting porcine reproductive and respiratory syndrome (PRRS) provided in an embodiment of the present application is prepared by sequencing a target genome library consisting of multiple representative strains of PRRSV American strains and PRRSV European strains, selecting the genome sequence of the representative strain of PRRSV as a reference sequence, and respectively comparing the variation information of the representative strains, the American strains, and the European strains, thereby designing a probe group comprising multiple probes in the conservative region and the variation region, which can completely cover the genomes of the American strain and the European strain of PRRSV, so that the PRRSV sequence in the sample to be tested can be captured by the designed probe group, thereby enriching PRRSV, and by detecting the enriched PRRSV, high-sensitivity detection of low-abundance viruses can be achieved on a culture-free basis.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

FIG1 is a schematic diagram of a process for preparing a probe set according to an embodiment of the present application;

FIG2 is a schematic diagram of a flow chart of a method for detecting PRRSV using a probe set provided in an embodiment of the present application;

FIG3 is a schematic diagram of the data analysis process of the probe set method for detecting PRRSV provided in an embodiment of the present application.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

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

The embodiment of the present application provides a probe group for detecting PRRSV, a pathogen of porcine blue ear disease. The nucleotide sequence of the probe group is shown in SEQ ID NOs. 1 to 53.

In some optional embodiments, the nucleotide sequence length of the probe set is 120 bp±30 bp.

In the embodiments of the present application, limiting the nucleic acid sequence length of the probe group can ensure that the probe can capture enough PRRSV sequences in the sample to be tested, thereby improving the effect of PRRSV enrichment.

In some optional embodiments, the probe set is designed based on the sequences of PRRSV American strain and PRRSV European strain.

In the examples of the present application, the specific design sources of the probe set are limited so that the probe set can completely cover the genomes of the American strain and the European strain of PRRSV, thereby improving the enrichment effect of PRRSV.

Based on a general inventive concept, the present application provides a kit for detecting PRRSV, a pathogen of porcine blue ear disease, and the kit includes the probe set.

The kit is implemented based on the above-mentioned probe group. The specific composition and sequence information of the probe group can be referred to the above-mentioned embodiments. Since the kit adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

In some optional embodiments, the detection limit of the kit is ≥10 ^-4 ng or the CT value of the sample identified by fluorescent PCR is ≤27.

In the examples of the present application, the detection limit of the test kit is limited. Since the probe group can completely cover the genomes of the American strain of PRRSV and the European strain of PRRSV, and the probe group is designed based on the conserved regions and variable regions of PRRSV, the test kit composed of the probe group can have a lower detection limit.

Based on a general inventive concept, as shown in FIG1 , the present application provides a method for preparing the probe set, the method comprising:

S1. Select genome sequences of representative strains of PRRSV American strains and PRRSV European strains in multiple public databases as target genome libraries;

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

S3. Compare the first candidate probe set with the target sequence set in the target genome library to obtain a second candidate probe set whose base difference rate with the target sequence set region does not exceed 10%;

S4: Taking the length of each probe capturing the target gene as ≥400 bp as the standard, 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.

In the examples of the present application, by comparing the genome sequences of the American strain of PRRSV and the European strain of PRRSV with the genome sequences of representative strains, and then based on the principle of full genome coverage, a highly sensitive probe set for PRRSV can be obtained.

The kit is implemented based on the above-mentioned probe group. The specific composition and sequence information of the probe group can be referred to the above-mentioned embodiments. Since the kit adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

In some optional embodiments, the PRRSV representative strain is ATCC VR-2332.

In the examples of the present application, the positive effect of limiting the representative strain of PRRSV to ATCC VR-2332 is that it is the strain that has been most widely studied, is easily accessible to ordinary people, and has a complete genome sequence.

Based on a general inventive concept, the present application provides an application of a probe group for detecting PRRSV, a pathogen of porcine reproductive and respiratory syndrome (PRRS). The application includes using the probe group for detecting PRRSV.

This application is implemented based on the above-mentioned probe group. The specific composition and sequence information of the probe group can refer to the above-mentioned embodiments. Since this application adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

In some optional embodiments, as shown in FIG2 , the specific steps of the detection are:

S1. Extract the nucleic acid of the sample to be tested, and then obtain a transcriptome library containing the adapter oligonucleotide sequence of the sample tag;

S2. Mixing the transcriptome library, and using the kit to capture the PRRSV sequence, followed by elution and PCR amplification to obtain a probe capture library;

S3. performing high-throughput sequencing on the probe capture library, performing quality control and analysis based on the high-throughput sequencing data, and calculating the genome alignment rate;

S4. According to the genome comparison rate, the genome sequence and type of the PRRSV strain that infects the sample to be tested are detected.

In the embodiments of the present application, specific detection steps are defined, a probe group is introduced to capture the PRRSV sequence, and the property that the probe group and the PRRSV sequence can specifically bind is utilized to enable PRRSV to be fully enriched, so that the genome alignment rate can be accurately measured and calculated in the subsequent high-throughput sequencing stage, and then the alignment rate can be used to clarify the situation of PRRSV strain infection in the sample to be tested, thereby achieving highly sensitive detection of PRRSV.

The genome alignment rate refers to the ratio of the number of sequencing sequences aligned to each genome to the total number of qualified quality control sequences of the sample when the sequencing sequence is aligned to the reference genome library formed by the representative strain of PRRSV in America.

In some optional embodiments, as shown in FIG. 3 , the nucleic acid of the sample to be tested is extracted to obtain a transcriptome library containing a linker oligonucleotide sequence of the sample tag, specifically comprising:

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

S102. The double-stranded cDNA is interrupted and connected to a linker oligonucleotide sequence containing a sample tag, and then PCR amplification is performed with an amplification primer to obtain a transcriptome library containing a linker oligonucleotide sequence containing a sample tag;

Wherein, the amplification primer is an amplification primer for amplifying a linker oligonucleotide sequence.

In the examples of the present application, the specific construction method of the transcriptome library is limited, and the use of linker oligonucleotide sequences can facilitate the calculation of the genome alignment rate in the subsequent high-throughput sequencing stage, thereby improving the accuracy of detection.

The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then carry out according to general international standards, normal conditions, or according to the conditions recommended by the manufacturer.

Example 1

Probe set design:

1131 genome sequences of PRRSV American strains and European strains published in public databases were collected, 9 of which were selected to form a target genome sequence group, and the genome sequence of the American strain ATCC VR-2332 (GenBank accession no. EF536003) was used as a reference series to obtain the variation information between each genome sequence and the reference sequence; a sequence library of the ATCC VR-2332 genome sequence with a step length of 120 bp was constructed, and the sequences with a GC content of 30% to 70% and the highest number in the target genome sequence group were screened as the first candidate probe group; the first candidate probe group was compared with the target sequence group in the target genome library to obtain a second candidate probe group with a base difference rate of no more than 10% with the target sequence group; each probe could capture a probe length of about 400 bp, and the minimum number that could cover all the target sequence groups was screened from the second candidate probe group to form the probe group.

In this example, a total of 53 PRRSV probe sets covering two subtypes of the American strain and the European strain were finally obtained. Each probe was about 120 bp long, and its nucleotide sequence is shown in SEQ ID NO. 1-53 in Table 1.

Table 1 Probe sequence distribution

Example 2

Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is:

CFWG-Seq method for detecting PRRSV:

1. Construction of transcriptome library:

After obtaining the nucleic acid of the sample to be tested, a commercial RNA-Seq kit is used to obtain the transcriptome library of the sample. The main steps include: using a commercial RNA-Seq kit to obtain cDNA, and then synthesizing double-stranded cDNA; after breaking the double-stranded cDNA, connecting it to the adapter oligonucleotide sequence containing the sample label; using primers that can amplify the adapter oligonucleotide sequences at both ends to perform PCR amplification to obtain the transcriptome library of the sample.

2. Construction of probe capture library:

Since the sample labels have been added in the first step, the transcriptome libraries of 1 to 10 samples to be tested can be mixed in this step to form a mixed library;

A capture kit containing a designed probe set is used, and the probe set and hybridization solution are added to the formed mixed library to capture the PRRSV sequence;

The hybridization product is bound to magnetic beads and unbound DNA is eluted to obtain the eluted product, which is the captured viral sequence;

The captured viral sequences are amplified by PCR using the aforementioned primers capable of amplifying the adapter sequences, and the captured viral sequences are further enriched to obtain a probe capture library, which is then sequenced on a high-throughput sequencing platform.

3. Sequencing data analysis:

After obtaining the sequencing data, firstly, the data were assigned to each sample according to the sample labels added in the first step; the sequencing data in each sample were quality controlled, and the sequencing sequences that passed the quality control were aligned to the reference genome library formed by the genome sequences of representative PRRSV strains (including 2 European strains, lena and LV, and 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 the number of sequencing sequences aligned to each genome to the total number of sequences that passed the quality control of the sample was calculated, that is, the genome alignment rate.

4. Result determination:

The genome with the highest matching rate is determined as the dominant strain in the sample.

5. Genome assembly:

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

6. Mutation Detection

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

Example 3

Comparing Example 3 with Example 2, the difference between Example 3 and Example 2 is:

Detection of virus culture of 3 representative PRRSV strains:

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

At the same time, each strain was tested three times, with three gradients of RNA, 10ng, 1ng and 0.1ng, respectively, and three sterile water templates were set as negative controls, for a total of 12 tests.

The results showed that in the three gradient samples of each strain, according to the determination and assembly method, as shown in Table 2, the strains marked in red were the strains with the highest genome matching rate, indicating that the method can accurately determine the strains in the samples to be tested; after sequence assembly, more than 99% of the genome sequences were assembled, while no sequence was assembled in the negative control, indicating the high efficiency of the set probe group.

Table 2. Comparison rate of genomes after identification and assembly of each strain

The RNA of the two strains, JXA1 and Lelystad virus, was further diluted 10-fold in a gradient, with ^10-2 ng, ^10-3 ng, ^10-4 ng, ^10-5 ng and ^10-6 ng of RNA as the starting amount, respectively, and two sterile water templates were set as negative controls, and a total of 12 tests were performed.

The results showed that both samples with a template amount of ^10-4 ng could accurately identify the strains in the tested samples and assemble more than 99% of the genome sequence. The ^10-5 ng and ^10-6 ng and negative control samples failed to obtain assembled sequences, indicating that the invented probe set, kit and CFWG-Seq method have a detection limit of ^10-4 ng for the amount of nucleic acid in viral culture samples.

Example 4

Comparing Example 4 with Example 3, the difference between Example 4 and Example 3 is:

Detection results of 11 diseased pig farm samples:

The invented probe set, kit and CFWG-Seq method were used to detect 8 samples of blue ear disease from different pig farms, as shown in Table 3, and the sampling sites included blood and lungs. The 8 samples were also confirmed by fluorescence quantitative PCR, with Ct values ranging from 18 to 34, as shown in Table 4.

Table 3 Pig ear disease sample collection table

Table 4 Results of fluorescence quantitative PCR for 8 samples

The results showed that PRRSV sequences could be detected in all 8 samples, of which 2 samples (S2 and S3) had relatively low genome alignment rates; after assembly, the sequences in the 6 samples with high genome alignment rates could obtain 99% of the viral genome sequences, indicating that the method can obtain PRRSV genome sequences directly from host cases without culture. For the 2 samples with low genome alignment rates, the assembled sequence length was less than 3000bp, and the assembly was considered to have failed. The CT values of S2 and S3 samples were 29.01 and 34.31, respectively, indicating that the detection limit of the method for clinical samples is CT value ≤ 27.

One or more technical solutions in the embodiments of the present application also have at least the following technical effects or advantages:

(1) The present application provides a probe group for detecting porcine reproductive and respiratory syndrome (PRRS) by sequencing multiple genome sequences of American PRRSV strains and European PRRSV strains, selecting the American PRRSV strain as a representative strain, and comparing the variation information of the representative strain, the American strain, and the European strain, respectively. A probe group comprising multiple probes is designed in the conservative region and the variation region, which can completely cover the genomes of the American strain and the European strain of PRRSV. Thus, the PRRSV sequence in the sample to be tested can be captured by the designed probe group, thereby enriching PRRSV. By detecting the enriched PRRSV, high-sensitivity detection of low-abundance viruses can be achieved on a culture-free basis.

(2) The method for detecting porcine reproductive and respiratory syndrome provided in the embodiment of the present application, after constructing the transcriptome library of the sample, adds a step of capturing the PRRSV sequence in the library with a PRRSV-specific probe group, which can achieve the enrichment of PRRSV. After high-throughput sequencing and analysis and assembly of the enriched viral sequence, the full genome sequence of PRRSV can be obtained. The addition of the probe capture step enables CFWG-Seq to detect low-abundance viruses without a large amount of data.

(3) The method for detecting porcine reproductive and respiratory syndrome provided in the embodiment of the present application, through the test of PRRSV nucleic acid standards with known copy numbers, the sensitivity of CGWG-Seq can be as low as 0.1pg of viral RNA, and the coverage range of the assembled genome is 98% to 100%.

Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values within the range. For example, the range description from 1 to 6 should be considered to have 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., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

In this application, unless otherwise specified, directional words such as "upper" and "lower" used refer specifically to the directions of the drawings in the accompanying drawings. In addition, in the description of the specification of this application, the terms "include", "comprising", etc. mean "including but not limited to".

In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and/or" describes the association relationship of associated objects, indicating that there can be three relationships, for example, A and/or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "multiple" refers to two or more. "At least one", "the following at least one (individual)" or similar expressions refer to any combination of these items, including any combination of single (individual) or plural (individual). For example, "at least one (individual) of a, b, or c", or "at least one (individual) of a, b, and c", can all represent: a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, where a, b, c can be single or multiple respectively.

The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims (10)

1. A probe set for detecting PRRSV, the pathogen of porcine blue-ear disease, characterized in that the nucleotide sequence of the probe set is as shown in SEQ ID NO.1-53. 2. The probe set according to claim 1, characterized in that the length of the nucleotide sequence of the probe set is 120bp±30bp. 3. The probe set according to claim 1, characterized in that the probe set is designed according to the sequences of PRRSV American strain and PRRSV European strain. 4. A test kit for detecting PRRSV, the pathogen of porcine blue-ear disease, characterized in that the test kit comprises the probe set according to any one of claims 1-3. 5 . The kit according to claim 4 , wherein the lower limit of detection of the kit is ≥ 10 ⁻⁴ ng of pure virus culture RNA or a CT value ≤ 27 of a sample identified by fluorescent PCR. 6 . 6. A method for preparing the probe set according to any one of claims 1-3, wherein the method comprises: Select the genome sequences of representative strains of PRRSV American strains and PRRSV European strains in multiple public databases as the target genome library; Select the genome sequence of a PRRSV representative strain as the reference sequence, obtain all 120bp sequences in the reference sequence, and screen the GC content in the obtained sequence to be 30% to 70% and compare it to the target genome library The sequence with the highest number of genomes is used as the first candidate probe set; comparing the first candidate probe set with the target sequence set in the target genome library, and obtaining a second candidate probe set whose base difference rate with the target sequence set is no more than 10%; The probe set is formed by selecting the minimum number that can cover all the target sequence sets from the second candidate probe set on the basis that the length of the target gene captured by each probe is ≥ 400 bp. 7. The method according to claim 6, wherein the representative strain of PRRSV is ATCC VR-2332. 8. An application of a probe set for detecting PRRSV, the pathogen of PRRSV in pigs, characterized in that the application includes using the probe set according to any one of claims 1-3 for detecting PRRSV in pigs. 9. The application according to claim 8, wherein the specific steps of the detection are: Extract the nucleic acid of the sample to be tested, and then obtain the transcriptome library containing the adapter oligonucleotide sequence of the sample tag; Mixing the transcriptome library, and using the kit as claimed in claim 4 or 5 to capture the PRRSV sequence, followed by elution and PCR amplification to obtain a probe capture library; performing high-throughput sequencing on the probe capture library, performing quality control and analysis according to the data of the high-throughput sequencing, and calculating the genome comparison rate; According to the genome comparison ratio, the genome sequence and type of the PRRSV strain infected by the sample to be tested are determined. 10. The application according to claim 9, wherein the nucleic acid of the sample to be tested is extracted, and then the transcriptome library comprising the adapter oligonucleotide sequence of the sample tag is obtained, specifically comprising: Extract the nucleic acid of the sample to be tested, and then perform RNA-seq to obtain double-stranded cDNA; breaking the double-stranded cDNA, and connecting the adapter oligonucleotide sequence containing the sample label, and then performing PCR amplification with the amplification primers to obtain a transcriptome library containing the adapter oligonucleotide sequence containing the sample label; Wherein, the amplification primer is an amplification primer for amplifying an adapter oligonucleotide sequence.

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