Reagent composition for detecting coronavirus porcine hemagglutination encephalomyelitis virus

Abstract

The invention relates to a reagent composition for detecting coronavirus porcine hemagglutination encephalomyelitis virus (PHEV), and belongs to the technical field of animal coronavirus molecular detection. The problems that an existing PHEV detection method or detection environment is high in requirement, long in consumed time or inaccurate in detection result are solved. The reagent composition disclosed by the invention comprises a primer pair, crRNA (Complementary Ribonucleic Acid) and an ssDNA (Deoxyribose Nucleic Acid) probe, the primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2; the nucleotide sequence of the crRNA is as shown in SEQ ID NO. 3; the nucleotide sequence of the ssDNA probe is TTATT, and two ends of the nucleotide sequence are respectively connected with a fluorophore or biotin. The reagent composition can realize high-sensitivity, rapid and anti-pollution field detection of PHEV.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology of animal coronaviruses, specifically relating to a reagent composition for detecting porcine hemagglutinating encephalomyelitis virus (PHEV), and more particularly to a reagent composition for detecting PHEV based on isothermal amplification combined with CRISPR / Cas12a. Background Technology

[0002] Porcine hemagglutinating encephalomyelitis (PHE) is an acute infectious disease primarily affecting suckling piglets, caused by porcine hemagglutinating encephalomyelitis virus (PHEV), a member of the coronavirus family. The disease occurs sporadically or endemically, especially in newborn piglets. PHEV is mainly transmitted through the respiratory and digestive tracts, with carrier sows and infected piglets being the primary sources of infection. The virus replicates in neurons, tonsils, respiratory tract, and intestinal epithelial cells, and is shed through nasal secretions and feces. The disease primarily affects piglets under 3 weeks of age, especially those aged 7-14 days, with mortality rates reaching 20%-100%. Susceptibility and mortality decrease significantly with age. Adult pigs often present with a subclinical course after infection but can become long-term carriers, continuously shedding the virus into the environment. The introduction of carrier breeding pigs is one of the main causes of PHE outbreaks in pig herds.

[0003] Reverse transcription recombinase-mediated strand substitution amplification (RT-RAA) is a rapid, isothermal nucleic acid amplification technique that primarily relies on reverse transcriptase, recombinase, single-strand binding proteins, and DNA polymerase. First, reverse transcriptase reverse-transcribes RNA into cDNA. The recombinase binds to primers to form a complex that scans the double-stranded template. With the assistance of single-strand binding proteins, the double strands unwind and locate complementary sequences. DNA polymerase then extends the primers to synthesize new strands, resulting in exponential growth of the amplified products. The reaction can be completed under isothermal conditions of 30-42℃ (optimal 37-39℃), with results obtained in 5-30 minutes. Due to its mild reaction conditions, rapid amplification (5-30 minutes), high sensitivity (10-100 copies / μL), portable equipment (no thermal cycler required), and simple operation (lyophilized reagents), it has been widely used in recent years for rapid detection of pathogens such as RNA viruses (e.g., influenza A virus) at the grassroots level and in the field, making it suitable for point-of-care testing (POCT) scenarios.

[0004] The CRISPR-Cas system originates from the adaptive immune system of bacteria and archaea, used to combat the invasion of foreign genetic factors such as bacteriophages. It mainly consists of CRISPR clusters and Cas proteins, and the immune process includes three stages: adaptation, expression, and interference. CRISPR / Cas12a belongs to the class 2, type V system and is an RNA-guided endonuclease. Under crRNA guidance, it recognizes the TTTV (V for A / C / G) PAM sequence of target DNA (located at the 5' end of the target DNA), cleaving double-stranded DNA through the RuvC domain to produce sticky ends. Upon activation, it also possesses trans-cleavage activity, capable of non-specifically cleaving single-stranded DNA (ssDNA). Based on this characteristic, researchers have combined it with isothermal amplification (such as RAA and LAMP) to develop detection technologies such as DETECTR and HOLMES, which can rapidly and sensitively detect viral nucleic acids and SNP typing of HPV, ASFV, etc., and are suitable for primary care and field diagnostics.

[0005] Currently, the main detection methods for PHEVs include RT-qPCR and the HA / HI test, but these methods have many limitations. While RT-qPCR has high sensitivity, it requires specialized laboratory equipment and technicians, and the testing process is time-consuming, often taking several hours or even longer from sample collection to results, failing to meet the needs of on-site, real-time diagnosis. The HA / HI test is susceptible to interference from external factors, affecting the accuracy and reliability of the results, and also requires a laboratory environment. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problems of high requirements for detection methods or detection environments, long detection time, or inaccurate detection results in the existing technology for PHEV detection. It provides a reagent composition for detecting coronavirus PHEV based on single-tube RT-RAA combined with CRISPR / Cas12a, which achieves highly sensitive (as low as 13 copies / μL), rapid, and contamination-resistant on-site detection of PHEV.

[0007] The objective of this invention is achieved through the following technical solution.

[0008] The reagent composition for detecting PHEV of the present invention includes primer pairs, crRNA and ssDNA probe; The primer pair includes an upstream primer and a downstream primer, the nucleotide sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.2. The nucleotide sequence of the crRNA is shown in SEQ ID NO.3; The nucleotide sequence of the ssDNA probe is TTATT, and the two ends of the nucleotide sequence of the ssDNA probe are respectively connected to a fluorescent group or biotin.

[0009] Preferably, the primer pair is designed to target the conserved region of the PHEV structural protein nucleocapsid protein N (PHEV-N) gene; More preferably, the primer pair is a specific primer pair obtained by screening highly conserved regions from 35 PHEV-N gene sequences in the GenBank database using SnapGene software, and then optimizing them using Prime Primer 5.0 and online tools (http: / / www.rgenome.net / ).

[0010] Preferably, the concentrations of the upstream and downstream primers are both 10 μM.

[0011] Preferably, the crRNA targets the conserved region of the PHEV-N gene; More preferably, the crRNA is used to determine four PHEV-N gene target sequences through sequence alignment, and the crRNA4 with the strongest Cas12a cleavage activity is screened out. Particularly preferred is that the crRNA is designed based on a sequence including the T7 promoter sequence, the Lachnospiraceae bacterial Cas12a (LbCas12a) scaffold sequence and the 23bp PHEV-N gene target sequence.

[0012] Preferably, the concentration of the crRNA is not less than 50 nM, and more preferably, the concentration of the crRNA is 200 nM.

[0013] Preferably, the concentration of the ssDNA probe is 500 nM.

[0014] Preferably, the fluorescent group is carboxyfluorescein (FAM) or black hole quencher (BHQ). More preferably, the 5' end of the nucleotide sequence of the ssDNA probe is labeled with FAM and the 3' end is labeled with BHQ, i.e., 5'FAM-TTATT BHQ 3', for use in fluorescence signal detection.

[0015] Preferably, it also includes RT-RAA amplification reagent, which comprises lyophilized enzyme powder, Buffer A and Buffer B in a volume ratio of 50:25:2.5, and the volume ratio of lyophilized enzyme powder, upstream primer and downstream primer is 50:2:2.

[0016] Preferably, it also includes a CRISPR / Cas12a reagent, which comprises LbCas12a protein and LbCas12a protein buffer in a volume ratio of 2:2, wherein the concentration of LbCas12a protein is 1250 nM, and the volume ratio of LbCas12a protein to crRNA and ssDNA probe is 2:1:1.

[0017] Preferably, it also includes diethyl pyrocarbonate (DEPC) water.

[0018] Preferably, it also includes an RNA extraction kit.

[0019] It should be noted that there are no special restrictions on the RNA extraction kit, which can be purchased from Beijing TransGen Biotech Co., Ltd., model number ER501-01-V2.

[0020] The method of using the reagent composition for detecting PHEV coronavirus of the present invention includes: Step 1, RNA extraction from samples Take the sample to be tested, use the RNA extraction kit, follow the instructions, elute with a volume of 50 μL, and obtain the RNA template with an RNA concentration ≥50 ng / μL. The sample to be tested is a nasal swab or a brain tissue homogenate; (2) Construction of a single-tube RT-RAA-CRISPR / Cas12a amplification system Add 25 μL of Buffer A, 2.5 μL of Buffer B, 2 μL of upstream primer (10 μM), and 2 μL of downstream primer (10 μM) to 50 μL of lyophilized enzyme powder, and bring the volume to 45 μL with DEPC water to obtain RT-RAA premix. Take 20 μL of RT-RAA premix and mix it with 5 μL of RNA template obtained in step one to obtain 25 μL of RT-RAA amplification system, and place it in an EP tube. Mix 5 μL of 1250 nM LbCas12a protein, 2.5 μL of 200 nM crRNA, 2.5 μL of 500 nM ssDNA probe and 5 μL of LbCas12a protein buffer, and add DEPC water to a final volume of 25 μL to obtain the CRISPR / Cas12a cleavage system. Place the CRISPR / Cas12a cutting system into an open PCR tube, invert it and place it into an EP tube containing the RT-RAA amplification system. Close the EP tube cap to ensure that the CRISPR / Cas12a cutting system and the RT-RAA amplification system are physically isolated in liquid. (3) Amplification The EP tube was placed in a 42℃ constant temperature water bath for 20 minutes to complete RNA reverse transcription and cDNA amplification. After centrifugation, the RT-RAA amplification system and the CRISPR / Cas12a cutting system were mixed and placed in a 37℃ constant temperature water bath for 30 minutes to obtain the test sample. (4) Detection Observe the fluorescence signal of the test sample under blue light excitation (such as a blue light meter or smartphone). If the test sample contains PHEV RNA, LbCas12a is activated and cuts the ssDNA probe, releasing a fluorescence signal. The results can be interpreted by the naked eye or by the instrument: positive samples show green fluorescence (FAM signal is released and BHQ is quenched), and negative samples (test sample does not contain PHEV RNA) show no fluorescence.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The reagent composition of the present invention for detecting coronavirus PHEV has high sensitivity, with a detection limit as low as 13 copies / μl for the PHEV-N gene.

[0022] The reagent composition for detecting PHEV coronavirus of the present invention has high specificity and no cross-reactivity with porcine deltacoronavirus (PDCoV), porcine circovirus type 2 and 3 (PCV2, PCV3), pseudorabies virus (PRV), etc.

[0023] The reagent composition for detecting PHEV coronavirus of the present invention is rapid and convenient, with the entire process completed within 50 minutes. It requires no complex or sophisticated instruments and is suitable for on-site environments such as farms. In farms, the immediate diagnosis of animal health is crucial, and traditional testing methods often fall short of the need for rapid on-site detection. The technology of this invention enables rapid on-site testing, providing strong support for farms to take timely prevention and control measures, thus preventing the spread of the epidemic.

[0024] The reagent composition for detecting PHEV coronavirus of the present invention employs a physical isolation design in a single-tube reaction system. The CRISPR / Cas12a cleavage system is placed in an open PCR tube, which is then inverted and placed inside an EP tube containing the RT-RAA amplification system. The EP tube cap is closed to separate the two systems. After amplification, the two systems are mixed by brief centrifugation. This single-tube stepwise design avoids exposure of amplification products, reduces the risk of aerosol contamination, and effectively avoids false positives caused by aerosols generated during the RAA reaction.

[0025] The single-tube RT-RAA-CRISPR / Cas12a detection system provided by this invention offers an efficient solution for rapid on-site diagnosis of PHEVs, accurately identifying PHEVs and effectively avoiding false positive and false negative results, thus having significant application value in PHEV prevention and control. Attached Figure Description

[0026] Figure 1 In Example 2, four crRNA target sequences were identified during the screening of crRNAs.

[0027] Figure 2 The fluorescence detection results of the CRISPR / Cas12a detection system for different concentrations of Cas12a protein in Example 2 are shown.

[0028] Figure 3 The results show the fluorescence detection of different concentrations of crRNA in the CRISPR / Cas12a detection system in Example 2.

[0029] Figure 4 The following are the fluorescence results of RT-RAA amplification at different temperatures in a single-tube RT-RAA-CRISPR / Cas12a array in Example 4. In this figure, A represents the fluorescence of PHEV-N plasmid samples amplified at 1.3 × 10⁻⁶ at three different RT-RAA amplification temperatures. 3 copies / μL - 1.3 × 10 1 The fluorescence intensity at the dilution factor of copies / μL, where B is the fluorescence signal value measured for the corresponding reaction solution.

[0030] Figure 5 The following are the fluorescence results of RT-RAA amplification at different times in a single-tube RT-RAA-CRISPR / Cas12a array in Example 5. In this array, A represents the fluorescence of the PHEV-N plasmid sample at three different RT-RAA amplification times at 1.3 × 10⁻⁶. 3 copies / μL - 1.3 × 10 1 The fluorescence intensity at the dilution factor of copies / μL, where B is the fluorescence signal value measured for the corresponding reaction solution.

[0031] Figure 6 The results of the sensitivity screening test of the single-tube RT-RAA-CRISPR / Cas12a detection system in Example 6 are shown; where A is a fluorescence image of the sensitivity screening test results of the RAA-CRISPR / Cas12a detection system, with the template concentration diluted to 1.3 × 10⁻⁶. 4 copies / μL - 1.3 × 10 1 The RAA reaction was performed using a concentration gradient of copies / μL to determine the detection sensitivity; B represents the fluorescence signal value measured in the corresponding reaction solution.

[0032] Figure 7 The results show the specificity detection of the single-tube RT-RAA-CRISPR / Cas12a detection system in Example 7. A is a fluorescence image, and B is the fluorescence signal value measured in the corresponding reaction solution. Detailed Implementation

[0033] Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions in the art, or as selected according to the product instructions. Reagents and raw materials in the following examples, unless otherwise specified, are commercially available. All quantitative experiments in the following examples were performed in triplicate.

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0035] In this embodiment, primer pairs were synthesized by Jilin Kumei Biotechnology Co., Ltd.; RT-RAA amplification reagent was purchased from Hangzhou Zhongce Biotechnology Co., Ltd., model S003ZC; DNA fragment purification kit was TAKARA, 9761; T7 in vitro transcription kit was New England Biolabs, T4130S; RNA purification kit was Monarch RNA Cleanup Kit, NEB, Beijing, China; LbCas12a protein was purified and preserved by the National Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases of Jilin University; and PHEV-N gene plasmid was preserved by the Pathological Anatomy Laboratory of the College of Veterinary Medicine of Jilin University at a concentration of 2.72 mg / ml.

[0036] Example 1 1.1 crRNA Design Thirty-five PHEV-N gene sequences were obtained from the GenBank database (https: / / www.ncbi.nlm.nih.gov / ). SnapGane software was used for rapid alignment to identify highly conserved sequence regions as primer targets for RAA (Rapid Alignment of Genetic Aqueous Activation). Specific crRNAs were then designed using Prime Primer 5.0 and an online tool (http: / / www.rgenome.net / ). Based on the sequence alignment results, four crRNA target sequences were identified. The crRNA4 with the strongest Cas12a cleavage activity was selected.

[0037] 1.2 crRNA preparation A target strand was constructed by combining the T7 promoter, the Lachnospiraceae bacterial Cas12a (LbCas12a) scaffold sequence, and the target sequence for subsequent transcription into crRNA. The synthesized target strand was incubated at 95°C for 5 min in hybridization buffer (20 mM Tris-HCl, pH 7.5; 100 mM KCl; 5 mM MgCl2), followed by a slow cooling to room temperature to obtain dsDNA of the crRNA. The dsDNA was purified using a DNA fragment purification kit. The purified dsDNA was mixed with NTP Buffer Mix and T7 RNA Ploymerase Mix from a T7 in vitro transcription kit and transcribed at 37°C for 16 h in a drying incubator to obtain crRNA. The obtained crRNA was purified using an RNA purification kit, and the crRNA concentration was determined using an Implen N60 UV-Vis spectrophotometer.

[0038] Example 2 2.1 Establishment of the CRISPR / Cas12a system First, a 20 μl CRISPR / Cas12a cleavage system was established, as shown in Table 1. This cleavage system contained 1000 nM LbCas12a protein, 100 nM crRNA, 500 nM 5'-carboxyfluorescein (FAM)-ttt-black hole quencher (BHQ)-3' probe, 100 ng PHEV-N gene plasmid, and LbCas12a protein buffer. Finally, DEPC water was added to bring the total volume to 20 μl.

[0039] Table 1 CRISPR / Cas12a cleavage system

[0040] 2.2 Optimization of the CRISPR / Cas12a System 2.2.1 crRNA screening To optimize the single-tube RT-RAA-CRISPR / Cas12a detection method, key components were optimized. In the crRNA screening experiment, four different crRNAs were prepared, and their activation efficiency for LbCas12a cleavage was compared to determine the optimal crRNA. The experimental method involved adding each of the four crRNAs separately to the CRISPR / Cas12a cleavage system, reacting under identical conditions, and then detecting the fluorescence signal intensity to reflect the cleavage efficiency. The results are as follows: Figure 1 The results showed that crRNA4 activated LbCas12a cleavage with the highest efficiency, indicating that it had the best targeting and activation ability for the PHEV-N gene.

[0041] 2.2.2 Optimization of LbCas12a protein concentration To achieve rapid detection using the RAA-CRISPR / Cas12a detection system, the concentrations of LbCas12a and crRNA were optimized based on the aforementioned crRNA screening. For LbCas12a protein concentration optimization, experiments were conducted with different concentrations of LbCas12a protein. The concentration of LbCas12a protein in the CRISPR / Cas12a cleavage system was set to 500 nM-1250 nM, and the PHEV-N gene plasmid was added before the reaction. The fluorescence signal intensity was detected to evaluate the cleavage activity. The experimental results are as follows: Figure 2 As shown, the cleavage activity was optimal and the strongest fluorescence signal was generated when the LbCas12a protein concentration was 1250 nM, indicating that the synergistic effect of LbCas12a protein with other components reached its best state at this concentration.

[0042] 2.2.3 Optimization of crRNA concentration In the crRNA concentration optimization experiment, different concentrations of crRNA were also set up for testing. The concentration of crRNA protein in the CRISPR / Cas12a cleavage system was set to 50 nM-200 nM, and the PHEV-N gene plasmid was added before the reaction. The fluorescence signal intensity was detected to evaluate the cleavage activity. The results are as follows: Figure 3 As shown, the results showed that the fluorescence signal was strongest when the crRNA concentration was 200 nM, which means that this concentration of crRNA can most effectively guide the LbCas12a protein to recognize and cleave the PHEV-N gene, thereby generating a significant detection signal.

[0043] Example 3: Design of a single-tube RT-RAA-CRISPR / Cas12a detection system RT-RAA reactions were performed using RT-basic nucleic acid amplification reagent (RAA method). 25 μl of Buffer A, 2.5 μl of Buffer B, 2 μl of upstream primer (10 µM), and 2 µl of downstream primer (10 µM) were added to 50 μl of lyophilized enzyme powder. The volume was then brought to 45 μl with DEPC water to obtain a premix, as shown in Table 2. 20 μl of the premix was transferred to a new tube, and 5 μl of the target RNA template was added for the RT-RAA reaction. 5 μL of 1250 nM LbCas12a protein, 2.5 μL of 200 nM crRNA, 2.5 μL of 500 nM ssDNA probe, and 5 μL of LbCas12a protein buffer were mixed, and the volume was increased to 25 μL with DEPC water to obtain the CRISPR / Cas12a cleavage system. The optimal concentrations of each component (LbCas12a and crRNA) in the final 50 μL mixture with the RT-RAA reaction system were ensured. The sequences of the primer pairs, crRNA, and ssDNA probe are shown in Table 3.

[0044] Table 2 Composition of the premixed solution

[0045] Table 3. Sequences of primer pairs, crRNA, and ssDNA probes

[0046] Example 4: Optimization of RT-RAA Reaction Temperature The optimal reaction temperature range for RT-RAA is 37℃-42℃. To investigate the effect of RT-RAA amplification temperature on detection sensitivity, the PHEV-N gene plasmid was used as a template, and its concentration was diluted to 1.3×10⁻⁶. 3 copies / μL - 1.3 × 10 1 Samples of different concentrations were incubated at 37℃, 39℃, and 42℃ for 20 min each to complete reverse transcription and isothermal amplification. After amplification, the samples were briefly centrifuged to thoroughly mix the amplified products with the CRISPR / Cas12a digestion system and incubated for an additional time. Once LbCas12a protein digestion was complete, the results were photographed under blue light using a mobile phone or camera, and analyzed using imaje J software. Based on the fluorescence intensity values ​​obtained from the analysis, bar graphs were plotted using Graphpadprism software to compare differences. The results showed that the fluorescence intensity of the single-tube reaction increased with increasing RT-RAA reaction temperature, confirming 42℃ as the optimal reaction temperature for this experiment. The results are shown below. Figure 4 .

[0047] Example 5: Optimization of RT-RAA Reaction Time After determining the optimal reaction temperature, the effect of different reaction times on detection sensitivity was further investigated. The PHEV-N gene plasmid was serially diluted tenfold to 1.3 × 10⁻⁶. 3 The three groups of samples were amplified in a water bath for 10 min, 20 min, and 30 min, respectively, using 1.3 × 10¹ copies / μL - 1.3 × 10¹ copies / μL. The remaining steps were the same as in Example 4. The results showed that the fluorescence intensity of the single-tube reaction increased with increasing RT-RAA reaction time, and the reaction sensitivity at 20 min and 30 min was not significantly different. Figure 5 As shown. This experiment aims to design a rapid detection method; therefore, 20 minutes was subsequently selected as the reaction time for the RT-RAA reaction in the one-tube method.

[0048] Example 6: Sensitivity Detection of a Single-Tube RT-RAA-CRISPR / Cas12a Detection System The optimized crRNA concentration, RT-RAA reaction time, and RT-RAA reaction temperature were applied to the detection system to evaluate the sensitivity of the PHEV-N gene plasmid. The PHEV-N gene plasmid was serially diluted 10-fold, with concentrations ranging from 1.3 × 10⁻⁶. 4 The reaction mixture consisted of 1.3 × 10¹ copies / μL to 1.3 × 10¹ copies / μL, with a negative control group included. After the reaction was complete, images were taken under blue light irradiation, and the results were analyzed using imaje J software. Based on the fluorescence intensity values ​​obtained from the analysis, bar charts were plotted using Graphpad Prism software, and differences were compared to determine the sensitivity of the RT-RAA-CRISPR / Cas12a method for the PHEV-N gene plasmid.

[0049] The results are as follows Figure 6 As shown, the initial template size is 1.3 × 10⁻⁶. 4 Fluorescent signals were detected at concentrations ranging from 1.3 × 10¹ copies / μL to 1.3 × 10¹ copies / μL, indicating that the limit of detection for this system is 1.3 × 10¹ copies / μL. This demonstrates that the single-tube RT-RAA-CRISPR / Cas12a detection system of this invention can achieve highly sensitive and visual detection of PHEVs.

[0050] Example 7: Specificity detection of a single-tube RT-RAA-CRISPR / Cas12a detection system To verify the specificity of the single-tube RT-RAA-CRISPR / Cas12a detection system, nucleic acids from other pathogens, including PDCOV, PCV2, PCV3, and PRV, were used. These are also viruses that infect pigs and exhibit similar clinical symptoms. The single-tube RT-RAA CRISPR / Cas12 detection method was used to detect these pathogens. Figure 7 As shown, the results revealed that only the PHEV-N gene plasmid and PHEV RNA group produced high fluorescence signals, while other groups and the negative control group did not produce fluorescence signals. This demonstrates that the detection method has good specificity for PHEV detection and can distinguish PHEV from other common porcine viruses.

[0051] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A reagent composition for detecting porcine hemagglutinating encephalomyelitis virus (PHEV), characterized in that, Includes primer pairs, crRNA, and ssDNA probes; The primer pair includes an upstream primer and a downstream primer, the nucleotide sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.

2. The nucleotide sequence of the crRNA is shown in SEQ ID NO.3; The nucleotide sequence of the ssDNA probe is TTATT, and the two ends of the nucleotide sequence of the ssDNA probe are respectively connected to a fluorescent group or biotin.

2. The reagent composition for detecting coronavirus PHEV according to claim 1, characterized in that, The primer pair was designed to target the conserved region of the PHEV structural protein nucleocapsid protein N (PHEV-N) gene. The crRNA targets the conserved region of the PHEV-N gene.

3. The reagent composition for detecting coronavirus PHEV according to claim 2, characterized in that, The primer pairs were specific primer pairs obtained by screening conserved regions from 35 PHEV-N gene sequences in the GenBank database using SnapGene software, and then optimizing them using Prime Primer 5.0 and online tools. The crRNA was used to identify four PHEV-N gene target sequences through sequence alignment, and the crRNA4 with the strongest LbCas12a cleavage activity was selected.

4. The reagent composition for detecting coronavirus PHEV according to claim 3, characterized in that, The crRNA was designed based on a sequence containing the T7 promoter sequence, the Lachnospiraceae bacterial Cas12a scaffold sequence, and a 23bp PHEV-N gene target sequence.

5. The reagent composition for detecting coronavirus PHEV according to claim 1, characterized in that, It possesses one or more of the following characteristics: The concentrations of the upstream and downstream primers were both 10 μM. The concentration of the crRNA is not less than 50 nM; The concentration of the ssDNA probe is 500 nM.

6. The reagent composition for detecting coronavirus PHEV according to claim 1, characterized in that, The fluorescent group is a carboxyfluorescein or a black hole quencher.

7. The reagent composition for detecting coronavirus PHEV according to claim 6, characterized in that, The ssDNA probe has a 5' end labeled with carboxyfluorescein and a 3' end labeled with a black hole quencher.

8. The reagent composition for detecting porcine hemagglutinating encephalomyelitis virus according to any one of claims 1-7, characterized in that, It also includes RT-RAA amplification reagent, which comprises lyophilized enzyme powder, Buffer A and Buffer B in a volume ratio of 50:25:2.5, and the volume ratio of lyophilized enzyme powder, upstream primer and downstream primer is 50:2:

2.

9. The reagent composition for detecting coronavirus PHEV according to any one of claims 1-7, characterized in that, It also includes a CRISPR / Cas12a reagent, which comprises LbCas12a protein and LbCas12a protein buffer in a volume ratio of 2:2, wherein the concentration of LbCas12a protein is 1250 nM, and the volume ratio of LbCas12a protein, crRNA and ssDNA probe is 2:1:

1.

10. The reagent composition for detecting coronavirus PHEV according to any one of claims 1-7, characterized in that, It also includes one or both of the following: diethyl pyrocarbonate water and RNA extraction kit.

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