A primer probe composition, kit and method for simultaneously detecting avian infectious bronchitis virus replicative and general types based on fluorescence type RPA

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to a primer probe composition, kit and method for simultaneously detecting avian infectious bronchitis virus (IBV) reproductive type and general type based on fluorescence type RPA. The present application designs IBV reproductive type primer and probe based on specific variation region related to reproductive type in S1 gene, and designs IBV general type primer and probe based on conservative region such as M gene, N gene or 3'UTR. The present application synthesizes the primer and probe of IBV reproductive type and general type, and utilizes RPA technology to establish a fluorescence probe detection method which can rapidly, sensitively and specifically identify IBV reproductive type and general type under constant temperature condition. The method is also suitable for establishing test strip detection method. The kit of the present application has the advantages of simple operation, no need of precise instrument, short detection time, high sensitivity, strong specificity and the like, and is suitable for on-site rapid screening in farms, and provides strong technical support for epidemic prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically relating to a primer-probe composition, kit, and method for simultaneously detecting the reproductive and universal forms of avian infectious bronchitis virus based on fluorescent RPA. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious disease caused by the infectious bronchitis virus (IBV). IBV belongs to the Coronaviridae family and primarily targets the respiratory, urinary, and reproductive systems of poultry, long hindering the healthy development of the poultry industry. Clinically, based on the differences in the major organs affected by different IBV strains, it is often classified into several types, including respiratory, renal, proventricular, and reproductive types. Among these, the reproductive type of IBV primarily affects the reproductive system of chickens, causing permanent damage to the oviduct, resulting in "false hens" after sexual maturity, a significant drop in egg production, or even complete loss of egg production, causing severe economic losses to the poultry industry.

[0003] Currently, multiple genotypes and serotypes of IBV coexist. Different strains exhibit varying tropisms for different organs, leading to complex and diverse clinical symptoms and posing significant challenges to early diagnosis and control of the disease.

[0004] Current detection methods for IBV mainly include traditional methods and molecular detection methods. Traditional methods (virus isolation, serological testing) are cumbersome, time-consuming, and lack sensitivity, making them unsuitable for rapid diagnosis. Among existing molecular detection technologies, conventional PCR relies on precise temperature control instruments and has a long detection time, failing to meet the needs of rapid on-site testing; real-time quantitative PCR (qPCR) requires expensive equipment and demands highly skilled operators, making it difficult to promote its application in grassroots farms. While loop-mediated isothermal amplification (LAMP) technology achieves isothermal amplification, it suffers from high false positive rates and complex primer design.

[0005] In recent years, recombinase polymerase amplification (RPA) technology, as a novel isothermal nucleic acid amplification technique, has attracted widespread attention from researchers. RPA technology boasts advantages such as ease of operation, rapid amplification speed, high specificity, and high sensitivity. It eliminates the need for sophisticated instruments and can complete nucleic acid detection under isothermal conditions (25–45°C). RPA technology is considered one of the most promising rapid molecular diagnostic tools currently available, with broad application prospects. However, there are currently no reports of applying RPA technology to simultaneously differentiate between reproductive and general IBV.

[0006] Therefore, establishing a highly sensitive, highly specific, easy-to-operate system that can rapidly identify the reproductive and general types of IBV (i.e., a universal IBV test that does not differentiate between types) is of great clinical significance for the early diagnosis, epidemic monitoring, and prevention and control of IBV infection.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and propose a primer-probe composition, kit, and method for simultaneously detecting the reproductive and universal forms of avian infectious bronchitis virus (IBV) based on fluorescent RPA. The IBV genome is highly variable; the M gene is the most conserved gene among IBV strains, and the S1 gene is the most important genotyping gene for distinguishing different IBV strains, and also the most variable segment in the entire genome. Therefore, this invention designs primers and probes for detecting the IBV reproductive form based on specific variant regions related to the reproductive form in the S1 gene, and primers and probes for detecting the IBV universal form based on conserved regions such as the M gene, N gene, or 3'UTR. This invention synthesizes primers and probes for both the IBV reproductive and universal forms and utilizes RPA technology to establish a fluorescent probe detection method that can rapidly, sensitively, and specifically identify the IBV reproductive and universal forms simultaneously under isothermal conditions; it is also applicable to establishing a test strip detection method. The reagent kit of this invention has the advantages of simple operation, no need for precision instruments, short detection time, high sensitivity and strong specificity. It is suitable for rapid on-site screening in farms and provides strong technical support for the identification of reproductive and general types of IBV and for the prevention and control of the epidemic.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a primer-probe composition for simultaneous detection of reproductive and universal types of avian infectious bronchitis virus based on fluorescent RPA. The primer-probe composition is used to simultaneously detect reproductive and universal types of avian infectious bronchitis virus, wherein the universal type of avian infectious bronchitis virus includes respiratory, renal, proventricular, and reproductive types of infectious bronchitis virus. The composition for detecting the reproductive avian infectious bronchitis virus includes a first upstream primer, a first downstream primer, and a first probe. The sequence of the first upstream primer is shown in SEQ ID NO:1, the sequence of the first downstream primer is shown in SEQ ID NO:2, and the sequence of the first probe is shown in SEQ ID NO:3. The composition for detecting the universal avian infectious bronchitis virus includes a second upstream primer, a second downstream primer, and a second probe. The sequence of the second upstream primer is shown in SEQ ID NO:7, the sequence of the second downstream primer is shown in SEQ ID NO:8, and the sequence of the second probe is shown in SEQ ID NO:9.

[0010] Furthermore, the first probe includes a fluorescent group and a quenching group from its 5' end to the alkyl carbon spacer end, and tetrahydrofuran (THF) is included between the fluorescent group and the quenching group; The second probe includes a fluorescent group and a quenching group from the 5' end to the 3' end, wherein the fluorescent group and the quenching group are sequentially composed of 5 bases, tetrahydrofuran (THF), and 5 bases. The fluorescent group labeled on the first probe is different from the fluorescent group labeled on the second probe.

[0011] Furthermore, the fluorescent group labeled on the first probe is selected from any one of FAM, HEX, TET, JOE, ROX, and Cy5; The quenching group on the first probe is selected from any one of BHQ1, BHQ2, and BHQ3; The alkyl carbon spacer is selected from any one of C3 Spacer, C2 Spacer, and C4 Spacer.

[0012] Furthermore, the fluorescent group labeled on the second probe is selected from any one of FAM, HEX, TET, JOE, ROX, and Cy5; The quenching group on the second probe is selected from any one of BHQ1, BHQ2, and BHQ3.

[0013] Preferably, the primer-probe composition described above can be selected from the following: The SEQ ID NO:1 is: 5'-CAAGATTTTGAGTGCGTATTGCTTGTGTTTA-3'; The SEQ ID NO:2 is: 5'-ACCTTGACCTACTCTACCATAGATATTATA-3'; The SEQ ID NO:3 is: 5'-TCACTGCTAATTTTGTAAATAA[dT-FAM][THF][dT-BHQ1]GGTTTTAGATAGGTGTGTTG-C3 spacer; Optionally, said SEQ ID NO:3 is: 5'-TCACTGCTAATTTTGTAAATAA[dT-HEX][THF][dT-BHQ2]GGTTTTAGATAGGTGTGTTG-C3 spacer; 5'-TCACTGCTAATTTTGTAAATAA[dT-TET][THF][dT-BHQ3]GGTTTTAGATAGGTGTGTTG-C3 spacer; 5'-TCACTGCTAATTTTGTAAATAA[dT-JOE][THF][dT-BHQ1]GGTTTTAGATAGGTGTGTTG-C4 spacer; 5'-TCACTGCTAATTTTGTAAATAA[dT-ROX][THF][dT-BHQ2]GGTTTTAGATAGGTGTGTTG-C2 spacer; 5'-TCACTGCTAATTTTGTAAATAA[dT-Cy5][THF][dT-BHQ1]GGTTTTAGATAGGTGTGTTG-C3 spacer.

[0014] Said SEQ ID NO:7 is: 5'-TGTGAACCAGACCACTTGCCTAAAGA-3'; Said SEQ ID NO:8 is: 5'-ACTGGCGAGCTAGAAAGTGTAGCAACA-3'; Said SEQ ID NO:9 is: 5'-ACTGGTGACCAAAGCG[dT-ROX]GAAAT[THF]AAGAA[dT-BHQ2]AAGGTTTGCTACATT-Spacer-3'.

[0015] Optionally, said SEQ ID NO:9 is: 5'-ACTGGTGACCAAAGCG[dT-TET]GAAAT[THF]AAGAA[dT-BHQ1]AAGGTTTGCTACATT-Spacer-3'; 5'-ACTGGTGACCAAAGCG[dT-HEX]GAAAT[THF]AAGAA[dT-BHQ3]AAGGTTTGCTACATT-Spacer-3'; 5'-ACTGGTGACCAAAGCG[dT-JOE]GAAAT[THF]AAGAA[dT-BHQ3]AAGGTTTGCTACATT-Spacer-3'; 5'-ACTGGTGACCAAAGCG[dT-Cy5]GAAAT[THF]AAGAA[dT-BHQ3]AAGGTTTGCTACATT-Spacer-3'; 5'-ACTGGTGACCAAAGCG[dT-FAM]GAAAT[THF]AAGAA[dT-BHQ3]AAGGTTTGCTACATT-Spacer-3'.

[0016] In a second aspect, the present invention also provides a kit for simultaneous detection of reproductive and universal types of avian infectious bronchitis virus based on fluorescent RPA, based on the primer and probe composition described in the first aspect. The kit includes a first primer and probe mixture for detecting reproductive avian infectious bronchitis virus, a second primer and probe mixture for detecting universal avian infectious bronchitis virus, an RPA amplification premix enzyme, and a nucleic acid release agent. The first primer-probe mixture comprises a first upstream primer, a first downstream primer, and a first probe in a molar ratio of 1:1:1. The second primer-probe mixture comprises a second upstream primer, a second downstream primer, and a second probe in a molar ratio of 1:1:1.

[0017] Furthermore, the kit also includes a positive control and a negative control.

[0018] Thirdly, the present invention also provides a method for using the kit for simultaneous detection of reproductive and universal types of avian infectious bronchitis virus based on fluorescent RPA as described above, specifically including the following steps: Step 1: Extract nucleic acid from the sample to be tested; Step 2: Using nucleic acid as a template, establish an RPA reaction system and perform the RPA reaction, followed by fluorescence detection; Step 3: Analyze the fluorescence signal curve to distinguish whether the avian infectious bronchitis virus in the sample is a reproductive strain or a universal strain.

[0019] Specifically, in step 3, if a fluorescence signal curve appears for the corresponding channel, it indicates that the sample contains the corresponding type of IBV (reproductive or universal); if no fluorescence signal curve appears, it indicates that the sample does not contain the corresponding type of IBV.

[0020] Furthermore, in step 2, the establishment of the RPA reaction system involves mixing the RPA amplification premixed enzyme, an equal volume of the first primer-probe mixture and the second primer-probe mixture, and nucleic acid.

[0021] Furthermore, in step 2, the conditions for the RPA reaction are: temperature 37–42°C, reaction time 5–20 minutes.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) Fast and efficient: The present invention uses RPA technology to achieve isothermal amplification. The reaction can be completed under isothermal conditions of 37-42℃, and the detection time is only 5-20 minutes, which is significantly better than the detection time of traditional PCR (requires 2-3 hours) and qPCR (requires 1-2 hours), greatly improving the detection efficiency.

[0023] (2) Simple operation: The present invention uses a nucleic acid release agent to treat the sample, which eliminates the need for a nucleic acid purification step, has stronger tolerance to sample inhibitors, and simplifies pretreatment. At the same time, the RPA reaction does not require sophisticated temperature-controlled instruments and a strict laboratory environment; ordinary constant temperature equipment can meet the requirements, making it very suitable for field application in grassroots farms.

[0024] (3) High sensitivity: The fluorescent detection primers of this invention have a detection sensitivity of up to 10⁻¹ ng / μL for both reproductive and general IBV types, and the detection sensitivity of the test strip primers can reach 10⁻¹ ng / μL, which can meet the rapid detection needs of early IBV infection.

[0025] (4) High specificity: The present invention designs specific primers and probes for the IBV reproductive S1 gene, the M gene, N gene or 3'UTR gene that are conserved for the IBV universal type, and there is no cross-reaction with other common avian pathogens (such as Newcastle disease virus, avian influenza virus, etc.), which ensures the accuracy of the detection results.

[0026] (5) Visual interpretation: The results can be interpreted intuitively through the fluorescence signal curve, without the need for complicated electrophoresis analysis or sequencing steps, which reduces the difficulty and subjectivity of result interpretation.

[0027] In summary, the primer-probe composition or kit of the present invention can solve the defects of existing IBV detection technologies, such as cumbersome operation, long time consumption, expensive instruments, inability to be used for rapid on-site detection, and inability to quickly distinguish between reproductive and general types of IBV. Attached Figure Description

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

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 This is a graph showing the screening results of the upstream primer for IBV reproductive types in Example 1 of this invention; Figure 2 This is a graph showing the screening results of IBV reproductive probes in Example 1 of the present invention; Figure 3 This is a diagram showing the screening results of IBV reproductive downstream primers in Example 1 of the present invention; Figure 4 This is a graph showing the screening results of the IBV universal upstream primer in Example 1 of the present invention; Figure 5 This is a graph showing the screening results of the IBV universal probe in Embodiment 1 of the present invention; Figure 6 This is a graph showing the screening results of the IBV universal downstream primer in Example 1 of the present invention; Figure 7 This is a diagram showing the typing results of different infectious bronchitis virus strains (positive controls for IBV reproductive type and IBV universal type) tested in this invention. Figure 8 This is a graph showing the results of the specificity test of this invention; Figure 9 This is a graph showing the results of the sensitivity test (IBV reproductive type) of this invention; Figure 10 This is a graph showing the results of the sensitivity test of the present invention (IBV general type). Detailed Implementation

[0031] Exemplary embodiments will now be described in detail. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples consistent with some aspects of the invention as detailed in the appended claims.

[0032] Example 1 This embodiment provides the design and screening of specific primers and probes. Based on the sequence characteristics of the reproductive type-specific gene sequence (S1 gene) and the universal conserved gene sequence (M gene) of avian infectious bronchitis virus (IBV), the following primers and probes were designed and synthesized: The following are the sequence alignments of the S1 gene from 9 reproductive bronchitis strains (genbank numbers) and the primers and probes used for screening: 1) Upstream primers such as Figure 1 Common gray bases: 2) Probes such as Figure 2 Common gray bases: 3) Downstream primers such as Figure 3 Gray base portion: (1) Fluorescent primers and probes for detecting IBV reproductive types: First upstream primer YG-IBV(GVI)-S1-F: 5'-CAAGATTTTGAGTGCGTATTGCTTGTGTTTA-3' (SEQ ID NO:1); The first downstream primer YG-IBV(GVI)-S1-R: 5'-ACCTTGACCTACTCTACCATAGATATTATA-3' (SEQ ID NO:2); The first probe YG-IBV(GVI)-S1-probe: 5'-TCACTGCTAATTTTGTAAATAA[dT-FAM][THF][dT-BHQ1]GGTTTTAGATAGGTGTGTTG-C3spacer (SEQ ID NO:3); The following are the sequence alignment results of the M gene from genbank numbers of 6 strains of the common bronchogenic strain: 1) Upstream primers such as Figure 4 Gray base portion: 2) Probes such as Figure 5 Gray base portion: 3) Downstream primers such as Figure 6 Gray base portion: (2) Universal fluorescent primers and probes for detecting IBV: The second upstream primer is YG-IBV(uni)-MF: 5'-TGTGAACCAGACCACTTGCCTAAAGA-3' (SEQ ID NO:7). The second downstream primer, YG-IBV(uni)-MR, is 5'-ACTGGCGAGCTAGAAAGTGTAGCAACA-3' (SEQ ID NO: 8). The second probe YG-IBV(uni)-M-probe: 5'-ACTGGTGACCAAAGCG[dT-ROX]GAAAT[THF]AAGAA[dT-BHQ1]AAGGTTTGCTACATT-Spacer-3' (SEQ ID NO: 9) Among them, the fluorescent group of the IBV(GVI) probe is FAM, the fluorescent group of the IBV(uni) probe is ROX; BHQ1 is a quenching group; THF is tetrahydrofuran; and C3 Spacer is an alkyl carbon spacer.

[0033] Example 2 This embodiment provides a kit for simultaneous detection of reproductive and universal types of avian infectious bronchitis virus based on fluorescent RPA. The kit includes: The first primer-probe mixture for detecting reproductive avian infectious bronchitis virus (including the first upstream primer, the first downstream primer, and the first probe from Example 1, with a molar ratio of 1:1:1). The second primer-probe mixture for detecting universal avian infectious bronchitis virus (including the second upstream primer, the second downstream primer, and the second probe from Example 1, with a molar ratio of 1:1:1). RPA amplification premixed enzyme; wherein, the RPA amplification premixed enzyme can be selected in two forms, specifically lyophilized powder and liquid premixed solution. If lyophilized powder is selected, it needs to be reconstituted. Both are commonly used in detection experiments.

[0034] Nucleic acid release agent (derived from potentially pathogenic organisms); Positive control.

[0035] Example 3 This embodiment provides a method for using a kit for simultaneous detection of reproductive and general types of avian infectious bronchitis virus based on fluorescent RPA. The kit from Example 2 is used to detect the virus using the RPA dual fluorescent probe detection method, specifically including the following steps: Step 1: Extraction of viral nucleic acid from the sample to be tested 1.1 Prepare reagents Remove the nucleic acid release agent and add it to the nucleic acid release kit. Equilibrate to room temperature, mix well, and set aside.

[0036] 1.2 Sample Processing Collect throat swabs from chickens to obtain the collection fluid. Use a rapid nucleic acid release agent (Easy-to-extract) to rapidly extract the collection fluid, following these steps: 1) Add 200 μl of the sample to be tested to a 1.5 ml centrifuge tube and centrifuge at 12,000 rpm for 10 min.

[0037] 2) Remove the supernatant, add 50 μl of nucleic acid release agent to the precipitate, vortex to mix, let stand for 10 min, centrifuge at 12,000 rpm for 1 min, and take 1~5 μl of supernatant for amplification reaction.

[0038] Step 2: Establishment of the isothermal amplification reaction system Using the nucleic acid (RNA) extracted in step 1 as a template, an RPA reaction was performed using the primer and probe composition designed in Example 1.

[0039] 2.1 Establishing the RPA reaction system Taking a 20 μL reaction system as an example, add the following to each reaction well: 16 μL of RPA amplification premixed enzyme; 1 μL of the first primer-probe mixture; 1 μL of the second primer-probe mixture; 2 μL of nucleic acid template.

[0040] The components of the RPA amplification premixed enzyme are: dry powder polymerase, 10 μL of Rehydration Buffer (2×), 2 μL of starter (10×), and 4 μL of enzyme-free water.

[0041] 2.2 RPA Reaction Procedure Set the temperature of the isothermal amplification fluorescence instrument to 37℃ (with the heated lid function turned off or the lid temperature set to 42℃), and the reaction time to 10–20 minutes. Fluorescence changes can be monitored continuously during this process to determine whether the nucleic acid test is positive.

[0042] Step 3: Result Interpretation Positive result: The appearance of a fluorescence signal curve indicates the presence of the target RNA template in the sample. Negative result: No fluorescence signal curve appeared, which is basically consistent with the negative control, indicating that the target RNA template was not detected in the sample.

[0043] The above applications or methods may be non-disease diagnostic applications or methods. The above applications or methods may not be directly aimed at obtaining disease diagnosis results or health status of living human or animal bodies. The sample to be tested may be a sample from a non-living human or animal body, or it may be an ex vivo sample.

[0044] To verify the effectiveness of the present invention, the following experiments were conducted: 1) Amplification of universal and reproductive strains Example 1: Reactions were performed on positive controls for IBV reproductive type and IBV general type, and the results are as follows... Figure 7 As shown, the positive controls are IBV (GVI) and IBV (general type). The positive controls are valid and the results are positive. The negative controls are consistent with the baseline and do not jump.

[0045] 2) Specificity test Example 2: Using the optimal conditions of the RPA dual fluorescent probe detection method constructed in Example 3, the positive control of the present invention was detected using IBV reproductive type (GVI), IBV general type, Newcastle disease virus, avian influenza virus, and avian adenovirus as nucleic acid templates.

[0046] See results Figure 8Both the reproductive and general forms of IBV were positive, indicating an upward trend. The other avian influenza viruses, Newcastle disease virus, and adenovirus were consistent with the baseline and did not rise, indicating negative results.

[0047] 3) Sensitivity test Recombinant plasmids containing IBV germline-specific genes and IBV universal conserved genes were constructed. The concentration of the extracted plasmids was determined, and they were serially diluted 10-fold with enzyme-free water. 4 ~10 1 Copies / μL were used to prepare standards. 2 μL of each gradient was taken as a reaction template, and RPA nucleic acid amplification was performed using the optimal reaction system and conditions constructed in Example 3. The results are as follows: Figure 9 and Figure 10 As shown.

[0048] See Figure 9 When the IBV reproductive response template is diluted to 10 4 ~10 1 Clear fluorescence amplification curves were observed when the template nucleic acid concentration was reduced to 10 copies / μL. No obvious amplification curves were observed when the template nucleic acid concentration was reduced to below 10 copies / μL, indicating that the detection limit of this method for IBV germline is 10 copies / μL.

[0049] See Figure 10 When the concentration of the IB V universal reaction template nucleic acid is 10 4 ~10 1 Clear fluorescence amplification curves were observed at template nucleic acid concentrations of 10 copies / μL. No significant amplification curves were observed when the template nucleic acid concentration was reduced to below 10 copies / μL, indicating that the detection limit for the general IBV strain is 10 copies / μL. Sensitivity assays showed that this method can meet the need for rapid early detection of both reproductive and general IBV infections.

[0050] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. 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 the invention.

[0051] It should be understood that the present invention is not limited to the content already described above, and various modifications and changes can be made without departing from its scope. The scope of the present invention is limited only by the appended claims.

Claims

1. A primer-probe composition for simultaneous detection of reproductive and universal forms of avian infectious bronchitis virus based on fluorescent RPA, characterized in that, The primer-probe composition is used to simultaneously detect reproductive and general types of avian infectious bronchitis virus, including respiratory, renal, proventricular, and reproductive types of avian infectious bronchitis virus. The composition for detecting the reproductive avian infectious bronchitis virus includes a first upstream primer, a first downstream primer, and a first probe. The sequence of the first upstream primer is shown in SEQ ID NO:1, the sequence of the first downstream primer is shown in SEQ ID NO:2, and the sequence of the first probe is shown in SEQ ID NO:

3. The composition for detecting the universal avian infectious bronchitis virus includes a second upstream primer, a second downstream primer, and a second probe. The sequence of the second upstream primer is shown in SEQ ID NO:7, the sequence of the second downstream primer is shown in SEQ ID NO:8, and the sequence of the second probe is shown in SEQ ID NO:

9.

2. The primer-probe composition according to claim 1, characterized in that, The first probe includes a fluorescent group and a quenching group from its 5' end to the alkyl carbon spacer end, with tetrahydrofuran (THF) between the fluorescent group and the quenching group; The second probe includes a fluorescent group and a quenching group from the 5' end to the 3' end, wherein the fluorescent group and the quenching group are sequentially composed of 5 bases, tetrahydrofuran (THF), and 5 bases. The fluorescent group labeled on the first probe is different from the fluorescent group labeled on the second probe.

3. The primer-probe composition according to claim 2, characterized in that, The fluorescent group labeled on the first probe is selected from any one of FAM, HEX, TET, JOE, ROX, and Cy5; The quenching group on the first probe is selected from any one of BHQ1, BHQ2, and BHQ3; The alkyl carbon spacer is selected from any one of C3 Spacer, C2 Spacer, and C4 Spacer.

4. The primer-probe composition according to claim 2, characterized in that, The fluorescent group labeled on the second probe is selected from any one of FAM, HEX, TET, JOE, ROX, and Cy5; The quenching group on the second probe is selected from any one of BHQ1, BHQ2, and BHQ3.

5. A kit for simultaneous detection of reproductive and universal forms of avian infectious bronchitis virus based on fluorescent RPA, characterized in that, Based on the primer and probe composition according to any one of claims 1 to 4, the kit comprises a first primer and probe mixture for detecting reproductive avian infectious bronchitis virus, a second primer and probe mixture for detecting universal avian infectious bronchitis virus, RPA amplification premix enzyme, and nucleic acid release agent. The first primer-probe mixture comprises a first upstream primer, a first downstream primer, and a first probe in a molar ratio of 1:1:

1. The second primer-probe mixture comprises a second upstream primer, a second downstream primer, and a second probe in a molar ratio of 1:1:

1.

6. The reagent kit according to claim 5, characterized in that, The kit also includes a positive control and a negative control.

7. A method of using a kit for simultaneous detection of reproductive and universal avian infectious bronchitis virus based on fluorescent RPA as described in claim 5 or 6, characterized in that, Specifically, the following steps are included: Step 1: Extract nucleic acid from the sample to be tested; Step 2: Using nucleic acid as a template, establish an RPA reaction system and perform the RPA reaction, followed by fluorescence detection; Step 3: Analyze the fluorescence signal curve to distinguish whether the avian infectious bronchitis virus in the sample is a reproductive strain or a universal strain.

8. The method of use according to claim 7, characterized in that, In step 2, the establishment of the RPA reaction system involves mixing the RPA amplification premixed enzyme, an equal volume of the first primer-probe mixture and the second primer-probe mixture, and nucleic acid.

9. The method of use according to claim 5, characterized in that, In step 2, the conditions for the RPA reaction are: temperature 37-42℃, reaction time 5-20 minutes.

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