A goose astrovirus RT-RAA detection primer probe set and application thereof

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to a primer-probe set for RT-RAA detection of goose astrovirus and its application. The primer-probe set includes an upstream primer F1, a downstream primer R2, and a probe P. This primer-probe set can simultaneously detect two genotypes of goose astrovirus, GoAstV-I and GoAstV-II; it exhibits high specificity and amplification efficiency, aiming to provide a rapid and convenient technical means for the early diagnosis, on-site monitoring, and epidemic prevention and control of goose astrovirus. Example results show that using GoAstV-I, GoAstV-II, GPV, GoCV, DTMUV, and NDV nucleic acids as templates for RT-RAA-LFD detection, positive bands were observed for GoAstV-I and GoAstV-II, while all other pathogens were negative, with no cross-reaction, indicating that the specificity of the primer-probe set is 100%.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a primer and probe set for the detection of goose astrovirus RT-RAA and its application. Background Technology

[0002] Goose astrovirus (GoAstV) exists in two genotypes, GoAstV-I and GoAstV-II. It is a major pathogen causing gout in goslings, resulting in severe economic losses for the global goose farming industry. This virus primarily infects goslings under 20 days old, causing an acute infectious disease characterized by gout, urate deposition in internal organs and joint cavities, with a mortality rate exceeding 50%. Furthermore, the virus can also infect other poultry such as chickens and ducks, indicating the potential for cross-species transmission of GoAstV, further contributing to the significant economic losses in the goose farming industry.

[0003] GoAstV belongs to the genus Astrovirus of the family Astroviridae. It is a non-enveloped, single-stranded, positive-sense RNA virus with a viral particle diameter of approximately 30 nm and a genome length of about 7 kb, containing three open reading frames (ORFs): ORF1a, ORF1b, and ORF2. Currently, two genotypes of GoAstV are known: GoAstV-I and GoAstV-II. GoAstV-II is the predominant circulating strain, although some cases of co-infection exist. Most current molecular detection methods only target GoAstV-II, posing a risk of missed detection of GoAstV-I. Furthermore, different conserved primers and probes need to be designed for each genotype, making the detection process cumbersome and costly. Therefore, to more effectively and rapidly monitor GoAstV incidence, a simpler method capable of simultaneously detecting both genotypes is needed. Summary of the Invention

[0004] The purpose of this invention is to provide a primer and probe set for the detection of goose astrovirus RT-RAA and its application. The primer and probe set can detect two genotypes simultaneously, with high specificity and high amplification efficiency.

[0005] This invention provides a primer and probe set for the detection of goose astrovirus RT-RAA, the primer and probe set comprising: upstream primer F1, downstream primer R2 and probe P; The sequence of the upstream primer F1 is shown in SEQ ID NO.1; the sequence of the downstream primer R2 is shown in SEQ ID NO.4; and the sequence of the probe P is shown in SEQ ID NO.5.

[0006] As a preferred embodiment, the 5' end of the upstream primer F1 is modified with biotin; the 5' end of the probe P is modified with a fluorescent reporter group, a tetrahydrofuran (THF) cleavage site is inserted in the middle, and the 3' end is modified with a C3Spacer blocking extension group.

[0007] As a preferred embodiment, the fluorescent reporter group is a FAM group.

[0008] This invention provides the application of the above-mentioned primer and probe set in the preparation of products for detecting goose astrovirus, wherein the goose astrovirus includes: GoAstV-I and / or GoAstV-II.

[0009] This invention provides a kit for detecting goose astrovirus RT-RAA, the kit comprising: the primer and probe set described above.

[0010] As a preferred embodiment, the working concentrations of the upstream primer F1, the downstream primer R2, and the probe P are 2~10 μmol / L, respectively.

[0011] As a preferred embodiment, the kit also includes: RT-RAA basic reaction solution, magnesium acetate, and RNase-FreeddH2O.

[0012] As a preferred embodiment, the kit further includes: an LFD test strip; the detection line (T line) of the LFD test strip is coated with anti-FAM antibody; and the control line (C line) of the LFD test strip is coated with streptavidin.

[0013] This invention provides a method for detecting goose astrovirus RT-RAA-LFD for non-diagnostic purposes, comprising the following steps: using the above-mentioned primer and probe set or the above-mentioned kit, RT-RAA isothermal amplification is performed using the RNA of the sample to be tested as a template, and the amplification product is detected by an LFD test strip and the colorimetric reaction is observed.

[0014] As a preferred embodiment, the RT-RAA isothermal amplification reaction temperature is 33~41℃, and the reaction time is 12~20min.

[0015] Beneficial Effects: This invention provides a primer and probe set for RT-RAA detection of goose astrovirus and its application. The primer and probe set of this invention can simultaneously detect two genotypes of goose astrovirus, GoAstV-I and GoAstV-II; it exhibits high specificity and amplification efficiency, aiming to provide a rapid and convenient technical means for the early diagnosis, field monitoring, and epidemic prevention and control of goose astrovirus. Example results show that using GoAstV-I, GoAstV-II, GPV (Goose parvovirus), GoCV (Goose circovirus), DTMUV (Duck tambusu virus), and NDV (Newcastle disease virus) nucleic acids as templates for RT-RAA-LFD detection, the results show a positive band for GoAstV, while the other pathogens are negative, with no cross-reaction, indicating that the specificity of the primer and probe set of this invention is 100%.

[0016] This invention provides a kit for detecting goose astrovirus RT-RAA, the kit comprising the primer and probe set described above. Recombinase-mediated isothermal amplification (RT-RAA) technology requires only a constant temperature to complete nucleic acid amplification, eliminating the need for a thermal cycler, making it simpler and faster. Example results show that the limit of detection of this invention is 10 copies / μL, with no cross-reactivity with other common avian viruses. The overall concordance rate between RT-RAA-LFD and RT-PCR detection methods is 95%, and the Kappa coefficient is 0.92 (K>0.75).

[0017] This invention provides a method for non-diagnostic detection of GoAstV RT-RAA-LFD, comprising the following steps: using the primer and probe set or the kit described above, RT-RAA isothermal amplification is performed using the RNA of the sample to be tested as a template; the amplification product is detected by an LFD test strip and the colorimetric reaction is observed. The detection method of this invention has good specificity, sensitivity, and repeatability, and features rapid detection, visualization, and suitability for rapid on-site testing. The RT-RAA amplification product can be observed using a horizontal lateral flow colloidal gold test strip (LFD), allowing for visual interpretation of the detection results. This provides a new technical means for rapid diagnosis and epidemiological investigation of GoAstV in farms, grassroots veterinary stations, and border quarantine facilities. Attached Figure Description

[0018] Figure 1 The results are from the RT-RAA-LFD primer assay; where A represents the LFD colorimetric result and B represents the gel electrophoresis result. Figure 2 Results of RT-RAA-LFD amplification reaction temperature test; Figure 3 Results of RT-RAA-LFD amplification reaction time assay; Figure 4 Results of working concentration experiments for RT-RAA-LFD primers and probes; Figure 5 Results of RT-RAA-LFD sensitivity test; Figure 6 These are the results of the RT-RAA-LFD specificity assay; where A represents the results of F1 / R2, and B represents the results of primer F2 / R1. Figure 7 The results are from the RT-RAA-LFD repeatability test. Detailed Implementation

[0019] This invention provides a primer and probe set for the detection of goose astrovirus RT-RAA, the primer and probe set comprising: upstream primer F1, downstream primer R2 and probe P; The sequence of the upstream primer F1 is shown in SEQ ID NO.1; the sequence of the downstream primer R2 is shown in SEQ ID NO.4; and the sequence of the probe P is shown in SEQ ID NO.5.

[0020] This invention preferably uses regions with high similarity to the ORF1b gene of GoAstV type I and GoAstV type II from 183 GoAstV whole genome sequences as target sequences (GoAstV type I AAstV / Goose / CHN / 2023 / C1: 4228~4411bp; GoAstV type II AstV / Goose / 2019 / HLJJX: 4116~4328bp). Primers and probes are designed using Primer Premier 5.0 and Oligo7, with degenerate base substitutions for differential bases, and modified according to LFD detection requirements to obtain the primer and probe set for RT-RAA-LFD detection of goose astrovirus. The upstream primer F1 of this invention is Biotin-ATMTTKTTACCAACTGGGGAGGTKTGTMMR (as shown in SEQ ID NO.1); the 5' end of the upstream primer F1 is modified with biotin; the downstream primer R2 is TGAKASMARCCTRTCATCKCCATAGCAAAT (as shown in SEQ ID NO.4); the probe P is (FAM)CAATWTTCAACMACWGTCSGACAAYAATW[THF]STGTAATGTMTGGCTYAC-C3Spacer (as shown in SEQ ID NO.5). In a preferred embodiment, the 5' end of the probe P is modified with a fluorescent reporter group, and the 30th base at the 5' end of the probe P is replaced by a tetrahydrofuran (THF) cleavage site, and the 3' end is modified with a C3Spacer blocking extension group. In another preferred embodiment, the fluorescent reporter group is the FAM group. The primer-probe set of this invention has high specificity and high amplification efficiency.

[0021] This invention provides the application of the aforementioned primer-probe set in the preparation of products for detecting goose astrovirus, including GoAstV-I and / or GoAstV-II. The primer-probe set of this invention can simultaneously detect both GoAstV-I and GoAstV-II genotypes of goose astrovirus; it shows no cross-homology with common avian viruses such as GPV, GoCV, DTMUV, and NDV. Example results show that using GoAstV-I, GoAstV-II, GPV, GoCV, DTMUV, and NDV nucleic acids as templates for RT-RAA-LFD detection, the results show positive bands for GoAstV-I and GoAstV-II, while the remaining pathogens are negative, indicating no cross-reaction. This demonstrates that the specificity of the primer-probe set of this invention is 100%.

[0022] This invention provides a kit for detecting goose astrovirus RT-RAA, the kit comprising the primer and probe set described above. The working concentrations of the upstream primer F1, downstream primer R2, and probe P can be any value within the range of 0.5~10 μmol / L, for example, 0.5, 1, 2, 4, 6, 8, or 10 μmol / L. In specific embodiments, the working concentrations of the upstream primer F1, downstream primer R2, and probe P can be the same or different. The kit may further comprise: RT-RAA basic reaction solution, magnesium acetate, and RNase-free ddH2O. The RT-RAA basic reaction solution is preferably RT-RAA Buffer A. In a specific embodiment of the present invention, the kit uses a 50 μL reaction system; the 50 μL reaction system includes: 25 μL RT-RAA basic reaction solution, upstream primer F1, downstream primer R2 and probe P at the above working concentrations, 2.5 μL magnesium acetate (solution), and finally the remaining RNase-free ddH2O. The sample described in this invention can be obtained from one of the following: liver, kidney, joint cavity exudate or anal swab. As a preferred embodiment, the kit further includes: an LFD test strip; the detection line T of the LFD test strip is coated with anti-FAM antibody; the control line C of the LFD test strip is coated with streptavidin. The present invention does not have a special limitation on the source of the LFD test strip; conventional commercially available products are acceptable. The preferred result determination criteria of this invention are: a positive result shows two colored bands, one on the test line (T) and the other on the control line (C); a negative result shows only one blue band on the control line, and no red band on the test line; if only the test line shows a red band, and no blue band on the control line, the result is invalid.

[0023] In a specific embodiment of this invention, gene fragments of GoAstV-I and GoAstV-II types were synthesized based on the complete genome sequences of GoAstV-I and GoAstV-II types (GoAstV-I type AAstV / Goose / CHN / 2023 / C1, GenBank: PP108251.1, 4228~4411 bp; GoAstV-O type AstV / Goose / 2019 / HLJJX, GenBank: MW505490.1, 4116~4328 bp) from GenBank, and cloned into the pUC57 vector to construct recombinant plasmids pUC57-GoAstV-I-ORF1b and pUC57-GoAstV-I-ORF1b. After the recombinant plasmids were confirmed to be correct by sequencing, their concentration was determined using a spectrophotometer to obtain the plasmid concentration. The formula is: Plasmid copy number (copies / μL) = 6.02 × 10⁻⁶. 23 × Plasmid concentration (ng / μL) × 10 -9Calculate the copy number of each plasmid by multiplying its length (bp) by 660. Dilute both plasmids separately with sterile TE buffer to a final concentration of 2.0 × 10⁻⁶. 9 The samples were then divided into copies / μL and mixed in a 1:1 volume ratio to obtain the mixed plasmid standard. After mixing, the final concentration of each plasmid in the standard was 1.0 × 10⁻⁶. 9 copies / μL, total copy number is 2.0 × 10⁻⁶ 9 copies / μL (sum of two targets). The above mixed plasmid standard was serially diluted 10-fold with sterile TE buffer to obtain 1.0×10 copies / μL. 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 1.0×10 1 and 1.0×10 0 Standard plasmids at concentrations of copies / μL were used as working solutions for subsequent performance evaluation tests, including sensitivity and repeatability, of the RT-RAA detection method.

[0024] This invention provides a method for detecting goose astrovirus RT-RAA-LFD, comprising the following steps: using the primer and probe set or the kit described above, RT-RAA isothermal amplification is performed using the RNA of the sample to be tested as a template; the amplification product is detected by an LFD test strip and the colorimetric reaction is observed. This invention is preferably a method for detecting goose astrovirus RT-RAA-LFD for non-diagnostic purposes. The reaction temperature of the RT-RAA isothermal amplification described in this invention can be any value within the range of 33~41℃, for example, 33, 35, 37, 39, or 41℃; the reaction time can be any value within the range of 12~20 min, for example, 12, 14, 16, 18, or 20 min. As a preferred embodiment, the detection limit for goose astrovirus GoAstV is 10 copies / μL. This invention can simultaneously detect both GoAstV-I and GoAstV-II genotypes of goose astrovirus, effectively avoiding the risk of missed detection of GoAstV-I.

[0025] To further illustrate the present invention, the primer and probe set for RT-RAA detection of goose astrovirus provided by the present invention and its application are described in detail below with reference to embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 (1) Design and synthesis of GoAstV RT-RAA-LFD primers and probes This invention preferably selects conserved regions with high co-similarity of RdRp in the ORF1b gene from 183 GoAstV whole genome sequences, based on GoAstV-I and GoAstV-II types, as target sequences (GoAstV-I AAstV / Goose / CHN / 2023 / C1 (GenBank: PP108251.1): 4228~4411bp; GoAstV-II AstV / Goose / 2019 / HLJJX (GenBank: MW505490.1): 4116~4328bp). Primers and probes were designed using Primer Premier 5.0 and Oligo 7, and modified according to LFD detection requirements. Upstream primer F1: Biotin-ATMTTKTTACCAACTGGGGAGGTKTGTMMR (as shown in SEQ ID NO.1); Downstream primer R2: TGAKASMARCCTRTCATCKCCATAGCAAAT (as shown in SEQ ID NO.4); Probe P: (FAM)CAATWTTCAACMACWGTGSGACAAYAATW[THF]STGTAATGTMTGGCTYAC-C3Spacer (as shown in SEQ ID NO.5).

[0027] The upstream primer F1, downstream primer R2, and probe were all purified by HPLC and stored at -20°C in the dark for later use.

[0028] (2) Viral nucleic acid extraction Take 200 μL of the sample to be tested (anal swab), add the collected anal swab to 500 μL of sterile PBS, vortex for 30 seconds, centrifuge at 7000 rpm for 8 minutes, and aspirate 200 μL of the supernatant. Extract total RNA according to the instructions of the Novozymes FastPure Viral DNA / RNA Mini Kit (Catalog No.: RC311; Batch No.: 7E0921A6). Dissolve the obtained RNA in RNase-free water and immediately store at -80°C. This is the template RNA.

[0029] (3) Construction of RT-RAA amplification system Based on the complete genome sequences of GoAstV-I and II types from GenBank (GoAstV-o type AAstV / Goose / CHN / 2023 / C1, GenBank: PP108251.1, 4228~4411 bp; GoAstV-o type AstV / Goose / 2019 / HLJJX, GenBank: MW505490.1, 4116~4328 bp), gene fragments were synthesized and cloned into the pUC57 vector to construct recombinant plasmids pUC57-GoAstV-I-ORF1b and pUC57-GoAstV-I-ORF1b. After the recombinant plasmids were confirmed to be correct by sequencing, their concentrations were determined using a spectrophotometer. The plasmid concentration was calculated using the formula: Plasmid copy number (copies / μL) = 6.02 × 10⁻⁶. 23 × Plasmid concentration (ng / μL) × 10 -9 Calculate the copy number of each plasmid by multiplying its length (bp) by 660. Dilute both plasmids separately with sterile TE buffer to a final concentration of 2.0 and then to a final concentration of 1.0 × 10⁻⁶. 9 The samples were then divided into copies / μL and mixed in equal volumes at a 1:1 volume ratio to obtain the mixed plasmid standard. After mixing, the final concentration of each plasmid in the standard was 1.0 μL. 9 copies / μL, total copy number is 2.0 B, and number of copies is 1.0 × 10⁻⁶. 9 copies / μL (sum of two targets). The above mixed plasmid standard was serially diluted 10-fold with sterile TE buffer to obtain 1.0×10 copies / μL. 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 1.0×10 1 and 1.0×10 0 Standard plasmids with concentrations in copies / μL series.

[0030] 50 μL reaction system: RT-RAA Buffer A 25 μL, upstream primer F (working concentration 10 μmol / L, stock solution concentration 10 mmol / L) 2 μL, downstream primer R (working concentration 10 μmol / L, stock solution concentration 10 mmol / L) 2 μL, probe P (working concentration 10 μmol / L, stock solution concentration 10 mmol / L) 0.6 μL, with a concentration of 1×10 55 μL of standard plasmid (copies / μL) was used as template, 2.5 μL of Buffer B (magnesium acetate solution) was added, and finally RNase-free ddH2O was added to bring the total volume to 50 μL. After the system was prepared, it was vortexed for 10 s and then centrifuged briefly. It was then placed in a constant temperature metal bath at 37℃ for 20 min to obtain the RT-RAA amplification product.

[0031] (4) LFD test strip testing and result interpretation Take 5 μL of RT-RAA amplification product and dilute it 1:20 with 95 μL of RNase-free water. Insert the lateral flow chromatography strip (LFD strip) into the dilution solution and react at room temperature for 3 min. The detection line (T line) of the LFD strip is coated with anti-FAM antibody; the control line (C line) is coated with streptavidin. The LFD strip was purchased from Hangzhou Zhongce Biotechnology Co., Ltd.; product number R103ZC, batch number 84831458.

[0032] Result determination: Positive: Red bands appear on both the control line (C line) and the test line (T line); Negative: Only the C line shows a band, and the T line shows no band; Invalid: No bands appear on line C.

[0033] Comparative Example 1 The procedure was carried out in accordance with Example 1, except that downstream primer R2 was replaced with downstream primer R1: ATARAKAATGTATRMMRATRTYTTTGGTATG (as shown in SEQ ID NO.3).

[0034] Comparative Example 2 The procedure was carried out in accordance with Example 1, except that upstream primer F1 was replaced with upstream primer F2: Biotin-CCAACTGGkGAGGTKTGTMMRGTYAAGAAR (as shown in SEQ ID NO.2).

[0035] Comparative Example 3 The procedure was carried out in accordance with Example 1, except that upstream primer F1 was replaced with upstream primer F2 (as shown in SEQ ID NO. 2), and downstream primer R2 was replaced with downstream primer R1 (as shown in SEQ ID NO. 3).

[0036] Experimental Example 1: Primer-Probe Combination Screening Two upstream primers (F1 / F2) and two downstream primers (R1 / R2) were used, forming a total of four primer pairs: primer pair F1 / R2 for Example 1, primer pair F1 / R1 for Comparative Example 1, primer pair F2 / R1 for Comparative Example 2, and primer pair F2 / R2 for Comparative Example 3. A concentration of 1.0 × 10⁻⁶ was used. 5 Standard plasmids of copies / μL were used as templates for RT-RAA amplification. The products were screened by a combination of 2% agarose gel electrophoresis and LFD colorimetric assay. The LFD results are shown in the figure. Figure 1 The gel electrophoresis results for A are shown in [reference needed]. Figure 1 In the B group, the band grayscale analysis was performed using Image J (Reference: Chen QY, Sun ZH, Chen RJ, Wu XM, Che YL, Wu RJ, Qiu JL, He B, Wang LB, Zhou LJ. A rapid visual detection method for porcineteschovirus through reverse transcription recombinase-aided amplification coupled with lateral flow dipstick. BMC Vet Res. 2024 Dec 30;20(1):588. doi:10.1186 / s12917-024-04442-9), and the results are shown in Table 1. "-" indicates a blank control without a template, i.e., performed according to Example 1, except that no standard plasmid was added.

[0037] Table 1. Gray-scale analysis results

[0038] The results showed that the primer pair F1 / R2 corresponding to Example 1 had the highest amplification efficiency, the highest gray value, the brightest gel electrophoresis band, and the deepest LFD color development, and was determined to be the optimal combination.

[0039] Example 2 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the temperature of the isothermal metal bath was replaced with 35°C.

[0040] Example 3 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the temperature of the isothermal metal bath was replaced with 39°C.

[0041] Example 4 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the temperature of the isothermal metal bath was replaced with 41°C.

[0042] Comparative Example 4 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the temperature of the isothermal metal bath was replaced with 33°C.

[0043] Experimental Example 2: Temperature Optimization With a concentration of 1, the concentration is 1.0 × 10⁻⁶. 5 Standard plasmids of copies / μL were used as templates for RT-RAA amplification. Temperature optimization experiments were conducted with ratios of 33 (Comparative Example 4), 35 (Example 2), 37 (Example 1), 39 (Example 3), and 41 (Example 4). The results of LFD colorimetric analysis of the products are shown below. Figure 2 .

[0044] The results showed that 37°C (Example 1) was the optimal reaction temperature, with the clearest color development and the lowest background.

[0045] Example 5 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the isothermal metal bath time was replaced with 16 min.

[0046] Example 6 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the isothermal metal bath time was replaced with 18 min.

[0047] Comparative Example 5 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the isothermal metal bath time was replaced with 12 min.

[0048] Comparative Example 6 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the isothermal metal bath time was replaced with 14 min.

[0049] Experiment Example 3: Time Optimization With a concentration of 1, the concentration is 1.0 × 10⁻⁶. 5 RT-RAA amplification was performed using standard plasmids of copies / μL as templates. Time optimization experiments were conducted with time settings of 12 min (Comparative Example 5), 14 min (Comparative Example 6), 16 min (Example 5), 18 min (Example 6), and 20 min (Example 1). The product was then analyzed by LFD colorimetry. Figure 3"Where " is a blank control without a template, that is, it is carried out in accordance with the method of Example 1, except that no standard plasmid is added.

[0050] The results showed that a strong positive signal could be obtained at 20 min (Example 1), and there was no significant improvement after further extending the time.

[0051] Example 7 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the working concentration of each primer and probe was replaced with 8 μmol / L.

[0052] Example 8 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the working concentration of each primer and probe was replaced with 6 μmol / L.

[0053] Comparative Example 7 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the working concentration of each primer and probe was replaced with 4 μmol / L.

[0054] Comparative Example 8 The procedure was carried out in accordance with Example 1, except that (3) in the construction of the RT-RAA amplification system, the working concentration of each primer and probe was replaced with 2 μmol / L.

[0055] Experiment 4: Primer / Probe Concentration Optimization With a concentration of 1, the concentration is 1.0 × 10⁻⁶. 5 Standard plasmids of copies / μL were used as templates for RT-RAA amplification. Concentrations were set at 2 μmol / L (Comparative Example 8), 4 μmol / L (Comparative Example 7), 6 μmol / L (Example 8), 8 μmol / L (Example 7), and 10 μmol / L (Example 1). Primer / probe concentration optimization experiments were performed. The product was analyzed by LFD colorimetry. Figure 4 .

[0056] The results showed that the bands were clearest at a working concentration of 10 μmol / L (Example 1), which was determined to be the optimal concentration.

[0057] Test Example 5 Sensitivity Test The content containing 1.0 × 10 9 The standard plasmid was serially diluted 10-fold with copies / μL, to a concentration range of 1.0 × 10⁻⁶. 7 ~1.0×10 0 The copies / μL were detected using the RT-RAA-LFD method described in Example 1, and the results are shown below. Figure 5"Where " is a blank control without a template, that is, it is carried out in accordance with the method of Example 1, except that no standard plasmid is added.

[0058] The results showed that the detection limit could reach as low as 10 copies / μL, which was significantly better than conventional RT-PCR and colloidal gold methods.

[0059] Test Example 6: Specificity Test Using GoAstV-Ⅰ, GoAstV-Ⅱ, GPV, GoCV, DTMUV, and NDV nucleic acids as templates, and with a negative control (RNase-free water), the F1 / R2 primer pair from Example 1 was used for RT-RAA-LFD detection. The results are shown in [Figure 1]. Figure 6 A.

[0060] The results showed that GoAstV-Ⅰ and GoAstV-Ⅱ were positive bands, while the remaining pathogens were negative, with no cross-reaction and 100% specificity.

[0061] Further, using F2 / R1 as templates for GoAstV-Ⅰ, GoAstV-Ⅱ, GPV, GoCV, DTMUV, and NDV nucleic acids, and setting up a negative control (RNase-free water), RT-RAA-LFD detection was performed in Example 1. The results are shown in [Figure 1]. Figure 6 B.

[0062] The results showed that GoAstV-Ⅰ and GoAstV-Ⅱ were still positive, while the remaining pathogens were negative. However, the band intensity was significantly weaker than that of the detection results using the F1 / R2 primer pair, suggesting that F1 / R2 is a better primer combination.

[0063] Test Example 7 Repeatability Test Select 1.0×10 7 copies / μL, 1.0×10 5 copies / μL and 1.0×10 3 Standard plasmids at three concentration gradients (copies / μL) were used for intra-batch replication experiments (three replicates of the same batch reaction) and inter-batch replication experiments (three replicates of different batches, at different times, and by different operators). Results are shown in [Figure number missing]. Figure 7 .

[0064] The results showed that the C-line and T-line were uniformly and stably colored, with a coefficient of variation (CV) of less than 5%, indicating that the method had good repeatability and strong stability.

[0065] Case 8: Clinical Sample Testing Seventy-two gosling samples suspected of having clinical gout were collected from goose farms in Sichuan and other regions. The RT-RAA-LFD method established in Example 1 and the method reported in the literature were used for parallel detection using the RT-qPCR method. (See Li M, Wang M, Wang J, LiH, Wang Q, Yan G, Huang Z. Development and validation of a quadruplex qPCRassay for simultaneous detection of GPV, GoCV, TMUV, and GAstV-Asin geese. Sci Rep. 2025 Dec 31;16(1):3992. doi: 10.1038 / s41598-025-34108-x.)

[0066] The results showed that the positive detection rate of RT-RAA-LFD was 58.33% (42 / 72), and the positive detection rate of RT-qPCR was 55.56% (40 / 72). A total of 70 samples showed consistent results between the two methods, including 40 double-positive samples, 30 double-negative samples, and 2 inconsistent samples (both RT-RAA-LFD positive / RT-qPCR negative). The overall concordance rate was 97.22% (70 / 72), with a Kappa value of 0.943 (P<0.001), indicating extremely high consistency between the two methods (Kappa>0.75). Two samples that were RT-RAA-LFD positive but RT-qPCR negative were identified by sequencing as GoAstV genotype I infection, confirming that this method can simultaneously detect two genotypes, providing a broader detection spectrum than RT-qPCR, which only targets genotype II.

[0067] Therefore, the RT-RAA-LFD detection method established in this invention has good specificity, sensitivity and stability, and features fast detection speed, visualization and suitability for rapid on-site testing, providing a new technical means for rapid diagnosis and epidemiological investigation of GoAstV in farms, grassroots veterinary stations, border quarantine, etc.

[0068] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer and probe set for the detection of goose astrovirus RT-RAA, characterized in that, The primer-probe set includes: upstream primer F1, downstream primer R2, and probe P; The sequence of the upstream primer F1 is shown in SEQ ID NO.1; the sequence of the downstream primer R2 is shown in SEQ ID NO.4; and the sequence of the probe P is shown in SEQ ID NO.

5.

2. The primer-probe set according to claim 1, characterized in that, The upstream primer F1 is modified with biotin at its 5' end; the probe P is modified with a fluorescent reporter group at its 5' end, with a tetrahydrofuran (THF) cleavage site inserted in the middle, and a C3Spacer blocking extension group modified at its 3' end.

3. The primer-probe set according to claim 2, characterized in that, The fluorescent reporter group is the FAM group.

4. The application of the primer-probe set according to any one of claims 1 to 3 in the preparation of products for detecting goose astrovirus, characterized in that, The goose starviruses include: GoAstV-I and / or GoAstV-II.

5. A kit for detecting goose astrovirus RT-RAA, characterized in that, The kit comprises: the primer and probe set as described in any one of claims 1 to 3.

6. The reagent kit according to claim 5, characterized in that, The working concentrations of the upstream primer F1, downstream primer R2, and probe P are 6~10 μmol / L, respectively.

7. The reagent kit according to claim 6, characterized in that, The kit also includes: RT-RAA basic reaction solution, magnesium acetate, and RNase-Free ddH2O.

8. The reagent kit according to claim 5, characterized in that, The kit also includes: an LFD test strip; the detection line (T line) of the LFD test strip is coated with anti-FAM antibody; and the control line (C line) of the LFD test strip is coated with streptavidin.

9. A method for detecting goose astrovirus RT-RAA-LFD for non-diagnostic purposes, characterized in that, The procedure includes the following steps: using the primer and probe set described in any one of claims 1 to 3 or the kit described in any one of claims 5 to 8, RT-RAA isothermal amplification is performed using the RNA of the sample to be tested as a template, and the amplification product is detected by an LFD test strip and the colorimetric reaction is observed.

10. The method according to claim 9, characterized in that, The reaction temperature for the RT-RAA isothermal amplification is 33~41℃, and the reaction time is 12~20 min.

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