A double rpa-pfago nucleic acid detection method and kit for simultaneously detecting rabbit hemorrhagic disease virus type I and type II

Abstract

The application discloses a double RPA-PfAgo nucleic acid detection method and kit for synchronously detecting rabbit hemorrhagic disease virus type I and type II, and belongs to the technical field of biology. The method is characterized in that specific RPA primers and guide DNA are designed according to the conserved regions of RHDV type I and type II VP60 genes, the nucleic acid cutting characteristics of recombinase polymerase amplification and Pyrococcus furiosus Argonaute protein are combined, and a double-channel fluorescence reporting system is introduced, so that the single-tube synchronous amplification, cutting and signal output of the two types of viruses are realized. The application has the advantages of constant-temperature rapidness, high sensitivity, strong specificity and visualized results, and is suitable for RHDV on-site screening and typing detection.

Description

Technical Field

[0001] This invention belongs to the field of pathogen nucleic acid detection in the field of biotechnology, specifically relating to a nucleic acid detection method based on recombinase polymerase amplification (RPA) and the Argonaute protein (PfAgo) of *Ficus pulverans*, particularly a dual fluorescence detection method and kit for simultaneously detecting and distinguishing between rabbit hemorrhagic virus type I (RHDV I) and type II (RHDV II). Background Technology

[0002] Rabbit hemorrhagic disease (RHD) is an acute, highly fatal infectious disease caused by rabbit hemorrhagic disease virus (RHDV). It is characterized by rapid spread and an extremely high mortality rate (often exceeding 80%). Its high frequency of genetic mutations and potential cross-species transmission risk cause enormous economic losses to the global rabbit farming industry. In recent years, two main genotypes of RHDV—classical RHDV I and variant RHDV II (also known as RHDV2 or RHDVb)—have co-circulated worldwide, with mixed infections occurring. RHDV I and RHDV II differ significantly in genomic sequence, antigenicity, host range, and age of onset. Therefore, accurate and rapid genotyping is crucial for epidemic monitoring, prevention and control strategy development, and vaccine selection.

[0003] Existing major molecular detection technologies all have certain limitations, as follows: (1) Conventional PCR and qRT-PCR techniques: Although sensitive and accurate, they rely on sophisticated thermal cyclers, with a detection cycle of about 1.5-2 hours. They also have high requirements for the operating environment and personnel skills, and are complex to operate. They are not suitable for on-site real-time testing in grassroots farms or in the wild.

[0004] (2) Isothermal amplification techniques (such as LAMP): Although they reduce equipment dependence, primer design is complex and nonspecific amplification and signal crosstalk are easy to occur during multiple detection.

[0005] (3) Serological methods: low sensitivity, long operation cycle, and cannot distinguish between infection and immunity.

[0006] (4) CRISPR-Cas system: Although it has been used for nucleic acid detection, its application is limited by the constraints of the prototype spacer neighbor motif (PAM) sequence, and the guide RNA (gRNA) is expensive and has poor stability.

[0007] (5) Existing patented technologies: For example, CN108315495A and CN108546780A disclose PCR primers and methods for simultaneous detection of different RHDV subtypes, but these methods still rely on traditional PCR temperature-controlled equipment and gel electrophoresis, which cannot meet the needs of rapid, on-site detection. Although CN116356077A realizes fluorescence quantitative PCR detection, it still requires a precision thermal cycler and is a stepwise single-phase or high-cost multiplex qPCR.

[0008] Therefore, there is an urgent need to establish a new on-site rapid detection technology that is highly sensitive, specific, easy to operate, requires no complex temperature-changing equipment, and can simultaneously distinguish between mixed RHDV type I and type II infections in a single reaction tube. Summary of the Invention

[0009] This invention aims to address the technical problems of existing RHDV genotyping detection methods, such as strong equipment dependence, cumbersome operation, and difficulty in achieving simultaneous, visualized, and rapid on-site genotyping detection. Furthermore, it provides a dual RPA-PfAgo nucleic acid detection method for the simultaneous detection of rabbit hemorrhagic disease virus types I and II. This method combines recombinase polymerase amplification (RPA) technology with the nucleic acid cleavage characteristics of the Argonaute protein (PfAgo) from *P. fibrillosa*, and integrates a dual-channel fluorescence reporter system, achieving simultaneous amplification, specific cleavage, and visualized interpretation of RHDV types I and II targets under isothermal conditions.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a dual RPA primer combination for simultaneously detecting or distinguishing rabbit hemorrhagic disease virus type I (RHDV I) and type II (RHDV II), comprising: (a) The first primer pair used for amplifying the conserved region of the RHDV type I VP60 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO:7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:16; and, (b) The second primer pair used to amplify the conserved region of the RHDV II VP60 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 24, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 27.

[0011] Secondly, the present invention provides a gDNA combination for simultaneously detecting or distinguishing RHDVⅠ and RHDVⅡ, comprising: (a) The first pair of gDNA used to guide the PfAgo protein to specifically cleave the RHDV type I RPA amplicons, the nucleotide sequences of which are shown in SEQ ID NO: 52 and SEQ ID NO: 53; and, (b) The second pair of gDNA used to guide the PfAgo protein to specifically cleave the RHDV II type RPA amplicon, the nucleotide sequences of which are shown in SEQ ID NO: 57 and SEQ ID NO: 58.

[0012] Thirdly, the present invention provides a fluorescent reporter probe combination for simultaneously detecting or distinguishing RHDVⅠ and RHDVⅡ, comprising: (a) A first probe for detecting RHDV type I, having the nucleotide sequence shown in SEQ ID NO: 54, and having a first fluorescent group labeled at the 5' end and a first quencher group labeled at the 3' end; and, (b) A second probe for detecting RHDV type II, the nucleotide sequence of which is shown in SEQ ID NO: 59, and the 5' end is labeled with a second fluorescent group and the 3' end is labeled with a second quenching group; Preferably, the first fluorescent group is ROX and the first quenching group is BHQ2; the second fluorescent group is FAM and the second quenching group is BHQ1.

[0013] Fourthly, this invention provides a dual RPA-PfAgo nucleic acid detection kit for simultaneously detecting or differentiating RHDVⅠ and RHDVⅡ, comprising: (a) RPA primer combinations; (b) gDNA combination; (c) Fluorescent reporter probe assemblies; (d) RPA amplification reaction reagents; (e) PfAgo protein and its reaction buffer; and, (f) Divalent metal ions.

[0014] Preferably, the final concentration of each primer in the RPA primer combination is 0.38~0.46μM, more preferably 0.42μM; and / or, the final concentration of RHDVⅠ-gDNA in the gDNA combination is 0.5~0.7μM, and the final concentration of RHDVⅡ-gDNA is 0.3~0.5μM; more preferably, the final concentration ratio of RHDVⅠ-gDNA to RHDVⅡ-gDNA is 1~2:1, more preferably 1.5:1; and / or, the final concentration of RHDVⅠ probe in the fluorescent reporter probe combination is 0.2~0.4μM, and the final concentration of RHDVⅡ probe is 0.5~0.7μM; and / or, the final concentration of PfAgo protein is 12~17pM; and / or, the final concentration of divalent metal ions is 4~8mM.

[0015] Preferably, the RPA amplification reaction reagent comprises: recombinase, single-stranded DNA-binding protein, strand displacement DNA polymerase, ATP, and dNTPs; and / or, the divalent metal ion is Mg. 2+ or Mn 2+ .

[0016] Preferably, the component providing divalent metal ions is magnesium acetate, magnesium chloride, manganese acetate, or manganese chloride.

[0017] More preferably, the kit further includes at least one of the following: RNA extraction reagent, reverse transcription reagent, positive control, negative control, and inorganic pyrophosphatase.

[0018] Fifthly, the present invention provides a dual RPA-PfAgo nucleic acid detection method for simultaneous detection or differentiation of RHDV I and RHDV II without diagnostic or therapeutic purposes, comprising the following steps: S1, Extract nucleic acid from the sample to be tested to obtain a template; S2, using the RPA primer combination, the template is subjected to recombinase polymerase amplification reaction under isothermal conditions to obtain the amplification product; S3, the amplification product of step S2 is mixed with gDNA, fluorescent reporter probe, PfAgo protein and divalent metal ions, incubated at the cutting temperature, and the signal of the first and / or second fluorescent channel is detected. S4. Determine whether RHDVⅠ and / or RHDVⅡ are present in the sample based on the presence or absence and / or color of the fluorescence signal.

[0019] Preferably, in step S2, the final concentration of each primer in the RPA primer combination is 0.38~0.46μM, more preferably 0.42μM; and / or, the isothermal condition is 37~38℃, and the reaction time is 18~22 minutes; preferably, the isothermal condition is 38℃, and the reaction time is 20 minutes.

[0020] Preferably, the final concentration of RHDVⅠ-gDNA in the gDNA combination is 0.5~0.7μM, and the final concentration of RHDVⅡ-gDNA is 0.3~0.5μM; more preferably, the ratio of the final concentration of RHDVⅠ-gDNA to the final concentration of RHDVⅡ-gDNA is 1~2:1, more preferably 1.5:1; the final concentration of RHDVⅠ probe in the fluorescent reporter probe combination is 0.2~0.4μM, and the final concentration of RHDVⅡ probe is 0.5~0.7μM; the final concentration of PfAgo protein is 12~17pM; and the final concentration of divalent metal ions is 3~6μM.

[0021] More preferably, the divalent metal ion is Mg. 2+ or Mn 2+The components that provide divalent metal ions are magnesium acetate, magnesium chloride, manganese acetate, or manganese chloride.

[0022] Preferably, in step S3, the cutting temperature is 94~96℃, more preferably 95℃, and the cutting time is 25~40 minutes, more preferably 30~35 minutes; and / or, The detection of signals from the first and / or second fluorescence channels is performed using a real-time quantitative PCR instrument or a chemiluminescence imaging system. When using a chemiluminescence imaging system, the infection type is determined by the detected fluorescence color: red fluorescence indicates RHDV I positivity, green fluorescence indicates RHDV II positivity, and yellow fluorescence resulting from the superposition of red and green fluorescence indicates a mixed positive result for both RHDV I and RHDV II.

[0023] In a sixth aspect, the present invention provides the use of the RPA primer combination described in the first aspect, the gDNA combination described in the second aspect, the fluorescent reporter probe combination described in the third aspect, or the kit described in the fourth aspect in the preparation of products for diagnosing or screening rabbit hemorrhagic disease virus.

[0024] Compared with the prior art, the present invention has the following significant advantages: Firstly, it is temperature-controlled, rapid, and has low equipment dependence: Utilizing RPA isothermal amplification technology, amplification can be completed in 20 minutes at a constant temperature of 37-38℃, eliminating reliance on precision PCR thermal cyclers. The entire detection process (excluding nucleic acid extraction) can be completed within 50 minutes.

[0025] Secondly, high specificity and single-base resolution: Guided by short-chain gDNA, PfAgo protein has extremely high recognition fidelity for target sequences, can accurately distinguish single nucleotide differences on RHDV type I and type II VP60 genes, and is not limited by PAM sequences.

[0026] Third, single-tube dual synchronous detection: For the first time, a dual-channel fluorescence reporting system is integrated into the RPA-PfAgo platform, which enables the simultaneous amplification, cleavage and signal output of RHDV type I and II in one reaction tube, effectively solving the problem of missed detection of mixed infection samples and simplifying the operation process.

[0027] Fourth, high sensitivity and accuracy: Clinical sample validation results show that the concordance rate between this method and TaqMan qPCR detection results is 100%, and the detection sensitivity is 10 copies / μL for RHDVⅠ-RPA pfAgo and 10 copies / μL for RHDVⅡ-RPA pfAgo.

[0028] Fifth, the results are easy to interpret: In addition to the fluorescence curve, a chemical imaging visualization solution is also provided, which can be directly interpreted by the naked eye through red (ROX), green (FAM) or yellow (overlay) signals, which is especially suitable for rapid on-site screening scenarios where real-time fluorescence quantitative PCR instruments are not available.

[0029] Sixth, cost is controllable and pollution is prevented: gDNA is a chemically synthesized stable single-stranded DNA, and its cost is lower than that of RNA. The high-temperature cleavage step after the reaction can inactivate the enzymes in the RPA system, effectively reducing the risk of aerosol pollution. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is an electrophoresis image of the full-length RHDV II-VP60 gene amplification in Example 1 of the present invention, where M is a molecular weight standard and 1-5 are the full-length RHDV II-VP60 amplification PCR products.

[0031] Figure 2 This is an electrophoresis image of partial gene amplification of RHDV type I-VP60 in Example 1 of the present invention, where M is a molecular weight standard, NC is a negative control, and 1 is the PCR product of partial gene amplification of RHDV type I-VP60.

[0032] Figure 3 The agarose gel electrophoresis results for identifying RHDV type I and RHDV type II-VP60 plasmids in Example 1 of this invention are shown. Wherein, M is the molecular weight standard, 1 is the RHDV type I-VP60 plasmid, and 2 is the RHDV type II-VP60 plasmid.

[0033] Figure 4 Figure 1 shows the screening results of downstream primers targeting RHDVⅠ-RPA in Example 1 of this invention. Figure a shows the electrophoresis results, M: 2000 bp DNA marker; Figure b shows the product concentrations of orthogonal amplification of different primer combinations for RPA; primer pairs numbered 1-6 correspond to F4 / R1, F4 / R2, F4 / R3, F4 / R4, F4 / R5, F4 / R6; primer pairs numbered 7-11 correspond to F4 / R7, F4 / R8, F4 / R9, F4 / R10, F4 / R11; NC: negative control.

[0034] Figure 5Figure 1 shows the screening results of upstream primers targeting RHDVⅠ-RPA in Example 1 of this invention. Figure a shows the electrophoresis results, M: 2000 bp DNA marker; Figure b shows the product concentrations amplified by orthogonal combinations of different primers for RPA; primer pairs numbered 1-4 correspond to F1 / R8, F2 / R8, F3 / R8, F4 / R8; primer pairs numbered 5-9 correspond to F5 / R8, F6 / R8, F7 / R8, F8 / R8, F9 / R8; NC: negative control.

[0035] Figure 6 The results of the primer verification for targeting RHDVⅠ-RPA in Example 1 of this invention are shown in Figure a. Figure a shows the electrophoresis results, M: 2000 bp DNA Marker; Figure b shows the product concentrations amplified by different primer combinations for RPA; primer pairs numbered 1-4 correspond to F4 / R2, F4 / R4, F4 / R7, and F4 / R8, NC: negative control.

[0036] Figure 7 Figure 1 shows the screening results of upstream primers targeting RHDVⅡ-RPA in Example 1 of this invention. Figure a shows the electrophoresis results, M: 600 bp DNA Marker; Figure b shows the product concentrations amplified by different primer combinations for RPA; primer pairs numbered 1-4 correspond to F1 / R4, F2 / R4, F3 / R4, and F4 / R4; primer pairs numbered 5-8 correspond to F5 / R4, F6 / R4, F7 / R4, and F8 / R4; NC: negative control.

[0037] Figure 8 Figure 1 shows the screening results of downstream primers targeting RHDVⅡ-RPA in Example 1 of this invention. Figure a shows the electrophoresis results, M: 600 bp DNA Marker; Figure b shows the product concentrations of orthogonal amplification of RPA with different primer combinations; primer pairs numbered 1-4 correspond to F2 / R1, F2 / R2, F2 / R3, and F2 / R4; primer pairs numbered 5-8 correspond to F2 / R5, F2 / R6, F2 / R7, and F2 / R8; NC: negative control.

[0038] Figure 9 The results of the second round of screening of upstream primers targeting RHDVⅡ-RPA in Example 1 of this invention are shown in Figure a. Figure a shows the electrophoresis results, M: 600 bp DNA Marker; Figure b shows the product concentrations of orthogonal amplification of RPA with different primer combinations; primer pairs numbered 1-4 correspond to F1 / R3, F2 / R3, F3 / R3, F4 / R3; primer pairs numbered 5-8 correspond to F5 / R3, F6 / R3, F7 / R3, F8 / R3; NC: negative control.

[0039] Figure 10The results of the screening and verification of the RHDVⅡ-RPA primer pairs in Example 1 of this invention are shown in Figure a. Figure a shows the electrophoresis results, M: 2000 bp DNA Marker; Figure b shows the product concentrations of orthogonal amplification of different primer combinations for RPA; primer pairs numbered 1-3 correspond to F2 / R4, F2 / R3, and F7 / R3; NC: negative control.

[0040] Figure 11 To optimize the RHDV-RPA reaction temperature in Example 1 of this invention, Figure a shows the RHDVⅠ-RPA electrophoresis results; Figure b shows the RHDVⅡ-RPA electrophoresis results; Figure c shows the concentration of RHDVⅠ-RPA amplification products; Figure d shows the concentration of RHDVⅡ-RPA amplification products; M: 2000 bp DNA marker, amplification conditions corresponding to numbers 1-6: 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃; NC: negative control.

[0041] Figure 12 To optimize the RHDV-RPA reaction time in Example 1 of this invention, Figure a shows the RHDVⅠ-RPA electrophoresis results; Figure b shows the RHDVⅡ-RPA electrophoresis results; Figure c shows the concentration of RHDVⅠ-RPA amplification products; Figure d shows the concentration of RHDVⅡ-RPA amplification products; M: 2000 bp DNA marker; Amplification conditions corresponding to numbers 1-6: 15 min, 20 min, 25 min, 30 min; NC: negative control.

[0042] Figure 13 The above are the electrophoresis results of the RHDV-RPA sensitivity test in Example 1 of this invention. Figure a shows RHDVⅠ-RPA; Figure b shows RHDVⅡ-RPA; M: 2000 bp DNA marker; numbers 1-6 represent 10... 1 ~10 6 10 serially diluted plasmids were used as templates for RPA amplification: 3 copies / µL, 10 2 copies / µL, 10 1 copies / µL, 10 0 copies / µL, 10 -1 copies / µL, 10 -2 copies / µL; NC: negative control.

[0043] Figure 14The electrophoresis results of the RHDVⅠ-RPA specific assay in Example 1 of this invention are shown. M: 2000 bp DNA marker; numbers 1 to 9 correspond to the amplified templates in sequence: rabbit hemorrhagic disease virus type I cDNA, rabbit hemorrhagic disease virus type II cDNA, rabbit calicivirus plasmid, feline calicivirus cDNA, rabbit-derived Escherichia coli nucleic acid, rabbit-derived Pasteurella nucleic acid, rabbit-derived Staphylococcus nucleic acid, rabbit-derived Salmonella nucleic acid, and rabbit-derived Klebsiella pneumoniae nucleic acid; NC: negative control.

[0044] Figure 15 The electrophoresis results of the RHDVⅠⅠ-RPA specificity assay in Example 1 of this invention are shown. M: 2000 bp DNA marker; numbers 1 to 9 correspond to the amplified templates in sequence: rabbit hemorrhagic disease virus type II cDNA, rabbit hemorrhagic disease virus type I cDNA, rabbit calicivirus plasmid, feline calicivirus cDNA, rabbit-derived Escherichia coli nucleic acid, rabbit-derived Pasteurella nucleic acid, rabbit-derived Staphylococcus nucleic acid, rabbit-derived Salmonella nucleic acid, and rabbit-derived Klebsiella pneumoniae nucleic acid; NC: negative control.

[0045] Figure 16 Figure 2 shows the gDNA screening results of RHDVⅠ-RPA-PfAgo in Example 2 of this invention. Figure a shows the fluorescence intensity detection results of different gDNA combinations; Figure b shows the RFU comparison results of different gDNA combinations; Figure c shows the chemical imaging results; Numbers 1-3 are RHDVⅠ-gDNA-1 / 2, RHDVⅠ-gDNA-3 / 4, and RHDVⅠ-gDNA-5 / 6, respectively; NC: negative control.

[0046] Figure 17 Figure 2 shows the gDNA screening results of RHDVⅠI-RPA-PfAgo in Example 2 of this invention. Figure a shows the fluorescence intensity detection results of different gDNA combinations; Figure b shows the RFU comparison results of different gDNA combinations; Figure c shows the chemical imaging results; Figures 1-2 are RHDVⅠI-gDNA-1 / 2 and RHDVⅠ-gDNA-3 / 4, respectively; NC: negative control.

[0047] Figure 18 To optimize the RHDVⅠ-RPA-PfAgo reaction temperature in Example 2 of this invention, Figure a shows the fluorescence intensity detection results at different PfAgo reaction temperatures (n=3 replicates); Figure b shows the comparison of fluorescence values ​​at different PfAgo reaction temperatures (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 91 ℃, 93 ℃, 95 ℃, 97 ℃, and 99 ℃ respectively; NC: negative control.

[0048] Figure 19To optimize the RHDVⅠI-RPA-PfAgo reaction temperature in Example 2 of this invention, Figure a shows the fluorescence intensity detection results at different PfAgo reaction temperatures (n=3 replicates); Figure b shows the comparison of fluorescence values ​​at different PfAgo reaction temperatures (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 91 ℃, 93 ℃, 95 ℃, 97 ℃, and 99 ℃ respectively; NC: negative control.

[0049] Figure 20 To optimize the RHDVⅠ-RPA-PfAgo reaction time in Example 2 of this invention, Figure a shows the fluorescence intensity detection results for different PfAgo reaction times (n=3 replicates); Figure b shows the comparison of fluorescence values ​​for different PfAgo reaction times (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 20 min, 25 min, 30 min, 35 min, and 40 min respectively; NC: negative control.

[0050] Figure 21 To optimize the RHDVⅠI-RPA-PfAgo reaction time in Example 2 of this invention, Figure a shows the fluorescence intensity detection results for different PfAgo reaction times (n=3 replicates); Figure b shows the comparison of fluorescence values ​​for different PfAgo reaction times (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 20 min, 25 min, 30 min, 35 min, and 40 min respectively; NC: negative control.

[0051] Figure 22 RHDVⅠ-PfAgo Mg in Example 2 of this invention 2+ The optimization, where Figure a shows different Mg 2+ Fluorescence intensity detection results at different concentrations (n=3 replicates); Figure b shows the fluorescence intensity at different concentrations of Mg. 2+ Comparison of fluorescence values ​​at different concentrations (n=3 repetitions); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM, respectively; NC: negative control.

[0052] Figure 23 RHDVⅠI-PfAgo Mg in Example 2 of this invention 2+ The optimization, where Figure a shows different Mg 2+ Fluorescence intensity detection results at different concentrations (n=3 replicates); Figure b shows the fluorescence intensity at different concentrations of Mg. 2+ Comparison of fluorescence values ​​at different concentrations (n=3 repetitions); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM, respectively; NC: negative control.

[0053] Figure 24 To optimize the concentration of RHDVⅠ-PfAgo gNDA in Example 2 of this invention, Figure a shows the fluorescence intensity detection results of different gNDA concentrations (n=3 replicates); Figure b shows the comparison of fluorescence values ​​of different gNDA concentrations (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 1.2 μM, 1.6 μM, 2 μM, 2.6 μM, and 2.8 μM respectively; NC: negative control.

[0054] Figure 25 To optimize the concentration of RHDVⅠI-PfAgo gNDA in Example 2 of this invention, Figure a shows the fluorescence intensity detection results of different gNDA concentrations (n=3 replicates); Figure b shows the comparison of fluorescence values ​​of different gNDA concentrations (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 1.2 μM, 1.6 μM, 2 μM, 2.6 μM, and 2.8 μM respectively; NC: negative control.

[0055] Figure 26 To optimize the ROX concentration in the RHDVⅠ-PfAgo system in Example 2 of this invention, Figure a shows the fluorescence intensity detection results of different ROX concentrations (n=3 replicates); Figure b shows the comparison of fluorescence values ​​of different ROX concentrations (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, and 0.6 μM, respectively; NC: negative control.

[0056] Figure 27 To optimize the FAM concentration in the RHDVⅠI-PfAgo system in Example 2 of this invention, Figure a shows the fluorescence intensity detection results of different FAM concentrations (n=3 replicates); Figure b shows the comparison of fluorescence values ​​of different FAM concentrations (n=3 replicates); Figure c shows the chemical imaging results; the reaction temperatures corresponding to numbers 1-5 are 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, and 0.6 μM, respectively; NC: negative control.

[0057] Figure 28 This is a sensitivity test of the RHDVⅠ-PfAgo system in Example 2 of the present invention. Figure a shows the fluorescence intensity detection results of the sensitivity test; Figure b shows the comparison of fluorescence values ​​in the sensitivity test; Figure c shows the chemical imaging results; plasmids numbered 1-6 correspond to 10... 3 copies / µL, 10 2 copies / µL, 10 1 copies / µL, 10 0copies / µL, 10 -1 copies / µL, 10 - 2 copies / µL; NC: negative control.

[0058] Figure 29 This is a sensitivity test of the RHDVⅠI-PfAgo system in Example 2 of the present invention. Figure a shows the fluorescence intensity detection results of the sensitivity test; Figure b shows the comparison of fluorescence values ​​in the sensitivity test; Figure c shows the chemical imaging results; plasmids numbered 1-6 correspond to 10... 3 copies / µL, 10 2 copies / µL, 10 1 copies / µL, 10 0 copies / µL, 10 -1 copies / µL, 10 - 2 copies / µL; NC: negative control.

[0059] Figure 30 This is a specificity test of the RHDVⅠ-PfAgo system in Example 2 of the present invention. Figure a shows the fluorescence intensity detection results of the specificity test; Figure b shows the comparison of fluorescence values ​​of the specificity test; Figure c shows the chemical imaging results; the amplification templates corresponding to numbers 1-5 are, in order: rabbit hemorrhagic disease virus type I cDNA, rabbit hemorrhagic disease virus type II cDNA, rabbit calicivirus plasmid, feline calicivirus nucleic acid, and rabbit-derived Escherichia coli nucleic acid; the amplification templates corresponding to numbers 6-9 are, in order: rabbit-derived Pasteurella nucleic acid, rabbit-derived Staphylococcus nucleic acid, rabbit-derived Salmonella nucleic acid, and rabbit-derived Klebsiella pneumoniae nucleic acid; NC: negative control.

[0060] Figure 31 This is a specificity test of the RHDVⅠI-PfAgo system in Example 2 of the present invention. Figure a shows the fluorescence intensity detection results of the specificity test; Figure b shows the comparison of fluorescence values ​​of the specificity test; Figure c shows the chemical imaging results; the amplification templates corresponding to numbers 1-5 are, in order: rabbit hemorrhagic disease virus type I cDNA, rabbit hemorrhagic disease virus type II cDNA, rabbit calicivirus plasmid, feline calicivirus nucleic acid, and rabbit-derived Escherichia coli nucleic acid; the amplification templates corresponding to numbers 6-9 are, in order: rabbit-derived Pasteurella nucleic acid, rabbit-derived Staphylococcus nucleic acid, rabbit-derived Salmonella nucleic acid, and rabbit-derived Klebsiella pneumoniae nucleic acid; NC: negative control.

[0061] Figure 32 This is a repeatability test of the RHDVⅠ-PfAgo system in Example 2 of the present invention. Figure a shows the fluorescence intensity detection results of the repeatability test (n=3 repetitions); Figure b shows the comparison of fluorescence values ​​of the repeatability test (n=3 repetitions); Figure c shows the chemical imaging results; NC: negative control.

[0062] Figure 33 This is a repeatability test of the RHDVⅠI-PfAgo system in Example 2 of the present invention. Figure a shows the fluorescence intensity detection results of the repeatability test (n=3 repetitions); Figure b shows the comparison of fluorescence values ​​of the repeatability test (n=3 repetitions); Figure c shows the chemical imaging results; NC: negative control.

[0063] Figure 34 The following is an example of the establishment of dual RPA in Example 3 of the present invention. Figure a shows the electrophoresis results of RHDVⅠ-RPA and RHDVⅡ-RPA amplification, M: 2000 bp DNA Marker; bands 1-2: RHDVⅡ amplification product bands; bands 3-4: RHDVⅠ amplification product bands; NC: negative control. Figure b shows the electrophoresis results of RHDV1 / RHDV2-dual RPA amplification, M: 2000 bp DNA Marker; bands 1-4: RHDVⅠ and RHDVⅡ dual RPA amplification product bands; NC: negative control.

[0064] Figure 35 The electrophoresis results for the optimized RHDV-double RPA primer concentration in Example 3 of this invention are shown below. M: 2000 bp DNA Marker; primer concentrations corresponding to numbers 1-4: 0.34 μM, 0.38 μM, 0.42 μM, 0.46 μM; NC: negative control.

[0065] Figure 36 The electrophoresis results of the RHDV-double RPA sensitivity assay in Example 3 of this invention are shown. M: 2000 bp DNA Marker; plasmids numbered 1-7 correspond to serially diluted plasmids: 10 3 copies / µL, 10 2 copies / µL, 10 1 copies / µL, 10 0 copies / µL, 10 -1 copies / µL, 10 -2 copies / µL, 10 -3 copies / µL; NC: negative control.

[0066] Figure 37The electrophoresis results of the RHDV-double RPA specificity assay in Example 3 of this invention are as follows: M: 2000 bp DNA Marker; 1: Rabbit hemorrhagic disease virus type I cDNA and rabbit hemorrhagic disease virus type II cDNA; 2: Rabbit calicivirus plasmid; 3: Feline calicivirus nucleic acid; 4: Rabbit-derived Escherichia coli nucleic acid; 5: Rabbit-derived Pasteurella nucleic acid; 6: Rabbit-derived Staphylococcus nucleic acid; 7: Rabbit-derived Salmonella nucleic acid; 8: Rabbit-derived Klebsiella pneumoniae nucleic acid; NC: Negative control.

[0067] Figure 38 This document outlines the preliminary establishment of the RPA-PfAgo dual detection system in Example 3 of the present invention. Figure a shows the fluorescence intensity detection results of the dual detection system (n=3 replicates); Figure b shows the fluorescence value comparison of the dual detection system (n=3 replicates); Figure c shows the chemical imaging results, with 1-3 from left to right representing: ROX channel, FAM channel, and ROX+FAM dual channel; NC: negative control.

[0068] Figure 39 For the first round of Reporter ratio optimization of the RPA-PfAgo dual detection system in Example 3 of the present invention, Figure a shows the fluorescence intensity detection results of the dual detection system; Figure b shows the fluorescence value comparison of the dual detection system; Figure c shows the chemical imaging results, NC: negative control.

[0069] Figure 40 The second round of Reporter ratio optimization for the RPA-PfAgo dual detection system in Example 3 of this invention is shown in Figure a, where Figure a is the fluorescence intensity detection result of the dual detection system; Figure b is the comparison of fluorescence values ​​of the dual detection system; Figure c is the chemical imaging result, NC: negative control.

[0070] Figure 41 For the first round of gDNA ratio optimization of the RPA-PfAgo dual detection system in Example 3 of the present invention, Figure a shows the fluorescence intensity detection results of the dual detection system; Figure b shows the comparison of fluorescence values ​​of the dual detection system; Figure c shows the chemical imaging results, NC: negative control.

[0071] Figure 42 For the second round of gDNA ratio optimization of the RPA-PfAgo dual detection system in Example 3 of the present invention, Figure a shows the fluorescence intensity detection results of the dual detection system; Figure b shows the comparison of fluorescence values ​​of the dual detection system; Figure c shows the chemical imaging results, NC: negative control.

[0072] Figure 43 The following are the results of the optimized gDNA ratio verification of the RPA-PfAgo dual detection system in Example 3 of the present invention. Figure a shows the fluorescence intensity detection results of the dual detection system; Figure b shows the comparison of fluorescence values ​​of the dual detection system; Figure c shows the chemical imaging results, NC: negative control.

[0073] Figure 44 The following are the sensitivity test results of the RPA-PfAgo dual detection system in Example 3 of this invention. Figure a shows the fluorescence intensity detection results of the dual detection system sensitivity test; Figure b shows the comparison of fluorescence values ​​of the dual detection system; Figure c shows the chemical imaging results, with plasmids numbered 1-7 corresponding to: 10 3 copies / µL, 10 2 copies / µL, 10 1 copies / µL, 10 0 copies / µL, 10 -1 copies / µL, 10 -2 copies / µL, 10 -3 copies / µL; NC: negative control.

[0074] Figure 45 The figures show the specificity test results of the RPA-PfAgo dual detection system in Example 3 of this invention. Figure a shows the fluorescence intensity detection results of the specificity test of the dual detection system; Figure b shows the comparison of fluorescence values ​​of the dual detection system; Figure c shows the chemical imaging results, where 1-8 correspond to templates: 1: Rabbit hemorrhagic disease virus type I cDNA and rabbit hemorrhagic disease virus type II cDNA; 2: Rabbit calicivirus plasmid; 3: Feline calicivirus nucleic acid; 4: Rabbit-derived Escherichia coli nucleic acid; 5: Rabbit-derived Pasteurella nucleic acid; 6: Rabbit-derived Staphylococcus nucleic acid; 7: Rabbit-derived Salmonella nucleic acid; 8: Rabbit-derived Klebsiella pneumoniae nucleic acid; NC: negative control.

[0075] Figure 46 Figure 3 shows the repeatability test results of the RPA-PfAgo dual detection system in Example 3 of this invention. Figure a shows the fluorescence intensity detection results of the dual detection system (n=3 repetitions); Figure b shows the comparison of fluorescence values ​​of the dual detection system (n=3 repetitions); Figure c shows the chemical imaging results; NC: negative control.

[0076] Figure 47 The images show the RT-qPCR clinical sample detection results in Example 3 of this invention. Figure a shows a pure RHDV type I sample; Figure b shows a pure RHDV type II sample; Figure c shows the ROX channel of a mixed infection sample; and Figure d shows the FAM channel of a mixed infection sample.

[0077] Figure 48 The results of the dual RPA-PfAgo clinical sample detection in Example 3 of the present invention are as follows: No. 1-6: pure RHDV type I samples; No. 7-18: pure RHDV type II samples; No. 19-24: mixed infection samples; NC: negative control. Detailed Implementation

[0078] The reagents used in this embodiment of the invention are as follows: Oriscience Prime RNA Extraction Kit (Oriscience Biotechnology (Chengdu) Co., Ltd.), Prime Script™ RT reagent Kit with gDNAEraser (TaKaRa Biotechnology (Dalian)), DL2000 DNA Marker (TaKaRa Biotechnology (Dalian)), TAKARA RNAiso Plus (TaKaRa Biotechnology (Dalian)), EZNAGel Extraction Kit (OMEGA), pEASY®-Blunt Cloning Kit (TransGenBiotech), Trans1-T1 clonal competent cells (TransGen Biotech), 2×T5Taq Super PCR Mix (Basic) (Beijing Qingke Biotechnology), 2×T8 High-Fidelity Master Mix (Beijing Qingke Biotechnology), TIANpure Midi Plasmid Kit (Tiangen Biotech (Beijing)), RPA nucleic acid amplification kit (Wuhan Dangkang Xingzhong Biotechnology), Universal DNA Purification Kit (Biosharp Biotechnology Co., Ltd.), Super Red nucleic acid dye (Biosharp Biotechnology Co., Ltd.), PfAgo protein microspheres (Shanghai Jiaohong Biotechnology Co., Ltd.), 10×PfAgo Reaction Buffer (Shanghai Jiaohong Biotechnology Co., Ltd.), 100mM Mg 2+ (Shanghai Jiaohong Biotechnology), PPi Inorganic Pyrophosphatase (Shanghai Jiaohong Biotechnology), 500bp DNA Marker Molecular Weight Standard (50-600bp) (TaKaRa Bioengineering (Dalian)), Biospin Bacterial Genomic DNA Extraction Kit (Shanghai Aiyan Biotechnology Co., Ltd.), STE Suspension (Shanghai Aiyan Biotechnology Co., Ltd.), SDS 10% (Shanghai Aiyan Biotechnology Co., Ltd.), Phenol-Chloroform-Isoamyl Alcohol (25:24:1) (Solarbio), Isoamyl Alcohol:Chloroform (24:1) (Solarbio).

[0079] In the description of this invention, the term "RPA" refers to recombinase polymerase amplification; the term "PfAgo" refers to Argonaute protein from *Firmiana simplex*; the term "gDNA" refers to guide DNA (5'-phosphorylated); and the term "Reporter" refers to a fluorescent reporter probe labeled with a fluorescent group and a quencher group at both ends.

[0080] Furthermore, in the description of this invention, it should be noted that unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0081] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0082] Example 1: Screening of detection targets and design of RPA primers for rabbit hemorrhagic disease virus

[0083] The rabbit hemorrhagic disease virus (RHDV) VP60 gene is the major capsid protein gene of the virus, and its encoded product possesses high immunogenicity. This gene also determines the composition of viral antigenic epitopes and participates in the regulation of viral infection and pathogenic mechanisms. The VP60 gene sequence contains conserved regions shared by different genotypes, as well as specific differential sites between RHDV I and RHDV II types, constituting the core target for virus detection and typing. Based on this, screening of detection targets and designing primers are prerequisites for improving the efficiency of the detection system. The sequence variation characteristics between different genotypes determine the rigorous requirements for target selection and primer design. If the target region is not selected properly or the primer design is unreasonable, it can easily lead to insufficient detection sensitivity, specificity bias, or even missed detections and false detections. Therefore, conducting VP60 gene sequence analysis and designing specific RPA primers is a necessary prerequisite for ensuring the reliability of the detection system.

[0084] I. Sample and Nucleic Acid Sources The suspected RHDV type I positive sample was collected from a commercial rabbit farm in Ziyang City, Sichuan Province. RNA was extracted and reverse transcribed into cDNA, and a portion of the VP60 gene was amplified by RT-PCR. The VP60 gene was then ligated into the blunt vector, screened to obtain positive clones, and sequenced to identify it as an RHDV type I-VP60 positive plasmid.

[0085] The suspected RHDV type II positive sample was collected from a commercial rabbit farm in Dongpo District, Meishan City, Sichuan Province. RNA was extracted and reverse transcribed into cDNA, the VP60 gene was amplified by RT-PCR, and positive clones were obtained by screening and sequencing and identified as RHDV type II-VP60 positive plasmids.

[0086] Negative controls and specific nucleic acid samples for other pathogens: Negative controls included liver tissue extracts from healthy rabbits lacking RHDV nucleic acid, as well as nucleic acid materials from various common rabbit-derived pathogens. Among these, viral nucleic acid materials included: rabbit calicivirus (RCV) plasmid (containing the full-length VP60 gene) and feline calicivirus (FCV) cDNA; the full-length RCV VP60 gene fragment was synthesized by Suzhou Hongxun Biotechnology Co., Ltd. based on a publicly available sequence, while the FCV cDNA was extracted and preserved from a clinical veterinary laboratory, and can also be obtained through artificial synthesis. Both of these viruses are existing viral strains.

[0087] Bacterial nucleic acid materials were extracted from clinical isolates, including *Escherichia coli*, *Pasteurella multocida*, *Staphylococcus spp.*, *Salmonella enterica*, and *Klebsiella pneumoniae*. All of the above nucleic acid samples were previously collected, isolated, and preserved by our clinical veterinary laboratory and have been identified as existing bacterial species.

[0088] II. Specific Operating Methods 2.1 Nucleic acid preparation of RHDV positive samples 2.1.1 Viral RNA Extraction and Reverse Transcription (1) Take about 50 mg of RHDV type I positive rabbit liver tissue and RHDV type II positive rabbit liver tissue stored at -80℃, add physiological saline and grind thoroughly. Then, extract total RNA using a nucleic acid extraction kit (Chengdu Aorui Sais Biotechnology Co., Ltd.). The specific operation is performed according to the instructions. The concentration and purity of the extracted RNA were determined by NanoDrop 2000. Immediately reverse transcribe or aliquot and store at -80℃.

[0089] (2) The reverse transcription reaction was performed using PrimeScript. TMThe RT reagent kit with gDNA Eraser (TaKaRa) was used according to the two-step procedure described in the instructions: First, 2.0 μL of 5×gDNA Eraser Buffer, 1.0 μL of gDNA Eraser, and 1.0 μg of total RNA were added to the reaction system, followed by RNase-free dH2O to a final volume of 10 μL. The mixture was incubated at 42°C for 2 min to remove genomic DNA. Then, 1.0 μL of PrimeScript RT Enzyme Mix I, 1.0 μL of RT Primer Mix (random hexamer primers), 4.0 μL of 5×PrimeScript Buffer 2, and 4.0 μL of RNase-free ddH2O were added. The mixture was incubated at 37°C for 15 min, and the reaction was terminated by heating at 85°C for 5 s. The synthesized RHDV I and RHDV II cDNAs were stored at -20°C for later use.

[0090] 2.1.2 Extraction of bacterial genomic DNA This embodiment involves the extraction of genomic DNA from five rabbit-derived bacteria (Escherichia coli, Pasteurella multocida, Staphylococcus aureus, Salmonella, and Klebsiella pneumoniae). Due to the differences in cell wall structure among the different bacteria, two methods were used: (1) Gram-negative bacteria (Escherichia coli, Pasteurella multocida, Salmonella, Klebsiella pneumoniae): Use the bacterial genomic DNA extraction kit (Shanghai Aiyan Biotechnology Co., Ltd.) according to the instructions. Take 1-5 mL of overnight culture, centrifuge at 10,000 rpm for 1 min to collect the bacterial cells, add 180 μL Buffer GL, 20 μL Proteinase K and 10 μL RNase A, incubate at 56 ℃ for 10 min, and then proceed according to the kit steps.

[0091] (2) Gram-positive bacteria (Staphylococcus): A modified phenol-chloroform extraction method was used. After collecting the bacterial cells, they were resuspended in 500 μL of TE buffer, and 50 μL of 50 mg / mL lysozyme was added. The cells were incubated at 37 °C for 1 h to break the cell walls. Then, 50 μL of 10% SDS and 10 μL of 20 mg / mL proteinase K were added, and the cells were incubated at 55 °C for 30 min. The cells were then extracted twice with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1). The DNA was precipitated in the supernatant by adding 0.6 times the volume of isopropanol, washed with 75% ethanol, and dissolved in TE buffer.

[0092] Bacterial DNA extracted by both methods was stored at -20 ℃ for later use.

[0093] 2.2 Construction of VP60 positive plasmid 2.2.1 Construction of RHDV type I-VP60 positive plasmid (1) Using RHDVⅠ cDNA from section 2.1.1 as a template, the target gene VP60 was amplified by PCR. The amplified fragment covered the full length of VP60 (approximately 940 bp). The PCR primers are shown in Table 5 (RHDVⅠ-F and RHDVⅠ-R). All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction system (Table 1) and amplification procedure (Table 2) are also shown. After separation by agarose gel electrophoresis, the gel block containing the target fragment was excised and purified using a gel extraction kit. The purified product was then temporarily stored at -20 °C after concentration determination.

[0094] Table 1 PCR reaction system

[0095] Table 2 PCR reaction procedure

[0096] (2) According to the Blunt vector kit instructions, the purified fragment was ligated into the Blunt vector and transformed into Trans T1 competent cells for cloning. Positive clones were obtained by PCR screening and sequenced for identification.

[0097] (3) Positive colonies verified by sequencing were expanded and cultured, and plasmid DNA was extracted using a plasmid extraction kit. The plasmid DNA was stored at -20 °C after concentration determination. The finally obtained RHDVⅠ-VP60 plasmid was used as a positive template for subsequent RPA-pfAgo method.

[0098] 2.2.2 Construction of RHDV II-VP60 positive plasmid (1) Using RHDV type II cDNA from 2.1.1 as a template, the target gene VP60 was amplified by PCR. The amplified fragment covered the full length of VP60 (approximately 1969 bp). The PCR primers are shown in Table 5 (RHDVⅠI-F and RHDVⅠI-R). The PCR reaction system (Table 1) and amplification program (Table 2) are also shown. After separation by agarose gel electrophoresis, the gel block containing the target fragment was excised and purified using a gel extraction kit. The purified product was then temporarily stored at -20 °C after concentration determination.

[0099] (2) According to the Blunt vector kit instructions, the purified fragment was ligated into the Blunt vector and transformed into Trans T1 competent cells for cloning. Positive clones were obtained by PCR screening and sequenced for identification.

[0100] (3) Positive colonies verified by sequencing were expanded and cultured, and plasmid DNA was extracted using a plasmid extraction kit. The plasmid DNA was stored at -20 °C after concentration determination. The finally obtained RHDVⅡ-VP60 plasmid was used as a positive template for subsequent RPA-pfAgo method.

[0101] 2.3 Primer Design and Target Selection Based on the selected conserved gene regions, RPA primers were designed.

[0102] 2.3.1 VP60 gene structure and target localization Based on the full-length VP60 sequence alignment of representative RHDV I (GI.1) and RHDV II (GI.2) strains from NCBI, conserved regions for RPA primer design were determined for both viral types: the design region for RHDV I was 59-498 nt, and the design region for RHDV II was 14-530 nt. Both regions are located within the relatively conserved S domain and P1 subregion, ensuring amplification stability. Furthermore, the two viral types share approximately 70% homology within this region, exhibiting stable inter-type differential sites, which can be used to design PfAgo genotyping gDNA.

[0103] 2.3.2 Primer Design and Synthesis Specific RPA primers were designed for the VP60 amplification region of RHDV I and RHDV II. Nine sets of RPA amplification primers were designed for RHDV I, and eight sets were designed for RHDV II, as shown in Tables 3 and 4. The RPA primers were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0104] Table 3 RHDV I type RPA primer sequences

[0105] Table 4. Primer sequences for RHDV II type RPA

[0106] Table 5 RHDV PCR and qPCR primer sequences

[0107] 2.4 Screening of optimal primer pairs for RPA The reaction system was prepared strictly according to the RPA reaction kit instructions. The constructed RHDVⅠ-VP60 and RHDVⅡ-VP60 positive plasmids were used as templates, and sterile, enzyme-free water was used as a negative control. For each upstream and downstream primer designed against the same pathogen, pairwise orthogonal combinations were performed, and premixed solutions were prepared for testing. Primer pairs with optimal amplification efficiency and specificity were screened. The premixed solution systems are shown in Table 6. Table 6 RPA isothermal amplification system

[0108] After thorough mixing, the premixed solution was aliquoted into reaction tubes containing lyophilized enzyme powder. Once the lyophilized enzyme dissolved, the volume was increased by 2 μL. Finally, 1.4 μL of 0.5 M magnesium ions was added to the tube cap, and the tubes were centrifuged to mix. The total reaction volume was 50 μL. The initial reaction program was set to 39 °C for 20 min. After the reaction, the reaction product was purified using a DNA purification kit and detected by electrophoresis. The optimal primer set was selected based on the position and intensity of the bands, and the concentration was determined.

[0109] 2.5 Optimization of RPA Reaction Conditions 2.5.1 Optimization of Reaction Temperature The optimal reaction temperature range for RPA is 37 ℃ to 41 ℃. Based on the initial RPA reaction system, six temperature gradients were set using a PCR instrument: 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, and 42 ℃. The optimal reaction temperature was determined by the concentration of the purified product and the position and brightness of the nucleic acid gel electrophoresis bands.

[0110] 2.5.2 Optimization of Reaction Time Based on the initial RPA reaction system and optimized reaction temperature, four time gradients were set at 5 min intervals: 15 min, 20 min, 25 min, and 30 min. The optimal reaction time was determined by the concentration of the purified product and the position and brightness of the nucleic acid gel electrophoresis bands.

[0111] 2.6 RPA Sensitivity and Specificity Test 2.6.1 Sensitivity Test This experiment, under optimized reaction conditions, targeted the RHDVⅠ-VP60 plasmid (10... 4 copies / µL) and RHDVⅡ-VP60 plasmid (10 copies / µL) 4 (copies / µL) were subjected to 10 1 Times ~10 6 The plasmid was serially diluted 10 times, with 6 different concentrations used as positive templates; ddH2O was used as a negative control for RPA reaction.

[0112] 2.6.2 Specificity Test In this experiment, RHDVⅠ and RHDVⅡ cDNA were used as positive controls, ddH2O as a negative control, and plasmids or nucleic acids of several common rabbit pathogens were used as negative samples: RCV rabbit calicivirus plasmid, FCV feline calicivirus nucleic acid, rabbit-derived Escherichia coli nucleic acid, rabbit-derived Pasteurella nucleic acid, rabbit-derived Staphylococcus nucleic acid, rabbit-derived Salmonella nucleic acid, and rabbit-derived Klebsiella pneumoniae nucleic acid. The reaction was carried out according to the optimized RPA reaction system and reaction conditions, and the specificity of the method was verified by electrophoresis analysis using 1.5% agarose gel.

[0113] 3 Results 3.1 Construction of positive plasmids Using cDNA extracted from RHDVⅠ and RHDVⅡ diseased rabbit tissues as a template, a partial fragment of the RHDVⅠ-VP60 target gene was obtained by PCR amplification, with a length of approximately 940 bp. Figure 2 The target gene fragment of RHDVⅡ-VP60 was obtained, with a length of approximately 1969 bp. Figure 1 After recovery, purification, and ligation cloning, RHDVⅠ-VP60 and RHDVⅡ-VP60 positive plasmids were successfully constructed. The plasmids were detected by 1.5% agarose gel electrophoresis. Figure 3 The plasmid concentrations were determined using NanoDrop 2000. The results showed that the concentration of RHDVⅠ-VP60 plasmid was 30 ng / µL and the concentration of RHDVⅡ-VP60 plasmid was 41 ng / µL.

[0114] 3.2 Screening of optimal primer pairs for RHDV RPA To screen for the optimal primer set suitable for RHDVⅠ and RHDVⅡ amplification, this embodiment configured a universal RPA reaction system according to the kit instructions. RHDVⅠ and RHDVⅡ positive plasmids were used as positive templates, and sterile, enzyme-free water was used as the negative template. The initial reaction conditions were set at 39 °C for 20 min. After amplification, the RPA products were purified using a gel extraction kit. The specificity of the product bands was detected by gel electrophoresis. Combined with the product concentration measurement results, the primer set with the highest amplification efficiency was selected for subsequent experiments.

[0115] 3.2.1 Screening of RHDV Type I primer pairs (1) First, upstream primer F4 was randomly fixed and combined with 11 downstream primers (R1-R11). Concentration determination showed that F4 / R1 (106.1 ng / μL), F4 / R2 (86.9 ng / μL), F4 / R3 (61.0 ng / μL), F4 / R4 (89.3 ng / μL), F4 / R5 (101.2 ng / μL), F4 / R6 (92.3 ng / μL), F4 / R7 (73.7 ng / μL), F4 / R8 (86.7 ng / μL), F4 / R9 (73.6 ng / μL), F4 / R10 (65.8 ng / μL), and F4 / R11 (31.3 ng / μL) were respectively... Figure 4 (a, b) The results showed that primer pairs F4 / R1, F4 / R2, F4 / R5, F4 / R6 and F4 / R8 had high amplification yields, but the amplification bands of F4 / R1, F4 / R5 and F4 / R6 were not uniform. Therefore, R8 was fixed in subsequent experiments.

[0116] (2) Subsequently, downstream primer R8 was fixed and combined with 9 upstream primers (F1-F9). Concentration determination showed that F1 / R8 (63.6 ng / μL), F2 / R8 (55.6 ng / μL), F3 / R8 (57.1 ng / μL), F4 / R8 (62.6 ng / μL), F5 / R8 (59.3 ng / μL), F6 / R8 (49.5 ng / μL), F7 / R8 (62.8 ng / μL), F8 / R8 (62.3 ng / μL), and F9 / R8 (48.7 ng / μL) were... Figure 5 (a) and (b) show that the amplification yields of primer pairs F1 / R8, F4 / R8, F7 / R8 and F8 / R8 are similar, but the amplification band of F8 / R8 is not singular. Therefore, F1 / R8, F4 / R8 and F7 / R8 were selected as backup options.

[0117] (3) Based on the results of the first and second rounds of primer screening, F1 / R8, F7 / R8, F4 / R2, and F4 / R8 were re-screened, and the results are as follows: Figure 6 In samples a and b, concentration measurements showed that F1 / R8 (128.5 ng / μL), F7 / R8 (66.9 ng / μL), F4 / R2 (60.6 ng / μL), and F4 / R8 (128.2 ng / μL) were significantly better than other combinations based on the combined results of band clarity and yield screening. Therefore, F4 / R8 was selected for subsequent experiments.

[0118] 3.2.2 Screening of RHDV II type primer pairs (1) First, the downstream primer R4 was randomly fixed and combined with 8 upstream primers (F1-F8). The concentration of amplified products was measured and showed that: F1 / R4 (13.0 ng / μL), F2 / R4 (29.1 ng / μL), F3 / R4 (23.0 ng / μL), F4 / R4 (21.0 ng / μL), F5 / R4 (23.2 ng / μL), F6 / R4 (19.9 ng / μL), F7 / R4 (16.3 ng / μL), and F8 / R4 (23.3 ng / μL). The results showed that F2 / R4 (29.1 ng / µL) had a relatively high yield (e.g., Figure 7 (a, b)

[0119] (2) Next, the upstream primer F2 was fixed and combined with 8 downstream primers (R1-R8), and the results are as follows. Figure 8 Concentration measurements in samples a and b showed the following results: F2 / R1 (15.1 ng / μL), F2 / R2 (20.0 ng / μL); F2 / R3 (29.6 ng / μL), F2 / R4 (22.5 ng / μL), F2 / R5 (21.6 ng / μL), F2 / R6 (22.9 ng / μL), F2 / R7 (24.4 ng / μL), and F2 / R8 (21.1 ng / μL). Among these, the F2 / R3 combination had the highest concentration and a single band. However, due to the generally low product concentrations, a downstream primer was randomly selected for a third round of primer screening.

[0120] (3) The downstream primer R3 was fixed and combined with 8 upstream primers. The results are as follows: Figure 9 Concentration measurements in samples a and b showed the following results: F1 / R3 (23.4 ng / μL), F2 / R3 (22.7 ng / μL), F3 / R3 (14.6 ng / μL), F4 / R3 (24.7 ng / μL), F5 / R3 (20.9 ng / μL), F6 / R3 (19.5 ng / μL), F7 / R3 (27.5 ng / μL), and F8 / R3 (14.2 ng / μL). The results indicated that primer pair F7 / R3 had the best amplification yield.

[0121] (4) Finally, F2 / R4, F2 / R3 and F7 / R3 were reviewed, and the results are as follows. Figure 10 The concentrations of samples a and b were measured to be 65.4 ng / µL, 71.6 ng / µL, and 82.3 ng / µL, respectively. Considering band clarity, specificity, and yield, the optimal primer pair for RHDV II was determined to be F2 / R3.

[0122] 3.3 Optimization of RPA reaction conditions and performance verification 3.3.1 Optimization of RPA reaction temperature A PCR instrument was used as the heating device. Optimal primer pairs were employed, and six different temperature gradients were set (37 ℃; 38 ℃; 39 ℃; 40 ℃; 41 ℃; 42 ℃). The amplification reaction time was 20 min. The concentrations of RHDVⅠ-RPA products at different reaction temperatures were 132.4, 103.1, 92.6, 87.6, 101.1, and 99.2 ng / µL, respectively. Figure 11 c). Gel electrophoresis results ( Figure 11 (a) The product concentration was high and the band was single when amplified at 37 °C, which was determined to be the optimal reaction temperature for RHDVⅠ-RPA; the purified concentrations of RHDVⅡ-RPA products were 84.0, 80.2, 79.2, 63.1, 27.2, and 30.6 ng / µL, respectively. Figure 11 (d). Gel electrophoresis results ( Figure 11 (b) Amplification at 38 °C resulted in the fewest nonspecific bands and the clearest target band, therefore 38 °C was selected as the optimal reaction temperature for RHDVⅡ-RPA.

[0123] 3.3.2 RPA Reaction Time Optimization Reaction time gradients of 15, 20, 25, and 30 min were set at their respective optimal reaction temperatures. The RHDVⅠ-RPA product concentrations were 85.7, 108.4, 64.5, and 71.2 ng / µL, respectively. Figure 12 c). Combining product concentration and band clarity ( Figure 12 (a) The amplification efficiency was highest at 20 min, which was determined to be the optimal reaction time. The concentrations of RHDVⅡ-RPA products were 39.8, 77.2, 70.6, and 79.0 ng / µL, respectively. Figure 12 (d). The overall product concentration and band clarity ( Figure 12 (b) Similarly, 20 minutes was selected as the optimal reaction time.

[0124] 3.4 RPA Sensitivity and Specificity Tests 3.4.1 RPA Sensitivity Test RHDVⅠ-VP60 plasmid (10 4 copies / µL) and RHDVⅡ-VP60 plasmid (10 copies / µL) 4 The copies / µL were serially diluted 10-fold to obtain 10 1 Up to 10 6 Template diluted 1 / 2. RPA amplification was performed under the established optimized conditions. Gel electrophoresis results ( Figure 13 As shown in a) and b), the detection limit of RHDVⅠ-RPA is 10 copies / µL (10 3(diluted 10 times), the detection limit of RHDVⅡ-RPA is 10 copies / µL (10 times dilution). 3 (Dilution times).

[0125] 3.4.2 RPA Specificity Test (1) RHDVⅠ-RPA specificity detection: RHDVⅠ cDNA was used as a positive template, and RPA amplification was performed using primer pair F4 / R8. Negative samples included RHDVⅡ cDNA, RCV plasmid, FCV cDNA, and nucleic acids from five rabbit-derived bacteria. Electrophoresis results ( Figure 14 The results showed that only the RHDVⅠ positive template lane showed a specific band of the expected size (approximately 258 bp), while the other lanes and the ddH2O negative control showed no amplification, indicating that the RHDVⅠ-RPA primers had good specificity and no cross-reactivity.

[0126] (2) RHDVⅡ-RPA specificity detection: RHDVⅡ cDNA was used as a positive template, and primer pair F2 / R3 was used for RPA amplification. Negative samples included RHDVⅠ cDNA, RCV, FCV, and nucleic acids from five rabbit-derived bacteria. Electrophoresis results ( Figure 15 The results showed that only the RHDVⅡ positive template lane showed a specific band of the expected size (approximately 352 bp), while the other lanes and the negative control showed no amplification, indicating that the RHDVⅡ-RPA primers had good specificity and no cross-reactivity.

[0127] Example 2: Establishment of RHDVⅠ-RPA-PfAgo and RHDVⅡ-RPA-PfAgo nucleic acid detection methods This embodiment will establish a single RPA-PfAgo nucleic acid detection method for RHDV I and RHDV II based on the RPA primers screened and designed in Example 1, and simultaneously design specific guide DNA (gDNA) targeting the inter-type differential sites of the two viral types. Combining RPA isothermal amplification technology with the specific cleavage characteristics of PfAgo, a single RPA-PfAgo nucleic acid detection method will be established. The key reaction conditions and component ratios for RPA amplification and PfAgo cleavage will be systematically optimized. The sensitivity, specificity, and repeatability of the two single systems will be comprehensively evaluated. The detection capability of the method on clinical samples will be preliminarily verified, and the usability of the detection method will be verified by comparison with the real-time PCR method. Finally, a stable and feasible single detection workflow will be established, providing key technical parameters for the subsequent integration of dual detection systems.

[0128] I. Sample Source Positive samples included: RHDV type I VP60 plasmid, RHDV type II VP60 plasmid, RHDV type I cDNA, and RHDV type II cDNA.

[0129] Negative samples included the full-length plasmid of rabbit calicivirus (RCV) VP60, feline calicivirus (FCV) cDNA, and nucleic acids from various rabbit-derived bacteria, including: *Escherichia coli*, *Pasteurella multocida*, *Staphylococcus spp.*, *Salmonella enterica*, and *Klebsiella pneumoniae*. All nucleic acid materials were extracted, purified, and stored at -20 °C for later use. The source was the same as in Example 1.

[0130] II. Specific Operating Methods 2.1 Design and Synthesis of gDNA and Reporter (1) RPA primers: The optimal primer pairs selected in Example 1 were used. For RHDV I type, F4 / R8 was used, and for RHDV II type, F2 / R3 was used.

[0131] (2) Design and synthesis of PfAgo-gDNA oligonucleotide sequence and Reporter probe Six candidate gDNAs were designed for RHDV type I, combined into three groups (gDNA1 / 2, gDNA3 / 4, and gDNA5 / 6); four candidate gDNAs were designed for RHDV type II, combined into two groups (gDNA1 / 2 and gDNA3 / 4). All gDNAs underwent 5' phosphorylation modification during synthesis. After the RPA-PfAgo cleavage system was established, the optimal gDNA combinations were screened through experimental evaluation.

[0132] Reporter probes: RHDV I type Reporter is a ROX-labeled probe; RHDV II type Reporter is a FAM-labeled probe. Both the gDNA and Reporter probes were synthesized by Suzhou Hongxun Biotechnology Co., Ltd. Detailed information on the gDNA and specific Reporter sequences is provided in Tables 7 and 8.

[0133] Table 7. Primer sequences for RHDVⅠ PfAgo

[0134] Table 8. Primer sequences for RHDVⅡ PfAgo

[0135] 2.2 Establishment and Optimization of the RHDV-PfAgo System The RPA-PfAgo detection system was prepared according to the following composition: 10×Buffer 2.5 μL, 100 mM Mg 2+2 μL of gDNA-1 (100 μM), 0.5 μL of gDNA-2 (100 μM), 0.5 μL of inorganic pyrophosphatase (PPi), 2 μL of Reporter probe (10 μM), 2 PfAgo microspheres, 25 μL of purified RPA product, and the remainder was made up to 50 μL with enzyme-free ddH2O. See Table 9 for specific components and amounts.

[0136] After preparation, the system was immediately placed in a real-time PCR instrument for signal detection. The reaction program was set to incubate at 95 °C for 30 minutes, with fluorescence data acquired every 30 seconds. After the reaction, the reaction products were visualized using an imaging system.

[0137] Table 9 RPA-PfAgo protein microsphere cleavage system

[0138] To obtain optimal fluorescence signal readings, this embodiment systematically optimized the key reaction parameters and component concentrations of the PfAgo microsphere cleavage system. The optimization focused on two aspects: first, reaction conditions, including the optimal reaction temperature and time for the Ago microspheres; and second, reaction components, including the screening of the optimal gDNA combination, gDNA concentration, and Mg content. 2+ Gradient testing of concentration and Reporter probe concentration was conducted, with optimization of different conditions and systems when reading fluorescence values ​​using a real-time PCR instrument, and three replicates for each sample.

[0139] 2.3 RHDV-PfAgo Sensitivity Test RHDVⅠ-VP60 plasmid (10 4 copies / µL) and RHDVⅡ-VP60 plasmid (10 copies / µL) 4 The copies / µL were serially diluted 10-fold to obtain 10 1 Up to 10 6 Six gradients of cDNA templates were prepared. After RPA amplification, the reaction products were purified using a DNA purification kit. The established RHDVⅠ-RPA-PfAgo and RHDVⅡ-RPA-PfAgo methods were used for detection, with ddH2O as a negative control. Changes in fluorescence signals were observed and recorded.

[0140] 2.4 RHDV-PfAgo Specificity Test Specificity assessment used RHDVⅠ and RHDVⅡ cDNA as positive templates and various rabbit-related pathogen nucleic acids as negative templates, including: rabbit calicivirus plasmid, feline calicivirus cDNA, rabbit-derived Escherichia coli nucleic acid, rabbit-derived Pasteurella nucleic acid, rabbit-derived Staphylococcus nucleic acid, rabbit-derived Salmonella nucleic acid, and rabbit-derived Klebsiella pneumoniae nucleic acid. A ddH2O negative control was also included. All samples were amplified by RPA, and the reaction products were purified using a DNA purification kit. The established RHDVⅠ-RPA-PfAgo and RHDVⅡ-RPA-PfAgo methods were used for detection, and changes in fluorescence signals were observed and recorded.

[0141] 2.5 RHDV-PfAgo Repeatability Test Take RHDVⅠ and RHDVⅡ positive plasmids (both diluted to 10). 3 Using copies / µL as templates, the established single nucleic acid RPA-PfAgo detection system was validated. Each experiment included three replicates and one negative control (ddH2O), and was independently repeated on three different dates. Fluorescence values ​​of the RHDVⅠ (ROX) and RHDVⅡ (FAM) channels were read separately, and the stability and reproducibility of the detection method were evaluated by comparing the differences in the positive control detection results across the three dates.

[0142] 3 Results 3.1 Screening and Validation of PfAgo-gDNA To screen for the optimal gDNA combination that can efficiently guide PfAgo to cleave the target, this embodiment uses the RHDVⅠ and RHDVⅡ plasmids constructed in Example 1 as templates and performs cleavage reactions with different gDNA combinations. The cleavage efficiency is evaluated by fluorescence signal intensity and chemical imaging results.

[0143] 3.1.1 RHDV type I gDNA screening results Six candidate gDNAs were designed targeting the RHDV I sequence and combined into three groups (gDNA1 / 2, gDNA3 / 4, and gDNA5 / 6) for testing. Real-time fluorescence monitoring results showed ( Figure 16 (a) and (b) all three gDNA groups could guide PfAgo to cleave the target and generate fluorescence signals, but the signal intensities differed significantly. The gDNA5 / 6 combination showed the fastest fluorescence increase, reaching its highest value at 30 min, significantly higher than the gDNA1 / 2 and gDNA3 / 4 combinations. Chemical imaging results ( Figure 16 (c) Consistent with quantitative fluorescence data, the gDNA5 / 6 combination produced the strongest imaging signal. Therefore, gDNA5 and gDNA6 were determined to be the optimal guide DNAs for RHDV I and used in subsequent experiments.

[0144] 3.1.2 RHDV II gDNA Screening Results Four candidate gDNAs were designed targeting the RHDV II sequence and combined into two groups (gDNA1 / 2 and gDNA3 / 4) for testing. Fluorescence monitoring results showed ( Figure 17 In samples a and b), the fluorescence signal rise rate and fluorescence value at 30 min for the gDNA3 / 4 combination were significantly better than those for the gDNA1 / 2 combination. (Chemical imaging results) Figure 17 (c) also shows a stronger imaging signal from the gDNA3 / 4 combination. Therefore, gDNA3 and gDNA4 were determined to be the optimal guide DNAs for RHDV type II.

[0145] 3.2 Optimization of the RHDV-PfAgo Single Detection System To obtain optimal fluorescence signal output, this embodiment systematically optimizes the key parameters of the RHDV I and RHDV II single detection systems, including reaction temperature, reaction time, and Mg. 2+ Concentration, gDNA concentration, and Reporter probe concentration.

[0146] 3.2.1 Optimization of PfAgo reaction temperature The recommended reaction temperature for PfAgo microspheres is 90–95 °C. In this study, five temperature gradients of 91, 93, 95, 97, and 99 °C were set for optimization.

[0147] (1) Temperature optimization results of RHDV I type like Figure 18 As shown in Figures a and b, the RHDV I system exhibits the strongest fluorescence signal at 95 °C, and the fluorescence signal curve shows the earliest rise and amplification. Fluorescence signals are weaker at 91 °C, 97 °C, and 99 °C. Chemical imaging results ( Figure 18 As shown in (c), the imaging signal is brightest at 95 °C. Therefore, the optimal reaction temperature for RHDVⅠ type PfAgo cutting is determined to be 95 °C.

[0148] (2) Temperature optimization results of RHDVⅡ type like Figure 19 As shown in Figures a and b, the RHDVⅡ system also exhibits the strongest fluorescence signal at 95 ℃ and the weakest signal at 99 ℃. The signal intensities at 91 ℃, 93 ℃, and 97 ℃ are all lower than those at 95 ℃. (Chemical imaging results) Figure 19 c) is consistent with the fluorescence data. Therefore, the optimal reaction temperature for RHDVⅡ is determined to be 95 °C.

[0149] 3.2.2 Optimization of PfAgo reaction time At a reaction temperature of 95 °C, five time gradients of 20, 25, 30, 35, and 40 min were set to monitor the time when the fluorescence signal reached the plateau phase.

[0150] (1) Time optimization results of RHDV Type I like Figure 20 As shown in Figures a and b, the fluorescence signal of the RHDV I system rapidly increases in the first 20 minutes of the reaction, reaches its peak at 30 minutes, plateaus after 30 minutes, and shows no significant difference at 40 minutes compared to 30 minutes. (Chemical imaging results) Figure 20 As shown in c), there was no significant difference in imaging signal brightness at 25 min, 30 min, and 40 min. The amplification curve showed the earliest upward movement at 30 min and the amplification effect was stable, thus determining that the optimal reaction time for RHDV I was 30 min.

[0151] (2) Time optimization results of RHDV II like Figure 21 As shown in a and b, the fluorescence signal of the RHDV II system increases slightly slower than that of the I system. The signal continues to rise after 25 min, reaches its peak at 30 min, and then stops increasing after 30 min. (Chemical imaging results) Figure 21 c) shows no significant difference in imaging signal brightness at 25 min, 30 min, and 40 min. The amplification curve at 35 min showed the earliest rise and the amplification effect was stable, confirming that the optimal reaction time for RHDV II was 35 min.

[0152] 3.2.3 Mg 2⁺ Concentration optimization Set five Mg molecules at concentrations of 2, 3, 4, 5, and 6 mM. 2⁺ The final concentration gradient was used to investigate its effect on cutting efficiency.

[0153] (1) RHDV I type Mg 2⁺ Concentration optimization results like Figure 22 As shown in Figures a and b, the fluorescence amplification curves show that the RHDV type I system in Mg 2⁺ The fluorescence signal is strongest at a concentration of 3 mM, Mg 2⁺ The fluorescence signal was weakest at a concentration of 2 mM. Mg 2⁺ The signal was slightly weaker at concentrations of 4 mM, 5 mM, and 6 mM than at 3 mM, but the difference was not statistically significant. (Chemical imaging results) Figure 22 As shown in (c), the imaging signal is brightest at 6 mM. Therefore, the optimal Mg for RHDVⅠ type is determined. 2⁺ The final concentration was 6 mM.

[0154] (2) RHDVⅡ type Mg2+ Concentration optimization results like Figure 23 As shown in Figures a and b, the fluorescence amplification curves show that the RHDV II system in Mg 2⁺ The fluorescence signal was strongest at a concentration of 6 mM, weakest at 2 mM, and increased with increasing concentration from 3 to 5 mM. (Chemical imaging results) Figure 23 As shown in c), the imaging signal brightness is similar between 2 and 6 mM. The optimal Mg for RHDVⅡ type was determined. 2+ The final concentration was 6 mM.

[0155] 3.2.4 Optimization of gDNA Concentration Five gDNA final concentration gradients of 0.6, 0.8, 1.0, 1.2, and 1.4 μM were set up to investigate their effect on cleavage efficiency.

[0156] (1) Optimization results of RHDV type I gDNA concentration like Figure 24 As shown in Figures a and b, the RHDV type I system (gDNA5 / gDNA6) exhibits the strongest fluorescence signal at a gDNA concentration of 1.0 μM. The signal is weaker at 0.6 μM and 0.8 μM, and decreases further at 1.2 μM and 1.4 μM. Chemical imaging results ( Figure 24 As shown in c), the imaging signal was brightest at 1.0 μM. Therefore, the optimal final concentration of gDNA for RHDV type I was determined to be 1.0 μM.

[0157] (2) Optimization results of RHDV II gDNA concentration like Figure 25 As shown in a and b, the RHDV II system (gDNA3 / gDNA4) exhibits the most stable fluorescence signal at a gDNA concentration of 1.0 μM. The fluorescence signal is stronger at 0.6 μM, but the chemical imaging results ( Figure 25 As shown in c), the stability is not as good as at 1.0 μM. Therefore, the optimal final concentration of gDNA for RHDV type II was determined to be 1.0 μM.

[0158] 3.2.5 Optimization of Reporter Probe Concentration Five final concentration gradients of the Reporter probe (0.2, 0.3, 0.4, 0.5, and 0.6 μM) were set up to investigate their effect on signal intensity.

[0159] (1) Optimization results of RHDV I Reporter concentration like Figure 26As shown in a and b, the RHDV I system (ROX probe) exhibits the strongest fluorescence signal at a probe concentration of 0.5 μM. The signal increases with increasing concentration from 0.2 to 0.5 μM. Chemical imaging results ( Figure 26 As shown in c), the imaging signal is brightest at 0.5 μM. Therefore, the optimal final concentration of the Reporter for RHDVⅠ is determined to be 0.5 μM.

[0160] (2) Optimization results of RHDVⅡ type Reporter concentration like Figure 27 As shown in Figures a and b, the RHDV II system (FAM probe) exhibits the strongest fluorescence signal at a probe concentration of 0.6 μM, but its stability is poor. The signal increases with increasing concentration from 0.2 to 0.5 μM. Chemical imaging results ( Figure 27 As shown in c), the imaging signal is optimal at 0.5 μM. Therefore, the optimal final Reporter concentration for RHDVⅡ is determined to be 0.5 μM.

[0161] 3.3 Sensitivity Test of RHDV-PfAgo Single Detection System RHDVⅠ-VP60 plasmid (10 4 copies / µL) and RHDVⅡ-VP60 plasmid (10 copies / µL) 4 The copies / µL were serially diluted 10-fold to obtain 10 l Dilute to 10 times 6 Six concentration gradients of template were prepared by dilution. Each gradient template was amplified by RPA and used as a cleavage template. The fluorescence signal was detected using an optimized singleton system.

[0162] (1) Results of RHDV Type I sensitivity test like Figure 28 As shown in a and b, the fluorescence signal intensity of the RHDVⅠ system gradually weakens with increasing template dilution factor (decreasing template concentration). 10 1 Diluted template (10 times) 3 The strongest fluorescence signal was observed at dilutions of 10 copies / µL, with the template signal decreasing progressively with increasing dilution. 3 Even at a 10-fold dilution of the template (10 copies / µL), a significantly higher fluorescence signal than the negative control could still be detected, while 10 4 Fluorescence signals from templates diluted 10 times or more showed no significant difference compared to the negative control. The negative control consistently showed no signal increase. Chemical imaging results ( Figure 28 c) shows that 10 3 The chemiluminescence signal corresponding to the template diluted by 10 times is still visible to the naked eye. 4The signal becomes indistinguishable after dilution. Therefore, the detection limit of the RHDVⅠ single-detection system is 10 copies / µL.

[0163] (2) Sensitivity test results of RHDV II like Figure 29 As shown in a and b, the fluorescence signal of the RHDVⅡ system also gradually decreases with increasing template dilution factor. 10 l Diluted template (10 times) 3 The strongest fluorescence signal was observed at dilutions of 10 copies / µL, with the template signal decreasing progressively with increasing dilution. 3 Even at a 10-fold dilution (10 copies / µL), the template still showed a significantly higher fluorescence signal than the negative control. 4 Fluorescence signals of templates diluted 10 times or more showed no significant difference compared to the negative control. (Chemical imaging results) Figure 29 c) shows that 10 3 The chemiluminescent signal corresponding to the diluted template is identifiable. Therefore, the detection limit of the RHDVⅡ single-detection system is 10 copies / µL.

[0164] 3.4 Specificity test of the RHDV-PfAgo single detection system To evaluate the detection specificity of the two singlet systems, RHDVⅠ and RHDVⅡ cDNA were used as positive controls, and RCV plasmid, FCV cDNA and nucleic acids of five rabbit-derived bacteria were used as negative samples, with ddH2O as a negative control. The optimized singlet system was used for detection.

[0165] (1) Specific detection results of RHDV type I like Figure 30 As shown in a and b, only the ROX channel of the RHDVⅠ positive template produced a strong fluorescence signal. The fluorescence signals of the RHDVⅡ template, RCV, FCV, and the five bacterial nucleic acids, although showing non-specific background signals, did not show any upward movement or amplification, and were not significantly different from the negative control. Chemical imaging results ( Figure 30 As shown in c), only the well corresponding to RHDVⅠ showed a red chemiluminescent signal, while the other wells showed no luminescence signal. The results indicate that the RHDVⅠ single-detection system has good specificity and no cross-reactivity with other rabbit pathogens.

[0166] (2) Specific detection results of RHDV type II like Figure 31 As shown in a and b, only the FAM channel of the RHDV II positive template produced a strong fluorescence signal. The fluorescence signals of the RHDV I template and other negative samples, although showing nonspecific background signals, did not exhibit any upward movement or amplification, and were not significantly different from the negative control. (Chemical imaging results) Figure 31As shown in c), only the well corresponding to RHDVⅡ showed a green chemiluminescent signal. The results indicate that the RHDVⅡ single-detection system also possesses good specificity.

[0167] 3.5 Repeatability Tests of the RHDV-PfAgo Single-Symmetric Detection System (1) RHDV Type I repeatability test results like Figure 32 As shown in Figures a, b, and c, the fluorescence signal intensity of the RHDVⅠ system remained stable in the three independent experiments on days 1, 5, and 10, with the fluorescence values ​​of the three replicates being essentially similar across each experiment. The CT values ​​were also similar across the three experiments. No signal was detected in the negative control in any of the experiments. These results indicate that the RHDVⅠ single-detection system has good repeatability.

[0168] (2) RHDV II type repeatability test results like Figure 33 As shown in Figures a, b, and c, the fluorescence signal intensity of the RHDVⅡ system remained stable in the three independent experiments on days 1, 5, and 10, with the fluorescence values ​​of the three replicates being essentially similar across each experiment. The overall fluctuation in fluorescence values ​​between the three experiments was small. The negative control consistently showed no signal. These results indicate that the RHDVⅡ single-detection system also exhibits good repeatability.

[0169] Example 3: Establishment and Preliminary Application of the RHDV-RPA-PfAgo Dual Detection Method This embodiment aims to integrate the research content of Embodiments 1 and 2 to construct a single-tube dual RPA-PfAgo detection platform capable of simultaneously identifying two types of viruses. Details are as follows: I. Sample Source Positive samples included: RHDV type I VP60 plasmid, RHDV type II VP60 plasmid, RHDV type I cDNA, and RHDV type II cDNA.

[0170] Negative samples included the full-length plasmid of rabbit calicivirus (RCV) VP60, feline calicivirus (FCV) cDNA, and nucleic acids from various rabbit-derived bacteria, including: *Escherichia coli*, *Pasteurella multocida*, *Staphylococcus spp.*, *Salmonella enterica*, and *Klebsiella pneumoniae*. All nucleic acid materials were extracted, purified, and stored at -20 °C for later use.

[0171] II. Specific Operating Methods 2.1 Establishment of Dual RPA Two pairs of optimal RPA primers for RHDV I and RHDV II were co-reacted in the same reaction tube (F4 / R8 for RHDV I and F2 / R3 for RHDV II). The constructed RHDV I and RHDV II positive plasmids were used as positive templates, and sterile, enzyme-free water was used as a negative control to simultaneously amplify the target DNA of both viruses. The components of the premix are shown in Table 10. Table 10 RHDV-Dual RPA Premixed Liquid System

[0172] After thorough mixing, the premixed solution was dispensed into reaction tubes containing lyophilized enzyme powder (the volume increases by 2 μL after dissolution). Then, 1.4 μL of 0.5 M magnesium acetate was added to the inside of the tube cap. After capping, the tubes were briefly centrifuged to mix. The reaction was then started with a total volume of 50 μL. The reaction program was 38°C for 20 min. The reaction temperature of 38°C was chosen because the optimal temperature for type 1 is 37°C, and for type 2 it is 38°C. However, considering that type 1 has a slightly higher amplification efficiency than type 2, the temperature for the dual RPA was set at 38°C.

[0173] 2.1.1 Optimization of the ratio of dual RPA primers To determine the optimal primer concentrations for the two genotypes in the dual RPA reaction, the upstream and downstream primer concentrations for RHDVⅠ and RHDVⅡ were simultaneously optimized. Following the RPA kit's recommended final primer concentration (0.42 μM), four different primer concentration gradients were set up, with the following addition amounts and corresponding concentrations: 1.7 μL (final concentration 0.34 μM), 1.9 μL (final concentration 0.38 μM), 2.1 μL (final concentration 0.42 μM), and 2.2 μL (final concentration 0.46 μM). The reaction system was placed in a PCR instrument and incubated at 38°C for 20 min. After the reaction, the products were purified using a DNA purification kit, and the amplification effect was detected by 1.5% agarose gel electrophoresis. The optimal primer concentration was selected based on the clarity and brightness of the bands and the consistency of the amplified bands for the two genotypes.

[0174] 2.1.2 Dual RPA Sensitivity and Specificity Tests Under optimized dual RPA reaction conditions, the detection sensitivity of this system for RHDVⅠ and RHDVⅡ was determined, respectively. RHDVⅠ-VP60 plasmid (10... 4 copies / µL) and RHDVⅡ-VP60 plasmid (10 copies / µL) 4The plasmids were serially diluted 10-fold using ddH2O to obtain six template dilutions. Each dilution served as a positive template, and ddH2O as a negative control for double RPA amplification. The reaction system contained two primer pairs, RHDVⅠ-RPA-F4 / R8 and RHDVⅡ-RPA-F2 / R3, with other components and reaction conditions as described above. The amplified products were purified using a DNA purification kit and detected by 1.5% agarose gel electrophoresis. The lowest template concentration at which a specific band was clearly detected was defined as the limit of detection.

[0175] 2.2 Dual RPA-PfAgo Nucleic Acid Detection System 2.2.1 Establishment of a dual RPA-PfAgo detection reaction system To achieve simultaneous single-tube detection of RHDV I and RHDV II, an RPA-PfAgo dual nucleic acid detection system was established based on the characteristics of the PfAgo protein. The system works as follows: First, target fragments of both viruses are isothermally amplified using RPA. Under 95°C thermal activation, the amplified products are guided by specific primary gDNA to PfAgo recognize and cleave the target double strand, releasing new secondary gDNA. The newly generated secondary gDNA further guides PfAgo to cleave reporter molecules modified with fluorophores and quenchers at both ends, thereby releasing a fluorescent signal. By detecting the fluorescence intensity of channels corresponding to different targets, simultaneous qualitative and semi-quantitative analysis of RHDV I and RHDV II is achieved.

[0176] The purified double RPA amplification product was used as a cleavage template for PfAgo cleavage. The reaction system was prepared according to the PfAgo enzyme instructions and the optimization results of the single system. The total system volume was 50 μL. The specific components and amounts are shown in Table 11. Table 11 Dual RPA-PfAgo protein microsphere cleavage system

[0177] 2.2.2 Optimization of the RPA-PfAgo Dual Detection Reaction System To balance the cleavage efficiency of RHDVⅠ and RHDVⅡ targets and achieve balanced output of dual fluorescence signals, this embodiment systematically optimized the ratio of gDNA to fluorescent reporter molecules while maintaining fixed RPA amplification conditions and the basic PfAgo reaction system. The concentrations of other components in the reaction system were based on the PfAgo enzyme instructions and the optimized PfAgo single nucleic acid reaction system.

[0178] (1) Reporter ratio optimization: First, the amount of FAM-Reporter was fixed (2 μL), and the molar ratio of ROX-Reporter to FAM-Reporter was set to 1:1, 2:1, 3:1, and 4:1, respectively; then, the amount of ROX-Reporter was fixed (2 μL), and the ratio of ROX-Reporter to FAM-Reporter was set to 1:1, 1:2, 1:3, and 1:4, respectively. The dual RPA amplification product was used as a template for detection. With the signal intensity ratio of the two fluorescence channels close to 1:1, the optimal Reporter ratio was determined based on the signal balance of the two channels, taking into account the results of the chemical imaging.

[0179] (2) gDNA ratio optimization: Different ratios of RHDVⅠ-gDNA to RHDVⅡ-gDNA were set, including 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:1, 1.5:1, 2:1, 2.5:1, and 3:1. The dual RPA amplification products were used as templates for detection. The optimal gDNA ratio was determined based on the results of the chemical imaging, with the signal intensity ratio of the two fluorescence channels approaching 1:1, and the balance of the dual-channel signals.

[0180] 2.3 Dual RPA-PfAgo Sensitivity Test The optimized RPA-PfAgo dual detection system was used to evaluate its detection sensitivity for RHDVⅠ and RHDVⅡ. RHDVⅠ-VP60 plasmid (10... 4 copies / µL) and RHDVⅡ-VP60 plasmid (10 copies / µL) 4 (copies / µL), and then performed a 10-fold serial dilution to obtain 10 1 Up to 10 6 Template was diluted 10 times. A mixture of plasmids at each dilution was used as a positive template (simulating double infection), and ddH2O was used as a negative control. RPA amplification and PfAgo cleavage reaction were performed according to the optimized reaction system, and the fluorescence values ​​of each channel were read using a real-time PCR instrument.

[0181] 2.4 Dual RPA-PfAgo Specificity Assay Using RHDVⅠ and RHDVⅡ cDNA as positive controls and ddH2O as a negative control, several common rabbit pathogen nucleic acids were selected as negative samples, including: rabbit calicivirus plasmid, feline calicivirus cDNA, rabbit-derived Escherichia coli nucleic acid, rabbit-derived Pasteurella multocida nucleic acid, rabbit-derived Staphylococcus aureus nucleic acid, Salmonella nucleic acid, and rabbit-derived Klebsiella pneumoniae nucleic acid. RPA amplification was performed using each sample as a template, and the optimized RPA-PfAgo dual system was used for detection. The fluorescence values ​​of the ROX and FAM channels were recorded to determine the presence of cross-reactivity.

[0182] 2.5 Dual RPA-PfAgo Repeatability Test To further verify the reproducibility of the RHDV dual RPA-PfAgo detection system, RHDVⅠ and RHDVⅡ positive plasmids were used as templates (final concentrations were 10⁻⁶ and 10⁻⁶ respectively). 3 copies / µL and 10 3 After double RPA amplification and product purification, the optimized detection system was used for fluorescence detection. Each experiment included three positive replicates and one negative control (ddH2O), and three independent replicates were performed on three different dates. After the reaction, the fluorescence values ​​of the ROX channel (RHDVⅠ) and FAM channel (RHDVⅡ) were simultaneously read. The stability and reproducibility of the detection method were evaluated by comparing the fluctuations of the positive control detection results in each channel across the three independent experiments.

[0183] 2.6 Clinical Sample Testing To evaluate the clinical applicability and reliability of the established dual RPA-PfAgo detection system, the method established in this embodiment was used to detect the collected clinical samples.

[0184] (1) Sample collection and processing: From March 2023 to June 2024, clinically suspected rabbit hemorrhagic disease (symptoms such as depression, anorexia, lethargy, sudden death, and bleeding from the mouth and nose) were collected from commercial rabbit farms in Meishan, Ziyang, and Chengdu, Sichuan Province. Autopsies were performed on these rabbits to obtain liver, lung, and kidney tissue samples from dying or dead rabbits, totaling 24 samples (18 liver samples, 3 lung samples, and 3 kidney samples). The samples were immediately stored at -80℃ for later use.

[0185] (2) Nucleic acid extraction: Take about 50 mg of each tissue sample, extract total RNA and reverse transcribe it according to the method in Example 1 to obtain cDNA template, and store it at -20℃ for later use.

[0186] (3) RT-qPCR detection: The specific RT-qPCR detection methods for RHDV types I and II (RHDVⅠ-OF, RHDVⅠ-OR, and RHDVⅠ-O-Probe, RHDVⅡ-CF, RHDVⅡ-CR, and RHDVⅡ-C-Probe in Table 5) reported in the reference (Wang Guanxuan, Chen Mengmeng, Qiu Rulong, et al. 2023. Establishment and application of dual TaqMan probe fluorescence quantitative PCR detection method for rabbit hemorrhagic disease virus types 1 and 2 [J]. Jiangsu Agricultural Sciences, (51)(18):40-44.) were used to perform parallel detection on 24 samples. The reaction system and procedure were performed according to the established protocol. The Ct value and genotyping results of each sample were recorded.

[0187] (4) Dual RPA-PfAgo detection: The RPA primers (RHDVⅠ-F4 / R8, RHDVⅡ-F2 / R3) optimized in Example 1 and the dual RPA-PfAgo detection system established in Example 2 were used to detect cDNA in 24 samples. The specific operation steps and reaction conditions were performed according to the optimization results of Examples 2 and 3. After the reaction, the results were read by fluorescence signal or gel electrophoresis, and the detection status of RHDVⅠ and RHDVⅡ and the mixed infection status of each sample were recorded.

[0188] (5) Compliance analysis: Using RT-qPCR detection results as the reference standard, the positive compliance rate, negative compliance rate and overall compliance rate of the dual RPA-PfAgo detection system were calculated.

[0189] 3 Results 3.1 Establishment of Dual RPA 3.1.1 Establishment of Dual RPA First, perform singlet RPA amplification to determine the positions of the RHDV type I and RHDV type II RPA product bands. Figure 34 (a) Double RPA amplification was performed using the selected RHDV I primers F4 / R8 and RHDV II primers F2 / R3. The two primer pairs and the template were placed in the same reaction tube for co-reaction. Electrophoresis results were then analyzed. Figure 34 As shown in b), both target bands of RHDVⅠ and RHDVⅡ were specifically amplified in the same reaction system, with clear boundaries between the two bands and no cross-reaction, indicating that the selected primer combination is suitable for dual RPA detection system.

[0190] 3.1.2 Optimization of the ratio of dual RPA primers The concentrations of RHDVⅠ and RHDVⅡ primers were adjusted and optimized. The effect of different primer concentrations on the double RPA amplification efficiency is as follows: Figure 35 As shown in the figure. Electrophoresis results showed that when the final primer concentration was 0.42 μM, the amplification bands for both genotypes were the clearest and brightest, and the brightness between the two bands was relatively uniform. When the primer concentration was lower (0.34 μM, 0.38 μM), the amplification bands were darker, and the product amount was significantly insufficient; when the primer concentration was higher (0.46 μM), the band clarity was not significantly improved. Therefore, the optimal final primer concentration for the dual RPA system was determined to be 0.42 μM.

[0191] 3.1.3 Dual RPA Sensitivity Test The sensitivity detection results of the dual RPA system for RHDVⅠ and RHDVⅡ are as follows: Figure 36As shown in the figure. Electrophoresis results showed that the amplification bands gradually faded as the template concentration decreased. RHDVI at a template concentration of 10 copies / μl (10 3 Even at a template concentration of 10 times (10 times dilution), a clear target band is still visible; below this concentration, no band is detected. Therefore, the detection limit for RHDVⅠ is 10 copies / μl; the detection limit for RHDVⅡ is 10 copies / μl (10 times dilution). 3 Even at a 10-fold dilution, specific bands could still be amplified, with a detection limit of 10 copies / μl. The results indicate that the established dual RPA method has comparable sensitivity to single RPA for detecting both viruses.

[0192] 3.1.4 Dual RPA Specificity Test Specific test results such as Figure 37 As shown, only lanes using RHDVⅠ and RHDVⅡ positive plasmids as templates showed clear specific amplification bands (RHDVⅠ band approximately 258 bp, RHDVⅡ band approximately 352 bp), while lanes containing RCV, FCV, and five rabbit-derived bacterial nucleic acid samples showed no amplification bands, and the negative control also showed no bands. The results indicate that the established dual RPA method can specifically detect RHDVⅠ and RHDVⅡ, and there is no cross-reactivity with other related pathogens.

[0193] 3.2 Establishment and optimization of the dual RPA-PfAgo reaction system 3.2.1 Establishment of the dual RPA-PfAgo reaction system Based on the endonuclease properties of the PfAgo protein, a dual nucleic acid detection system of RPA-PfAgo was successfully constructed. After the reaction, the sample was placed in a chemical imaging system, and the ROX channel (corresponding to RHDVⅠ) and FAM channel (corresponding to RHDVⅡ) were activated separately, and color overlay images were acquired. The results showed ( Figure 38 (a, b, c): Samples containing only the RHDV I template showed red fluorescence in the ROX channel and no signal in the FAM channel; samples containing only the RHDV II template showed green fluorescence in the FAM channel and no signal in the ROX channel; samples containing both RHDV I and RHDV II templates showed signals in both channels, which, when superimposed, appeared as yellow fluorescence. The negative control showed no fluorescence signal. The results indicate that the established dual detection system can specifically identify and distinguish between RHDV I and RHDV II.

[0194] 3.2.2 Reporter Proportion Optimization (1) With a fixed amount of FAM-Reporter, different ratios of ROX-Reporter and FAM-Reporter were set for optimization (Table 12). Fluorescence monitoring results showed that ( Figure 39(a, b, c) When the ROX:FAM ratio is 1:1, the signal strengths of the two channels are closest, with a signal ratio close to 1:1. As the ROX ratio increases, the RHDV I channel signal weakens. When the ROX:FAM ratio is 4:1, the signal strength deviation between the two channels is most significant, with the RHDV I channel signal being significantly weaker than the RHDV II channel.

[0195] Table 12 Reporter Proportion Optimization in Round 1

[0196] (2) With a fixed amount of ROX-Reporter, the ROX:FAM ratio was optimized at 1:1, 1:2, 1:3, and 1:4 (Table 13). The results show ( Figure 40 (a, b, c) As the FAM ratio increases, the RHDVⅡ channel signal gradually strengthens. When the ROX:FAM ratio is 1:3, the dual-channel signal strength is most balanced, and the difference between the dual-channel signal strength is the smallest.

[0197] Table 13 Reporter Proportion Optimization in Round 2

[0198] 3.2.3 Optimization of gDNA Ratio (1) Different ratios of RHDVⅠ-gDNA and RHDVⅡ-gDNA were set for optimization (Table 14). Fluorescence monitoring results showed that ( Figure 41 (a, b, c) When the ratio of the two types of gDNA is 1:3, the RHDVⅠ channel signal is slightly weaker than the RHDVⅡ channel signal. As the RHDVⅡ-gDNA ratio decreases, the RHDVⅡ channel signal becomes relatively stable, while the RHDVⅠ channel signal gradually increases. When the RHDVⅠ:RHDVⅡ gDNA ratio is 1:1, 1:1.5, and 1:2, the signal intensities of the two channels are relatively close. Considering the overall chemical imaging results, a ratio of 1:1.5 is more appropriate.

[0199] Table 14. Optimization of gDNA Ratio in Round 1

[0200] (2) Further verification of the reverse ratio optimization results was performed by setting different ratios of RHDVⅠ-gDNA and RHDVⅡ-gDNA for optimization (Table 15). Fluorescence monitoring results showed that ( Figure 42(a, b, c) When the ratio of the two types of gDNA is 3:1, the RHDV II channel signal is slightly weaker than the RHDV I channel; as the RHDV I-gDNA ratio decreases, the RHDV II channel signal shows a stable trend; when the RHDV I:RHDV II gDNA ratio is 1.5:1, 2:1 and 2.5:1, the dual-channel signal balance is better. Considering the chemical imaging results, 2:1 is more appropriate.

[0201] Table 15. Second round of gDNA ratio optimization

[0202] (3) The results of the gDNA ratio optimization were reviewed (Table 16), and the review results were further verified. Figure 43 (a, b, c) When the RHDVⅠ:RHDVⅡ gDNA ratio is 1.5:1, the dual-channel signal balance is optimal. Therefore, the optimal gDNA ratio is determined to be RHDVⅠ-gDNA : RHDVⅡ-gDNA = 1.5:1 (i.e., final RHDVⅠ-gDNA concentration of 0.6 μM and final RHDVⅡ-gDNA concentration of 0.4 μM).

[0203] Table 16 Review of gDNA Proportion Optimization

[0204] 3.3 Dual RPA-PfAgo Sensitivity Test An optimized dual detection system was used to detect mixed cDNA templates at different dilutions. Fluorescence monitoring results showed ( Figure 44 (a, b, c) As the template dilution factor increases, the fluorescence signal intensity of both the ROX channel (RHDVⅠ) and the FAM channel (RHDVⅡ) gradually decreases.

[0205] RHDVⅠ at a template concentration of 10 copies / μl (10 3 At a template concentration of 10 copies / μl, the fluorescence value of the corresponding channel was still significantly higher than that of the negative control (P<0.01), and the detection limit was 10 copies / μl; RHDVⅡ at a template concentration of 10 copies / μl (10 3 Even at a 10-fold dilution, a positive signal could still be detected, with a detection limit of 10 copies / μl. The results indicate that the established dual RPA-PfAgo method has the same detection sensitivity for both viruses as the single-detection system, and dual detection did not significantly sacrifice sensitivity.

[0206] 3.4 Dual RPA-PfAgo Specificity Assay Specific test results such as Figure 45As shown in a, b, and c. The RPA purification product using a mixed RHDVⅠ and RHDVⅡ cDNA as a template was detected using dual RPA-PfAgo. The positive control showed significant signal enhancement in both the ROX and FAM channels. While non-specific background signals were present in the fluorescence signals of RCV, FCV, and the five bacterial nucleic acids, no upward movement or amplification was observed, showing no significant difference from the negative control. Chemical imaging results ( Figure 46 As shown in c), the positive control wells exhibited a yellow chemiluminescent signal, while the other wells showed no luminescence signal. The results indicate that the dual RPA-PfAgo detection system has good specificity and shows no cross-reactivity with other rabbit pathogens.

[0207] 3.5 Dual RPA-PfAgo Repeatability Test The results of the repeatability test showed that ( Figure 46 In the three independent experiments (a, b, and c) on days 1, 5, and 10, the fluorescence signal intensities of the ROX and FAM channels remained stable. The fluorescence values ​​of the three replicates within each experiment were generally similar, and the overall fluctuation in fluorescence values ​​between the three experiments was small. No signal was detected in the negative control in any of the experiments. The results indicate that the dual RPA-PfAgo detection system has good intra- and inter-batch repeatability.

[0208] 3.6 Clinical Sample Testing We performed parallel testing on 24 clinical samples (18 liver samples, 3 lung samples, and 3 kidney samples) using both RT-qPCR and dual RPA-PfAgo methods. The results are as follows: (1) RT-qPCR detection results are as follows Figure 47 In the samples a, b, c, and d: among the 24 samples, a total of 6 samples were positive for pure RHDV I, 12 samples were positive for pure RHDV II, and 6 samples were mixed infection samples (i.e., samples that tested positive for both RHDV I and RHDV II).

[0209] (2) Dual RPA-PfAgo detection results: The dual detection system established in this embodiment was used to detect 24 samples, and the results showed that ( Figure 48 ): 6 samples were positive for pure RHDVⅠ, 12 samples were positive for pure RHDVⅡ, and 6 samples were mixed infection samples. The results were completely consistent with the RT-qPCR test results. The CT values ​​corresponding to RT-qPCR are shown in Tables 17, 18 and 19.

[0210] Table 17 Results of RT-qPCR detection of pure RHDV type I samples

[0211] Table 18 Results of RT-qPCR detection of pure RHDV type II samples

[0212] Table 19 Results of RT-qPCR RHDV pooled sample detection

[0213] (3) Concordance rate analysis: Using RT-qPCR as the gold standard, the positive concordance rate, negative concordance rate, and overall concordance rate of the dual RPA-PfAgo detection system were all 100% (24 / 24). The specific test results are summarized in Table 20.

[0214] Table 20 Summary of Clinical Sample Test Results

[0215] In summary, this invention successfully establishes a dual RPA-PfAgo nucleic acid detection method for the simultaneous detection of RHDV types I and II. This method integrates the advantages of RPA isothermal amplification and PfAgo specific cleavage, achieving single-tube, rapid, highly sensitive, and highly specific genotyping detection. Results can be intuitively interpreted through dual-channel fluorescence or chemical imaging. This method provides strong technical support for RHDV field screening, early warning, epidemiological monitoring, and precise prevention and control.

[0216] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. A dual RPA-PfAgo nucleic acid detection kit for simultaneous detection or differentiation of RHDVⅠ and RHDVⅡ, characterized in that, include: (a) A dual RPA primer combination; including: (a1) The first primer pair used for amplifying the conserved region of the RHDV type I VP60 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO:7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:16; and, (a2) The second primer pair used to amplify the conserved region of the RHDV II VP60 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 24, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 27; (b) gDNA combinations; including: (b1) The first pair of gDNA used to guide the PfAgo protein to specifically cleave the RHDV type I RPA amplicons, the nucleotide sequences of which are shown in SEQ ID NO: 52 and SEQ ID NO: 53; and, (b2) The second pair of gDNA used to guide the PfAgo protein to specifically cleave the RHDV II type RPA amplicon, the nucleotide sequences of which are shown in SEQ ID NO: 57 and SEQ ID NO: 58; (c) Fluorescent reporter probe assemblies; including: (c1) A first probe for detecting RHDV type I, the nucleotide sequence of which is shown in SEQ ID NO: 54, and the 5' end is labeled with a first fluorescent group and the 3' end is labeled with a first quencher group; and, (c2) A second probe for detecting RHDV type II, the nucleotide sequence of which is shown in SEQ ID NO: 59, and the 5' end is labeled with a second fluorescent group and the 3' end is labeled with a second quenching group; (d) RPA amplification reaction reagent; (e) PfAgo protein and its reaction buffer; and, (f) Divalent metal ions; The kit is used to simultaneously amplify the VP60 gene target of RHDV type I and RHDV type II under isothermal conditions, and to achieve single-tube simultaneous detection and differentiation of the two types of viruses through guided cleavage of PfAgo protein and dual-channel fluorescence signal output.

2. The reagent kit according to claim 1, characterized in that, In the fluorescent reporter probe assembly, the first fluorescent group is ROX, the first quencher group is BHQ2, the second fluorescent group is FAM, and the second quencher group is BHQ1.

3. The kit according to claim 1 or 2, characterized in that, The final concentration of each primer in the RPA primer set is 0.38~0.46 μM; and / or, the final concentration of RHDVⅠ-gDNA in the gDNA set is 0.5~0.7 μM, and the final concentration of RHDVⅡ-gDNA is 0.3~0.5 μM; and / or, the final concentration of RHDVⅠ probe in the fluorescent reporter probe set is 0.2~0.4 μM, and the final concentration of RHDVⅡ probe is 0.5~0.7 μM; and / or, the final concentration of PfAgo protein is 12~17 pM; and / or, the final concentration of divalent metal ions is 4~8 mM.

4. The reagent kit according to claim 3, characterized in that, In the gDNA combination, the final concentration ratio of RHDVⅠ-gDNA to RHDVⅡ-gDNA is 1~2:

1.

5. The reagent kit according to claim 1, characterized in that, The RPA amplification reaction reagents include recombinase, single-stranded DNA binding protein, strand displacement DNA polymerase, ATP, and dNTPs.

6. The reagent kit according to claim 1, characterized in that, The kit also includes at least one of the following: RNA extraction reagent, reverse transcription reagent, positive control, negative control, and inorganic pyrophosphatase.

7. A dual RPA-PfAgo nucleic acid detection method for simultaneous detection or differentiation of RHDV I and RHDV II without diagnostic or therapeutic purposes, characterized in that, Includes the following steps: S1, Extract nucleic acid from the sample to be tested to obtain a template; S2, using a dual RPA primer combination, the template is subjected to a recombinase polymerase amplification reaction under isothermal conditions to obtain the amplification product; the dual RPA primer combination includes: (a1) The first primer pair used for amplifying the conserved region of the RHDV type I VP60 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO:7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:16; and, (a2) The second primer pair used to amplify the conserved region of the RHDV II VP60 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 24, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 27; S3, the amplification product from step S2 is mixed with the gDNA combination, the fluorescent reporter probe combination, the PfAgo protein, and divalent metal ions, incubated at the cleavage temperature, and the signal of the first and / or second fluorescent channel is detected; the gDNA combination includes: (b1) The first pair of gDNA used to guide the PfAgo protein to specifically cleave the RHDV type I RPA amplicons, the nucleotide sequences of which are shown in SEQ ID NO: 52 and SEQ ID NO: 53; and, (b2) The second pair of gDNA used to guide the PfAgo protein to specifically cleave the RHDV II type RPA amplicon, the nucleotide sequences of which are shown in SEQ ID NO: 57 and SEQ ID NO: 58; The fluorescent reporter probe assembly includes: (c1) A first probe for detecting RHDV type I, the nucleotide sequence of which is shown in SEQ ID NO: 54, and the 5' end is labeled with a first fluorescent group and the 3' end is labeled with a first quencher group; and, (c2) A second probe for detecting RHDV type II, the nucleotide sequence of which is shown in SEQ ID NO: 59, and the 5' end is labeled with a second fluorescent group and the 3' end is labeled with a second quenching group; (d) RPA amplification reaction reagent; S4. Determine whether RHDVⅠ and / or RHDVⅡ are present in the sample based on the presence or absence and / or color of the fluorescence signal. The constant temperature conditions for step S2 are 37-38℃, and the reaction time is 18-22 minutes; The cutting temperature in step S3 is 94-96℃, and the cutting time is 25-40 minutes.

8. The method according to claim 7, characterized in that, In step S2, the final concentration of each primer in the RPA primer combination is 0.38~0.46 μM.

9. The method according to claim 8, characterized in that, In step S3, the detection of the signals of the first and / or second fluorescence channels is performed by a real-time quantitative PCR instrument or a chemiluminescence imaging system. When using a chemiluminescence imaging system, the infection type is determined by the detected fluorescence color: red fluorescence indicates RHDVⅠ positive, green fluorescence indicates RHDVⅡ positive, and yellow fluorescence with red and green superposition indicates a mixed positive result for RHDVⅠ and RHDVⅡ.

10. The use of the kit according to any one of claims 1-6 in the preparation of products for the diagnosis or screening of rabbit hemorrhagic disease virus.

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