Compositions, kits and uses thereof for detecting porcine reproductive disorder pathogens

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to a composition, method and use for simultaneous detection and differentiation of swine fever virus, psittacosis chlamydia, porcine reproductive and respiratory syndrome virus and porcine circovirus type II. The composition for simultaneous detection provided by the present application can realize rapid detection of four pathogen target genes in one tube of reaction solution by using multiplex fluorescence RPA detection technology, and has the advantages of high throughput, short time and low cost. On one hand, the present application can accurately detect pathogens and analyze mixed infection of pathogens, so as to achieve the effect of precise medication, reduce the use of antibiotics, save costs, and reduce the cost and increase the benefit of enterprises, and on the other hand, compared with fluorescence PCR, the detection time is greatly shortened, and the purpose of rapid detection is achieved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection, specifically to the detection of porcine reproductive disorder pathogens; more specifically, to the detection of four porcine reproductive disorder pathogens: classical swine fever virus, Chlamydia psittaci, porcine reproductive and respiratory syndrome virus, and porcine circovirus type II. Background Technology

[0002] Swine farming is the core of my country's livestock industry. In recent years, the frequent occurrence of reproductive disorders in pigs has severely impacted the industry. These disorders reduce swine reproductive capacity, and in severe cases, can lead to infertility. Reproductive disorders have become one of the most significant and prevalent diseases in swine farming, caused by various pathogens such as classical swine fever virus, chlamydia psittaci, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type II. To promote the large-scale development of the swine farming industry, it is essential to further improve feeding and management practices to reduce the incidence and prevalence of reproductive disorders in pigs.

[0003] Classical swine fever (CSF) can occur year-round and can infect pigs of all breeds, ages, and sexes. Maternal infection rates are high, and morbidity and mortality rates are even higher in young piglets. The disease is more prevalent during the winter and spring farrowing seasons, often first appearing in sow pens during peak farrowing periods, with a high incidence rate. Infected pregnant sows can transmit the virus to their fetuses via the placenta, causing newborn piglets to become ill and develop tearing. Affected sows mainly exhibit fever, lethargy, and decreased appetite; pregnant sows may experience reproductive problems such as abortion, stillbirth, and mummified fetuses; postpartum sows may experience reduced milk production or agalactia.

[0004] Chlamydia psittaci (CPS) infection has a high morbidity and wide prevalence, affecting pigs of different breeds and growth stages, but posing a significant threat to pregnant sows. It can lead to abortion, weak or stillbirths, and reduced piglet survival rates, impacting the healthy development of the pig farming industry and reducing farm profitability. Porcine chlamydial disease was first reported in the United States in 1955, and subsequently, many countries, including the United Kingdom, Germany, and the Soviet Union, reported its occurrence and spread. In 1982, Chlamydia psittaci was first isolated from samples of aborted sows and infected piglets. Since then, the disease has occurred and spread in many regions, and its incidence has increased with the development of the pig farming industry. Symptoms of Chlamydia psittaci infection are diverse, generally presenting as a chronic disease, sporadic or endemic, but some cases present as an acute outbreak.

[0005] Porcine reproductive and respiratory syndrome (PRRS) is a global, viral, and highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). It can cause reproductive disorders in pregnant sows, such as abortion, stillbirth, and mummified fetuses, as well as emaciation, growth retardation, and increased mortality in weaned piglets. The disease can cause fever and respiratory distress in pigs, leading to cyanosis of the ears, hence its alternative name, "blue ear disease." PRRS first gained attention in 1987 due to a "miscarriage storm" in the United States. At that time, because the cause was unknown, this disease that caused abortions in sows was called "mystery swine disease" (MSD).

[0006] Since its outbreak, porcine circovirus 2 (PCV2) has been considered a major pathogen causing reproductive disorders in sows. PCV2 nucleic acid has been detected in samples from aborted fetuses and stillborn piglets. Currently, PCV2-induced reproductive disorders in sows have become a major pain point for the pig farming industry. Reproductive performance is one of the important indicators in pig farming, and reproductive disorders lead to reduced reproductive performance in sows, resulting in frequent stillbirths and weak piglets. The disease spreads rapidly; a single infected case in a pig herd can cause large-scale transmission and subsequent mass infections. Therefore, the prevention and control of PCV2 pathogens is of paramount importance.

[0007] Reproductive disorders in pigs are a common problem in large-scale pig farming, and their infectious nature can lead to economic losses if not controlled promptly. Therefore, relying solely on conventional testing methods and clinical experience is often insufficient to identify the pathogen, potentially delaying optimal disease control. Analysis of pig reproductive disorders reveals that most are caused by environmental and husbandry practices, increasing the probability of disease occurrence. Effective diagnostic techniques and scientific prevention and control are crucial, combining advanced methods such as laboratory testing with a focus on prevention, treatment, and environmental optimization to minimize disease severity and reduce unnecessary economic losses.

[0008] Therefore, there is a need in the field for a product that can detect the pathogens causing reproductive disorders in pig farming, which can easily and efficiently identify the causes of reproductive disorders in pigs and can help provide targeted prevention strategies. Summary of the Invention

[0009] In view of the above-mentioned deficiencies, the present invention provides a composition for detecting porcine reproductive disorder pathogens, comprising at least one group of primers and probes for detecting the following pathogens:

[0010] The upstream and downstream primers and probes for detecting classical swine fever virus are shown in SEQ ID NO: 1-3;

[0011] The upstream and downstream primers and probes for detecting Chlamydia psittaci, as shown in SEQ ID NO: 4-6;

[0012] The upstream and downstream primers and probes for detecting porcine reproductive and respiratory syndrome virus are shown in SEQ ID NO: 7-9;

[0013] The upstream and downstream primers and probes for detecting porcine circovirus type II are shown in SEQ ID NO: 10-12.

[0014] Furthermore, a composition for detecting porcine reproductive disorder pathogens includes at least two groups of primers and probes for detecting the following pathogens:

[0015] The upstream and downstream primers and probes for detecting classical swine fever virus are shown in SEQ ID NO: 1-3;

[0016] The upstream and downstream primers and probes for detecting Chlamydia psittaci, as shown in SEQ ID NO: 4-6;

[0017] The upstream and downstream primers and probes for detecting porcine reproductive and respiratory syndrome virus are shown in SEQ ID NO: 7-9;

[0018] The upstream and downstream primers and probes for detecting porcine circovirus type II are shown in SEQ ID NO: 10-12.

[0019] Furthermore, a composition for detecting porcine reproductive disorder pathogens includes at least three groups of primers and probes for detecting the following pathogens:

[0020] The upstream and downstream primers and probes for detecting classical swine fever virus are shown in SEQ ID NO: 1-3;

[0021] The upstream and downstream primers and probes for detecting Chlamydia psittaci, as shown in SEQ ID NO: 4-6;

[0022] The upstream and downstream primers and probes for detecting porcine reproductive and respiratory syndrome virus are shown in SEQ ID NO: 7-9;

[0023] The upstream and downstream primers and probes for detecting porcine circovirus type II are shown in SEQ ID NO: 10-12.

[0024] Furthermore, a composition for detecting porcine reproductive disorder pathogens includes primers and probes for detecting the following pathogens:

[0025] The upstream and downstream primers and probes for detecting classical swine fever virus are shown in SEQ ID NO: 1-3;

[0026] The upstream and downstream primers and probes for detecting Chlamydia psittaci, as shown in SEQ ID NO: 4-6;

[0027] The upstream and downstream primers and probes for detecting porcine reproductive and respiratory syndrome virus are shown in SEQ ID NO: 7-9;

[0028] The upstream and downstream primers and probes for detecting porcine circovirus type II are shown in SEQ ID NO: 10-12.

[0029] The combined detection composition provided by this invention mainly utilizes multiplex fluorescent RPA detection technology, enabling rapid detection of four pathogen target genes in a single reaction tube. This method boasts high throughput, short detection time, and low cost. On the one hand, this invention can accurately detect pathogens and analyze mixed infections of chlamydia and viruses, thereby achieving precision medicine, reducing antibiotic use, saving costs, and increasing efficiency for enterprises. On the other hand, compared to fluorescent PCR, it significantly shortens the detection time, achieving the goal of rapid detection.

[0030] Furthermore, the probe is labeled with a fluorescent group, a quenching group, and a 3' C3-spacer, and modified with tetrahydrofuran.

[0031] Furthermore, the probe SEQ ID NO:3, after being marked and modified, is as follows:

[0032] 5'-CGGGGGTCGCTAGGGTGAAATCACACCAGG[P-dT]G[THF][B-dT]GGGGGGTACGACCTGA-C3Spacer-3';

[0033] The probe SEQ ID NO: 6, after being marked and modified, is:

[0034] 5'-ACCGTTAATTGCAGATCCTCGCCAGGTAACAAA[P-dT][THF]G[B-dT]GCGGGCATTCGTTT-C3 Spacer-3';

[0035] The probe SEQ ID NO: 9, after being marked and modified, is:

[0036] 5'-TTGAAAAGCCTCGTGTTGGGTGGCAGAAGAGC[P-dT][THF][B-dT]CAAACAGGGAGTGGT-C3 Spacer-3';

[0037] The probe SEQ ID NO: 12, after being marked and modified, is:

[0038] 5'-CAGTCAAAACGCCCTCTTGGGCGGTGGACA[P-dT]G[THF][B-dT]GAGATTCAATATTAATGA-C3 Spacer-3';

[0039] Wherein, P is a fluorescent group, THF is tetrahydrofuran, and B is a quenching group; the fluorescent groups of the probes used in the composition to detect different pathogens are different from each other and do not interfere with each other.

[0040] It should be noted that the "P" above only refers to the fluorescent group and does not limit the fluorescent groups of the above four probes to be the same, and the same applies to "B". For example, the P of probe SEQ ID NO:3 can be any one of ATTO425, Quasar705, FAM, HEX, ROX and CY5; the P of probe SEQ ID NO:6 can be any one of ATTO425, Quasar705, FAM, HEX, ROX and CY5; the P of probe SEQ ID NO:9 can be any one of ATTO425, Quasar705, FAM, HEX, ROX and CY5; the P of probe SEQ ID NO:12 can be any one of ATTO425, Quasar705, FAM, HEX, ROX and CY5.

[0041] In this invention, "different and non-interfering" means that each probe in the composition uses a different fluorescent group, and these groups do not affect each other's detection; that is, different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values, allowing for the selection of different channels and thus preventing mutual interference.

[0042] Furthermore, in some embodiments, the composition of the present invention may simultaneously comprise one or more sets of the primer and probe pairs described above. In the present invention, a "set" refers to a mutually matched upstream primer, downstream primer, and probe for detecting a target.

[0043] The compositions of this invention can be arbitrarily combined to detect any combination of four corresponding targets. Those skilled in the art can combine them as needed, determining which targets to detect by combining the primer and probe pairs corresponding to those targets. All such combinations are included in this invention.

[0044] For example, it may include any 3 sets of the above 4 sets of primers and probes, any 2 sets of the above 4 sets of primers and probes, or any 1 set of the above 4 sets of primers and probes.

[0045] In some specific embodiments, the compositions of the present invention are used in fluorescent RPA technology.

[0046] In one specific implementation, the fluorescent group of the classical swine fever virus probe is FAM, the fluorescent group of the psittacosis chlamydia probe is HEX, the fluorescent group of the porcine reproductive and respiratory syndrome virus probe is ROX, and the fluorescent group of the porcine circovirus type II probe is CY5.

[0047] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.

[0048] Furthermore, the 3' end of the probe is a C3 spacer.

[0049] In one specific embodiment, each component of the composition of the present invention is contained in a separate package.

[0050] In one specific embodiment, the components of the composition of the present invention are contained in a single package.

[0051] Furthermore, the components of the composition of the present invention exist in a mixed form.

[0052] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for detecting and differentiating porcine reproductive disorder pathogens, wherein the porcine reproductive disorder pathogen is one or more of classical swine fever virus, psittacosis chlamydia, porcine reproductive and respiratory syndrome virus, and porcine circovirus type II.

[0053] Thirdly, the present invention provides a kit for detecting and differentiating porcine reproductive tract pathogens, the kit comprising the composition of the present invention as described above.

[0054] Furthermore, the kit also includes a reaction buffer and magnesium ions.

[0055] In one specific implementation, the magnesium ions are derived from magnesium acetate.

[0056] Furthermore, the reaction buffer comprises Tris-HCl, dNTPs, KCl, glycerol, and proclin 300.

[0057] Furthermore, the reaction buffer comprises recombinase, reverse transcriptase, DNA polymerase, single-stranded binding protein, and exonuclease III.

[0058] In one specific implementation, the reaction buffer comprises Tris-HCl, dNTPs, KCl, glycerol, proclin 300, recombinase, reverse transcriptase, DNA polymerase, single-stranded binding protein, and exonuclease III.

[0059] Furthermore, the kit also includes negative and positive controls.

[0060] In one specific implementation, the negative control is at least one of DEPC H2O and physiological saline. The positive control is at least one of classical swine fever virus, Chlamydia psittaci, porcine reproductive and respiratory syndrome virus, a cloning plasmid of the target gene of porcine circovirus type II, a standard, and a pseudovirus.

[0061] Furthermore, the kit also includes at least one of a nucleic acid release reagent and a nucleic acid extraction reagent.

[0062] Furthermore, the system of the kit for RPA detection, in 50 μL, includes the following components: 20 μl to 30 μl of reaction buffer, 0.5 μl to 1 μl / strip of 20 μM upstream primer, 0.5 μl to 1 μl / strip of 20 μM downstream primer, 0.25 μl to 0.5 μl / strip of 20 μM probe, 5 μl to 15 μl of template, and 2.2 μl to 2.7 μl of 280 mmol / L magnesium acetate.

[0063] Furthermore, the system for RPA detection using the kit, in 50 μL volume, includes the following components: 25 μL reaction buffer, 1 μL / 20 μM upstream primer, 1 μL / 20 μM downstream primer, 0.375 μL / 20 μM probe, 10 μL template, and the remainder is made up with DEPC H2O. Before starting the experiment, 2.5 μL of magnesium acetate (280 mmol / L) is added to initiate the experiment.

[0064] Fourthly, a method for preparing a diagnostic reagent for porcine reproductive disorder pathogens using a composition is provided, wherein the detection includes the following steps:

[0065] S1. Extract or release the nucleic acid from the sample to be tested;

[0066] S2. Perform a fluorescent RPA reaction on the nucleic acid obtained in step S1 using the composition of the present invention as described above or the kit of the present invention as described above.

[0067] S3. Obtain and analyze the results.

[0068] In this invention, the samples used for detection can be blood, excrement, vaginal swabs, oral and nasal swabs, breast milk, semen, etc., but are not limited to these.

[0069] Furthermore, the reaction conditions for the fluorescent RPA reaction are as follows:

[0070] Amplification at 42℃ for 30 seconds, for a total of 40 cycles. Attached Figure Description

[0071] Figure 1 The image shows the detection results of the composition of the present invention.

[0072] Figures 2-5 The graphs show the sensitivity test results of the compositions of the present invention (in order: classical swine fever virus, Chlamydia psittaci, porcine reproductive and respiratory syndrome virus, and porcine circovirus type II).

[0073] Figure 6 This is a graph showing the specificity detection results of the composition of the present invention;

[0074] Figures 7-8 This is a diagram showing the comparative single-sample test results of this invention;

[0075] Figure 9 This is a diagram showing the comparative test results of the present invention. Detailed Implementation

[0076] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0077] Example 1: Primers and probes used in this invention

[0078] The primers and probes used in this invention are shown in Table 1:

[0079] Table 1:

[0080]

[0081]

[0082] The fluorescent group of the probe for classical swine fever virus is FAM; the fluorescent group of the probe for Chlamydia psittaci is HEX; the fluorescent group of the probe for porcine reproductive and respiratory syndrome virus is ROX; and the fluorescent group of the probe for porcine circovirus type II is CY5.

[0083] SEQ ID NO:3, after being marked and modified, is:

[0084] 5'-CGGGGGTCGCTAGGGTGAAATCACACCAGG[FAM-dT]G[TH F][BHQ1-dT]GGGGGGTACGACCTGA-C3 Spacer-3';

[0085] SEQ ID NO:6, after being marked and modified, is:

[0086] 5'-ACCGTTAATTGCAGATCCTCGCCAGGTAACAAA[HEX-dT][T HF]G[BHQ1-dT]GCGGGCATTCGTTT-C3 Spacer-3';

[0087] SEQ ID NO:9, after being marked and modified, is:

[0088] 5'-TTGAAAAGCCTCGTGTTGGGTGGCAGAAGAGC[ROX-dT][T HF][BHQ1-dT]CAAACAGGGAGTGGT-C3 Spacer-3';

[0089] SEQ ID NO:12, after being marked and modified, is:

[0090] 5'-CAGTCAAAACGCCCTCTTGGGCGGTGGACA[CY5-dT]G[THF][BHQ1-dT]GAGATTCAATATTAATGA-C3 Spacer-3';

[0091] Example 2: Method for detecting pathogens causing porcine reproductive disorders

[0092] The test samples were nasal and oral swab samples. Nucleic acid was extracted from the test samples using the Sansure Biotech magnetic bead method. The nucleic acid extraction was performed in the sample processing room as follows:

[0093] 1.1 Take out the pre-aliquoted 96-well plate reagent from the kit, equilibrate to room temperature, shake off any liquid that may adhere to the sealing membrane or well walls of the deep well plate to the bottom, and let stand for 1–3 minutes.

[0094] 1.2 Remove the sealing film; add the sample and proteinase K to wells A2-H2 and A8-H8 of the deep well plate; the recommended sample volume is 200 μL / well and proteinase K volume is 40 μL / well.

[0095] 1.3 Turn on the extractor, place the deep hole plate and magnetic sleeve into the corresponding positions on the instrument, and start the corresponding program.

[0096] 1.4 The extraction process ends after approximately 15 to 30 minutes.

[0097] 1.5 Remove the deep well plate and transfer the nucleic acid for later use.

[0098] The real-time fluorescence RPA reaction system is shown in Table 2:

[0099] Table 2:

[0100]

[0101] Reaction amplification program: 42℃, 30S amplification, for a total of 40 cycles.

[0102] The main components of the above reaction buffer are Tris-HCl, dNTPs, KCl, glycerol, proclin300, recombinase, reverse transcriptase, DNA polymerase, single-stranded binding protein, and exonuclease III.

[0103] Results analysis:

[0104] (1) The target detection signal is the amplification curve of the FAM, HEX, ROX and CY5 channels;

[0105] (2) Baseline settings: The baseline is generally set to 3-15 cycles, which can be adjusted according to the actual situation. The adjustment principle is: select a region where the fluorescence signal is relatively stable before exponential amplification, avoid signal fluctuations at the beginning of fluorescence acquisition, and reduce the endpoint by 1-2 cycles compared to the earliest sample Ct that shows exponential amplification. Threshold settings: The principle is to set the threshold line so that it just exceeds the highest point of the normal negative control.

[0106] (3) Determination of positive and negative results, and interpretation of test results are shown in Table 3:

[0107] Table 3:

[0108]

[0109]

[0110] Example 3: Detection results of the composition of the present invention on the test sample

[0111] The primers and probes shown in Example 1 were used to detect and differentiate a mixed sample of classical swine fever virus, Chlamydia psittaci, porcine reproductive and respiratory syndrome virus, and porcine circovirus type II, according to the method in Example 2. The results are as follows: Figure 1 As shown, by Figure 1 It is understood that the composition of the present invention can simultaneously detect and differentiate classical swine fever virus, psittacosis chlamydia, porcine reproductive and respiratory syndrome virus, and porcine circovirus type II.

[0112] Example 4: Sensitivity of the composition of the present invention

[0113] The LOD (sensitivity) of each virulence gene was measured. The results showed that the composition could accurately detect samples with a density as low as 1000 copies / mL in each channel, indicating that the sensitivity of the composition of this invention is 1000 copies / mL. Figures 2-5 As shown.

[0114] Example 5: Specificity of the composition of the present invention

[0115] Cross-reactivity was assessed using nucleic acid samples from porcine pseudorabies virus, porcine parvovirus, Japanese encephalitis virus, and porcine circovirus types 1 and 3, which exhibited homology in their nucleic acid sequences and were likely to induce similar or identical clinical symptoms. Specific detection was performed, and the results were as follows: Figure 6 As shown in the figure, its specificity is good.

[0116] Comparative Example 1: Other primers and probes designed in this invention that do not perform well.

[0117] Due to the principle of complementary base pairing, primers and / or probes can form dimers, but this probability is low and can be eliminated at the initial design stage. However, when detecting multiple pathogens together, there are numerous primers and probes, and dimers can easily form between primers, probes, or between different primers and probes. To ensure the conservation of the design (conservatism is crucial for detection accuracy) while also considering the mutual interference between different primers and probes, careful primer and probe design is required.

[0118] Therefore, the inventors also designed other primers and probes (primers and probes for detecting classical swine fever virus, and primers and probes for detecting Chlamydia psittaci, sequences not shown) to form different detection systems. The aforementioned primers and probes for detecting classical swine fever virus and Chlamydia psittaci were used individually to detect classical swine fever virus and Chlamydia psittaci, respectively, with the following results: Figures 7-8 As shown. By Figures 7-8It can be seen that some targets (FAM channel for classical swine fever virus target, HEX channel for Chlamydia psittaci target) showed good performance when detected individually. When the above two sets of primers and probes were combined with primers and probes (SEQ ID NO: 7-12) for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type II (PCVII), the results were as follows: Figure 9 As shown, by Figure 9 It can be seen that the performance is reduced when using combined detection, the Ct value is delayed, the fluorescence increment decreases, and the overall detection effect is poor.

[0119] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A composition for detecting porcine reproductive disorder pathogens, characterized in that, Includes at least one set of primers and probes for detecting the following pathogens: The upstream and downstream primers and probes for detecting classical swine fever virus are shown in SEQ ID NO: 1-3; The upstream and downstream primers and probes for detecting Chlamydia psittaci, as shown in SEQ ID NO: 4-6; The upstream and downstream primers and probes for detecting porcine reproductive and respiratory syndrome virus are shown in SEQ ID NO: 7-9; The upstream and downstream primers and probes for detecting porcine circovirus type II are shown in SEQ ID NO: 10-12.

2. The composition according to claim 1, characterized in that, The composition includes upstream and downstream primers and probes as shown in SEQ ID NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9 and SEQ ID NO: 10-12.

3. The composition according to claim 1 or 2, characterized in that, The probe is labeled with a fluorescent group, a quenching group and a 3' C3-spacer, and modified with tetrahydrofuran.

4. The composition according to claim 3, characterized in that, The probe SEQ ID NO:3, after being marked and modified, is: 5'-CGGGGGTCGCTAGGGTGAAATCACACCAGG[P-dT]G[THF][B-dT]GGGGGGTACGACCTGA-C3Spacer-3'; The probe SEQ ID NO: 6, after being marked and modified, is: 5'-ACCGTTAATTGCAGATCCTCGCCAGGTAACAAA[P-dT][THF]G[B-dT]GCGGGCATTCGTTT-C3Spacer-3'; The probe SEQ ID NO: 9, after being marked and modified, is: 5'-TTGAAAAGCCTCGTGTTGGGTGGCAGAAGAGC[P-dT][THF][B-dT]CAAACAGGGAGTGGT-C3Spacer-3'; The probe SEQ ID NO: 12, after being marked and modified, is: 5'-CAGTCAAAACGCCCTCTTGGGCGGTGGACA[P-dT]G[THF][B-dT]GAGATTCAATATTAATGA-C3Spacer-3'; Wherein, P is a fluorescent group, THF is tetrahydrofuran, and B is a quenching group; the fluorescent groups of the probes used in the composition to detect different pathogens are different from each other and do not interfere with each other.

5. The composition according to claim 4, characterized in that, The probe SEQ ID NO:3, after being marked and modified, is: 5'-CGGGGGTCGCTAGGGTGAAATCACACCAGG[FAM-dT]G[TH F][BHQ1-dT]GGGGGGTACGACCTGA-C3 Spacer-3'; The probe SEQ ID NO: 6, after being marked and modified, is: 5'-ACCGTTAATTGCAGATCCTCGCCAGGTAACAAA[HEX-dT][THF]G[BHQ1-dT]GCGGGGCATTCGTTT-C3 Spacer-3'; The probe SEQ ID NO: 9, after being marked and modified, is: 5'-TTGAAAAGCCTCGTGTTGGGTGGCAGAAGAGC[ROX-dT][THF][BHQ1-dT]CAAACAGGGAGTGGT-C3 Spacer-3'; The probe SEQ ID NO: 12, after being marked and modified, is: 5'-CAGTCAAAACGCCCTCTTGGGCGGTGGACA[CY5-dT]G[THF][BHQ1-dT]GAGATTCAATATTAATGA-C3 Spacer-3'.

6. The composition according to any one of claims 1 to 5, characterized in that, The components of the composition exist in a mixed form.

7. The use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting porcine reproductive disorder pathogens, characterized in that, The pathogen causing porcine reproductive disorders is one or more of classical swine fever virus, Chlamydia psittaci, porcine reproductive and respiratory syndrome virus, and porcine circovirus type II.

8. A kit for detecting porcine reproductive disorder pathogens, characterized in that, The kit comprises the composition according to any one of claims 1 to 6.

9. The reagent kit according to claim 8, characterized in that, The kit also includes a reaction buffer comprising Tris-HCl, dNTPs, KCl, glycerol, proclin 300, recombinase, reverse transcriptase, DNA polymerase, single-stranded binding protein, and exonuclease III.

10. The use of a composition for preparing a diagnostic reagent for porcine reproductive disorder pathogens, characterized in that, The detection includes the following steps: S1. Extract or release the nucleic acid from the sample to be tested; S2. Perform fluorescent RPA analysis on the nucleic acid obtained in step S1 using the composition as described in any one of claims 1 to 6 or the kit as described in claim 8 or 9; S3. Obtain and analyze the results.

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