RPA primer, probe and detection method for detecting H9 subtype avian influenza virus

Abstract

The invention relates to the technical field of molecular detection, in particular to an RPA (recombinase polymerase amplification) primer, a probe and a detection method for detecting H9 subtype avian influenza virus. The RPA primer is divided into an upstream primer RPA-F and a downstream primer RPA-R, and the nucleotide sequence of the upstream primer RPA-F is any one as shown in SEQ ID NO.1-4; the nucleotide sequence of the downstream primer RPA-R is any one as shown in SEQ ID NO.5-8; the nucleotide sequence of the probe is as shown in SEQ ID NO. 9; wherein the RPA primer is designed on the basis of the HA gene sequence of the H9 subtype avian influenza virus, the specificity is relatively high, and the detection accuracy is high; the detection method has high detection sensitivity, isothermal amplification of the H9 subtype AIV can be completed at 39 DEG C within 20 min, and the problems that a conventional detection method needs long time, dependence on instruments and equipment is high, and the false positive result of the detection result is high are solved.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to an RPA primer, probe and detection method for detecting H9 subtype avian influenza virus. Background Technology

[0002] Avian influenza viruses (AIV) belong to the genus Influenza A (type A) of the family Orthomyxoviridae, and were first reported in 1878. Based on their pathogenicity in birds and the molecular marker of the HA protein, AIVs are classified into highly pathogenic avian influenza viruses (HPAIV) and low pathogenic avian influenza viruses (LPAIV). HPAIV includes some H5 or H7 subtypes of avian influenza viruses, while the rest are LPAIVs. The H9N2 subtype AIV is the most prevalent in poultry and also poses the greatest threat to public health. This virus was first discovered and isolated from turkeys in Wisconsin, USA in 1966 (A / turkey / Wisconsin / 1 / 1966(H9N2)), and subsequently spread rapidly worldwide.

[0003] Although H9N2, as an LPAIV (low-level artificial insemination virus), does not directly cause high mortality rates in poultry, it can induce host immunosuppression and lead to co-infection with other pathogens, thereby increasing mortality and causing severe economic losses to the poultry industry. H9N2 has a strong transmissibility, allowing the virus to continuously circulate in migratory birds, free-range poultry, and the ecological environment, making complete eradication impossible. Furthermore, AIVs are prone to gene mutation and rearrangement, and frequent contact between migratory birds and poultry accelerates viral gene recombination, continuously expanding the virus's host range. Initially, H9N2's hosts were limited to turkeys and quails, but it later also affected chickens and even humans.

[0004] In addition to directly infecting humans, the H9N2 subtype AIV also provides some or all of its internal gene fragments for a variety of emerging H5N1, H7N9, H10N8 and H5N6 viruses. These variant strains pose a risk of infecting humans and causing death, seriously threatening public health security. Therefore, it is necessary to strengthen the prevention and control of the H9N2 subtype AIV.

[0005] Currently, common H9N2 detection methods fall into three main categories: virus isolation and identification, molecular biology methods, and serological methods. Among these, traditional avian influenza diagnostic methods, such as virus isolation and identification, hemagglutination inhibition (HI) tests, and enzyme-linked immunosorbent assays (ELISA), while possessing high accuracy, are cumbersome and time-consuming, making them unsuitable for rapid diagnosis and on-site detection in the early stages of an outbreak. Although PCR and RT-qPCR methods offer high sensitivity and specificity, making them the most commonly used methods for pathogen detection in laboratories, they still require expensive specialized equipment and skilled personnel, and must be performed under laboratory conditions, hindering their rapid widespread application at the grassroots level and in the field.

[0006] Isothermal amplification (RPA) is a novel nucleic acid amplification technique based on the principle of exponential amplification of target nucleic acids using enzymes of different activities and specific primers at a constant temperature. It includes loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and exponential amplification reaction (EXPAR). Among these, RPA technology offers higher sensitivity, specificity, and simpler operation compared to other similar detection techniques, making it of significant research value. The RPA reaction system includes recombinase UvsX and its cofactor UvsY, single-stranded binding protein (SSB), DNA polymerase, MgCl2, and detection template and specific primers. The reaction conditions are convenient, typically achieving exponential amplification at 37–42°C; the reaction speed is fast, with detection completed within 20–30 minutes after sample addition; it has strong anti-interference capabilities and is suitable for complex sample detection. Currently, many biotechnology companies both domestically and internationally have successfully developed mature RPA reaction reagents. RPA has also been successfully applied to the detection of various bacterial infectious diseases such as Salmonella, Staphylococcus aureus, and Brucella, and methods for detecting various avian and livestock viral pathogens such as fowlpox virus and Muscovy duck parvovirus have been established. Therefore, providing RPA primers, probes, and detection methods for detecting H9 subtype avian influenza virus is of great significance for the rapid clinical diagnosis of H9 subtype AIV. Summary of the Invention

[0007] Based on the deficiencies in the existing technologies, this invention, based on RPA technology, designs RPA primers and probes by searching for conserved sequences on the HA gene fragment of H9 subtype AIV, and optimizes reaction conditions to establish a fluorescent RT-RPA detection technology for H9 subtype AIV. The aim is to provide a new detection method and approach for the rapid clinical diagnosis of H9 subtype AIV.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an RPA primer and probe for detecting H9 subtype avian influenza virus. The RPA primer consists of an upstream primer RPA-F and a downstream primer RPA-R, wherein: The nucleotide sequence of the upstream primer RPA-F is any one of those shown in SEQ ID NO. 1~4; The nucleotide sequence of the downstream primer RPA-R is any one of those shown in SEQ ID NO. 5~8; The nucleotide sequence of the probe is shown in SEQ ID NO.9; The nucleotide sequence shown in SEQ ID NO.9 is as follows: ATATGGGCATATAATGCAGAATTGCTAGTTCTGCTTGAAAACCAGA.

[0009] Preferably, the probe shown in SEQ ID NO.9 is modified by using a fluorescent group at 29 bp of the 5' end of the probe; replacing the 31 bp of the probe with tetrahydrofuran (THF); using a quenching group at 32 bp of the probe; and using C3Spacer to modify the 3' end of the probe.

[0010] Preferably, the fluorescent group is one of FAM, HEX, TET, JOE, and VIC; and the fluorescence quenching group is one of BHQ1, BHQ2, and BHQ3.

[0011] Preferably, the modified probe sequence is: 5'-ATATGGGCATATAATGCAGAATTGCTAG-i6FAMdT-T-THF-iBHQ1dT-GCTTGAAAACCAGA-C3Spacer-3'.

[0012] The present invention also provides a kit for detecting H9 subtype avian influenza virus, comprising the aforementioned RPA primers and probes.

[0013] Preferably, the kit further includes at least one of a reaction buffer, a positive control, and a negative control.

[0014] This invention also provides a method for detecting H9 subtype avian influenza virus for non-diagnostic purposes, comprising the following steps: Using H9 subtype avian influenza virus as template DNA, RPA amplification reaction was performed using the aforementioned RPA primers and probes. The amplification products were detected by agarose gel electrophoresis. If an amplification band appeared at 119-384 bp, it was H9 subtype avian influenza virus; otherwise, it was not.

[0015] Preferably, the RPA amplification reaction system is: 29.4 μL of buffer A, 10 μmol·L⁻¹ -1 Upstream primer 2 μL, 10 μmol·L -1 Downstream primer 2 μL, 10 μmol·L-1 0.6 μL of fluorescent probe, 3 μL of template RNA, and 10.5 μL of ddH2O were used.

[0016] Preferably, the RPA amplification reaction is carried out at a temperature of 35–45°C for 10–20 minutes.

[0017] The present invention has the following technical effects and advantages: This invention establishes a fluorescent RT-RPA detection technology for H9 subtype AIV by identifying conserved sequences on the HA gene fragment of H9 subtype AIV, designing RPA primers and probes, and optimizing reaction conditions. This technology features high specificity, high sensitivity, practicality, and low requirements for equipment, providing a new method for rapid clinical diagnosis of H9 subtype AIV. The RPA primers are designed based on the HA gene sequence of H9 subtype avian influenza virus, exhibiting high specificity and accuracy. The detection method has high sensitivity, completing isothermal amplification of H9 subtype AIV within 20 minutes at 39℃, overcoming the limitations of existing conventional techniques such as long detection times, high dependence on equipment, and high false-positive rates. Attached Figure Description

[0018] Figure 1 RT-RPA amplification results of the H9 subtype AIV HA gene; M: DL1000 DNA marker; 1-16: primer combinations F1+R1, F1+R2, F1+R3, F1+R4, F2+R1, F2+R2, F2+R3, F2+R4, F3+R1, F3+R2, F3+R3, F3+R4, F4+R1, F4+R2, F4+R3, and F4+R4, respectively; N: negative control; M: DL1000 DNA marker; 1-16: primer combinations F1+R1, F1+R2, F1+R3, F1+R4, F1+R5, F2+R1, F2+R2, F2+R3, F2+R4, F3+R1, F3+R2, F3+R3, F3+R4, F4+R1, F4+R2, F4+R3, and F4+R4 respectively.R4; N: negative control; Figure 2 Results of temperature optimization for fluorescence RT-RPA of the H9 subtype AIV HA gene; where A: amplification curves at temperatures of 35, 37, 39, 41, 43 and 45℃ respectively; B: maximum fluorescence value of fluorescence RT-RPA amplification reaction at different temperatures; Figure 3The fluorescence signal of the 10-minute reaction of the H9 subtype AIV HA gene fluorescent RT-RPA; Figure 4 The fluorescence signal of the 15-minute reaction of the H9 subtype AIV HA gene fluorescent RT-RPA. Figure 5 The fluorescence signal of the 20-minute reaction RT-RPA of the H9 subtype AIV HA gene; Figure 6 The maximum fluorescence signal values ​​for different amplification reaction times of the H9 subtype AIV HA gene fluorescent RT-RPA are shown. Figure 7 The results show the specificity of the fluorescence RT-RPA detection method for the H9 subtype AIV HA gene; where A: fluorescence signal curves of different pathogens amplified by the fluorescence RT-RPA detection method in this study; B: bar chart of maximum fluorescence signal. Figure 8 The results show the sensitivity of the fluorescence RT-RPA method for detecting the H9 subtype AIV HA gene; Figure 9 This provides repeatability results for the fluorescence RT-RPA detection method of the H9 subtype AIV HA gene; Figure 10 To evaluate the accuracy of real-time fluorescent RT-RPA and RT-qPCR in detecting positive and negative samples, respectively. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1 1. Materials and Methods 1.1 Virus strain samples and clinical samples The avian influenza virus strains (H9N2 AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), fowlpox virus (FPV), infectious laryngotracheitis virus (ILTV), avian leukosis virus (ALV), avian adenovirus type 4 (FAdV-4), and turkey herpesvirus (HVT) used in this study were isolated, identified, and preserved by the Poultry Disease Research Laboratory of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences. The standard antigens for the H5 and H7 subtypes of avian influenza virus strain (H5N1) were purchased from the Harbin Veterinary Research Institute. Thirty-two clinical swab samples were collected from suspected diseased chicken and duck farms in Zhejiang Province.

[0021] 1.2 Experimental Materials, Reagents and Instruments DNA Marker, gel / fragment recovery kit, and one-step reverse transcription kit were all purchased from TaKaRa; magnetic bead DNA / RNA rapid extraction kit was purchased from Changzhou Xingchun Biotechnology; T vector pEASY-T&B Zero (including T7 Promoter) and Trans-T1 competent cells were both purchased from Beijing TransGen Biotech Co., Ltd.; T7 High Yield RNA Transcription kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; EZNA MicroElute RNAClean-up kit was purchased from Omega; RNA isothermal rapid amplification kit (basic type)-II and RNA isothermal rapid amplification kit (fluorescent type)-II were both purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd. 1.3 Design and Synthesis of Primers and Probes for Fluorescent RPA Isothermal Detection The HA gene sequence of H9 subtype avian influenza virus published in GenBank was screened. Based on the RPA primer design principles, RPA-specific amplification primers and probes used in this study were designed using Oligo 7 software, as shown in Table 1. Multiple primer pairs were initially designed and BLAST-aligned with H9 subtype AIV and common avian pathogens (H5N1, NDV, IBV, etc.). Amplification was then performed using a standard RPA reaction, and primer sequences with good specificity were selected. All primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0022] Table 1 Primer and probe sequences used in the experiment In the primer sequences described in SEQ ID NOs 1-8 of Table 1, the Y, R, N, W, and H bases are all degenerate base codes. Specifically, Y = C or T; R = A or G; N = A, C, G, or T; W = A or T; H = A, C, or T. The HA-Probe1 probe is modified with the nucleotide sequence shown in SEQ ID NO.9. The 29 bp at the 5' end is modified with a FAM fluorescent group; the 31 bp at the 5' end is replaced with tetrahydrofuran (THF); the 32 bp at the 5' end is modified with a BHQ1 quencher group; and the 3' end is modified with C3Spacer.

[0023] The nucleotide sequence shown in SEQ ID NO.9 is as follows: ATATGGGCATATAATGCAGAATTGCTAGTTCTGCTTGAAAACCAGA.

[0024] 1.4 Viral DNA / RNA Extraction and Preparation of H9 Subtype AIV Standards Mix 200 μL of virus solution with 20 μL of proteinase K and nucleic acid extraction solution, place the mixture in a nucleic acid extractor to extract viral DNA / RNA, aliquot the extracted DNA / RNA into 1.5 mL centrifuge tubes, label them and store them at -80℃.

[0025] The H9 subtype AIV HA gene fragment was amplified using the PrimeScript One Step RT-PCR Kit one-step reverse transcription reagent and primers H9HA-F / R (H9HA-F (SEQ ID NO.10): 5'-ATGGAGACAGTATCAC TAATAACTAT-3', H9HA-R (SEQ ID NO.11): 5'-TTATATACAAATG TTGCATCTGC-3'). The 50 μL reaction system consisted of 2 μL PrimeScript 1 Step Enzyme Mix, 25 μL 2×1 Step Buffer, 1 μL each of forward and reverse primers, 2 μL RNA, and RNase-free H2O was added to bring the total volume to 50 μL. The RT-PCR reaction program was as follows: reverse transcription at 50 ℃ for 30 min, pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 30 s, annealing at 53 ℃ for 30 s, extension at 72 ℃ for 2 min, for a total of 35 cycles; the reaction was terminated at 4 ℃ after a 10-min extension at 72 ℃. The PCR products were identified by 1% agarose gel electrophoresis, and the fragments were excised and recovered. The recovered fragments were ligated into the T vector pEASY-T&B Zero and transformed into Trans-T1 competent cells. After selection with ampicillin (Amp) and sequencing identification at Beijing Qingke Biotechnology Co., Ltd., the positive plasmid was named pT-H9HA.

[0026] After identifying the correct positive clones, plasmids were extracted from the culture overnight. Linearized plasmids were digested with Pst I, and the fragments were recovered and their concentrations determined. RNA was transcribed in vitro according to the instructions of the T7 High Yield RNA Transcription kit. The total transcribed length was 1978 nt. DNase I was then added to the system to eliminate template DNA. The RNA fragments were purified using EZNA MicroEluteRNA Clean-up kits, and the size and integrity of the transcribed RNA were determined by 2% agarose gel electrophoresis. The RNA concentration was then measured three times, and the average value was taken. The formula was: Copy number concentration (copies / μL) = 10-1. -9 ×RNA concentration (ng / μL) × 6.022 × 10 23 / (length of in vitro transcribed RNA × 340) calculates the fragment copy number.

[0027] 1.5 Establishment of the fluorescent RT-RPA reaction system According to the instructions for the RNA Isothermal Rapid Amplification Kit (Fluorescent)-II, a fluorescent RPA isothermal amplification reaction system with a total volume of 50 μL was prepared. 29.4 μL of A buffer and 10 μmol·L⁻¹ upstream primer were added to each dry powder reaction tube. -1 2 μL of downstream primer (10 μmol·L⁻¹) -1 2 μL, probe (10 μmol·L⁻¹) -1 0.6 μL of template RNA, 3 μL of template RNA, and 10.5 μL of ddH2O were added to the inside of the reaction tube cap. Finally, 2.5 μL of start-up reaction buffer B was added to the inside of the cap. After capping tightly, the tube was shaken to mix. After a brief centrifugation, the tube was quickly placed into a real-time quantitative PCR instrument to detect the signal. The program was set to collect the fluorescence signal every 30 seconds.

[0028] 1.6 Screening of fluorescent RT-RPA reaction primers To screen for the optimal primer combination, H9 subtype AIV was amplified using the RNA Isothermal Rapid Amplification Kit (Basic)-II and different upstream and downstream primer combinations; the amplification system was prepared with a total volume of 50 μL: A buffer 29.4 μL, upstream primer (10 μmol·L⁻¹) -1 2 μL of downstream primer (10 μmol·L⁻¹) -1 2 μL of template RNA and 3 μL of ddH2O were added to the reaction tube. Finally, 2.5 μL of starter buffer B was added to the inside of the tube cap, the tube was tightly sealed, and the mixture was shaken to mix. After a brief centrifugation, the tube was quickly placed in a PCR instrument and incubated at 40°C for 30 min. After the reaction, 50 μL of DNA extraction buffer was added to the amplified product, and the mixture was vortexed and centrifuged at 12000 rpm for 5 min. 5 μL of the supernatant was mixed with 1 μL of 6× Loading Buffer and subjected to 2% agarose gel electrophoresis to screen for primer combinations with high sensitivity and specificity.

[0029] 1.7 Optimization of Fluorescent RT-RPA Reaction Temperature Using H9 subtype AIV nucleic acid as a template and the selected optimal primers, the same reaction system was configured, and RT-RPA reactions were performed at 35, 37, 39, 41, 43 and 45 °C for 20 min. The fluorescence intensity was observed to screen for the optimal reaction temperature.

[0030] 1.8 Optimization of Fluorescent RT-RPA Reaction Time Using H9 subtype AIV nucleic acid as a template, the same reaction system and negative control were configured according to the optimized conditions. The RT-RPA reaction was carried out at the optimized reaction temperature and incubated for 10, 15 and 20 min respectively. The fluorescence intensity was observed to screen the optimal reaction time.

[0031] 1.9 Fluorescent RT-RPA Specificity Assay Nucleic acids extracted in section 1.4, including H5 subtype AIV, NDV, IBV, IBDV, FPV, ILTV, ALV, FADV-4, and HVT, were used as detection targets. H9 subtype AIV nucleic acid was set as a positive control and ddH2O as a negative control. Fluorescent RT-RPA amplification was performed according to the optimized conditions and reaction procedures. The fluorescence intensity was observed to evaluate the specificity of this experimental method.

[0032] 1.10 Fluorescent RT-RPA Sensitivity Test The H9 subtype AIV standard prepared in section 1.4 was serially diluted 10-fold using ddH2O, and a copy concentration of 1.25 × 10⁻⁶ was selected. 7 10 6 10 5 10 4 10 3 10 2 10 1 Using 100 copies / μL of H9 subtype AIV standard as a template, the reaction system and reaction procedure were configured according to the optimized conditions for fluorescent RT-RPA amplification. The fluorescence intensity was observed, and the minimum detection RNA concentration was determined to evaluate the sensitivity of this experimental method.

[0033] 1.11 Fluorescent RT-RPA Repeatability Test Using the H9 subtype AIV nucleic acid extracted in section 1.4 as a template, fluorescent RT-RPA amplification was performed according to the optimized reaction conditions and procedures. Intra-batch and inter-batch repeatability tests were conducted. Three replicates were performed for each batch, with a one-day interval between batches, and detection was carried out continuously for three days. Fluorescence intensity was observed to determine the repeatability of this experimental method.

[0034] 1.12 Detection of Clinical Samples with Fluorescent RT-RPA Thirty-two swab samples suspected of being H9N2 subtype avian influenza were processed and centrifuged. 200 μL of the supernatant was collected and viral nucleic acid was extracted using a magnetic bead-based DNA / RNA rapid extraction kit. Using each nucleic acid as a template, the established fluorescent RT-RPA method was used for identification. Simultaneously, the same nucleic acid was detected using RT-qPCR, and the difference in detection rates for clinical samples was analyzed to determine the clinical applicability of this method.

[0035] 2. Results and Analysis 2.1 Preparation of H9 subtype AIV standards The post-transcriptional length of the single-stranded RNA was 1978 nt, and its concentration, measured by UV spectrophotometer, was 1352 ng / μL. The concentration of the H9 subtype AIV standard, calculated using the formula, was 1.25 × 10⁻⁶. 12 copies / μL.

[0036] 2.2 Optimization of reaction primers To screen for the optimal primers for fluorescent RT-RPA amplification, different primer combinations were used to perform conventional RT-RPA amplification on H9 subtype AIV positive samples. Nucleic acids were purified using DNA extraction buffer (phenol:chloroform:isoamyl alcohol = 25:24:1, pH > 7.8). After centrifugation at 12000 rpm for 5 min, 5 μL of the supernatant was collected for 1% agarose gel electrophoresis for identification. Results are as follows: Figure 1 As shown, all primer combinations yielded specific target genes, with fragment sizes ranging from 119 to 384 bp, consistent with predictions. The primer combination of upstream primer F2 and downstream primer R1 exhibited the highest amplification efficiency, producing a target fragment size of 164 bp, and no obvious non-specific bands were observed. Therefore, F2 and R1 were selected as the optimal primers for establishing the fluorescent RT-RPA detection method.

[0037] 2.3 Optimization of reaction temperature To screen the optimal temperature for fluorescent RT-RPA amplification, amplification reactions were performed at different temperatures (35, 37, 39, 41, 43, and 45°C). The amplification of the target product was determined by observing the fluorescence signal. The results are as follows: Figure 2 As shown, significant fluorescence signals were observed at all temperature gradients. With increasing temperature, the time of fluorescence signal appearance shifted earlier, but the maximum fluorescence value continuously decreased. The strongest fluorescence signal was observed at 39℃, with the fluorescence appearing earlier in the timeframe. Therefore, 39℃ was selected as the optimal reaction temperature for establishing the fluorescence RT-RPA detection method.

[0038] 2.4 Optimization of reaction time To screen for the shortest reaction time in fluorescent RT-RPA amplification, different reaction times (10, 15, and 20 min) were used to amplify positive H9 subtype AIV samples with high and low nucleic acid concentrations. The results are as follows: Figures 3-6 As shown, low-concentration templates can be detected by fluorescent RT-RPA amplification in just 15 minutes, with the fluorescence value showing an upward trend. For high-concentration templates, the detection method can detect them in just 10 minutes, demonstrating that fluorescent RT-RPA is a highly efficient and rapid detection method for H9 subtype AIV. To ensure the detection of low-concentration positive nucleic acid samples as much as possible, this study set the amplification reaction time to 20 minutes as the optimal reaction time for establishing the fluorescent RT-RPA detection method.

[0039] 2.5 Specificity Test To test the specificity of fluorescent RT-RPA amplification reactions, nucleic acids of various avian pathogens were amplified. The results are as follows: Figure 7 The results showed that a significant fluorescent signal could be detected only when H9 subtype AIV nucleic acid was used as a template, and no cross-reaction occurred with other pathogens, proving that the established detection method has high specificity.

[0040] 2.6 Sensitivity Test To detect the sensitivity of the fluorescent RT-RPA amplification reaction, a concentration of 1.25 × 10⁻⁶ was used. 7 ~ 1.25×10 0 Nucleic acid was detected by fluorescent RT-RPA amplification reaction using H9 subtype AIV standard RNA at a concentration of copies / μL. The results are as follows: Figure 8 As shown, when the RNA concentration is as low as 1.25 × 10⁻⁶ 1 A weak fluorescence signal was still detected at RNA concentrations of 1.25 copies / μL, and a significant fluorescence signal was produced at higher RNA concentrations; no fluorescence signal was detected at an RNA concentration of 1.25 copies / μL and in the negative control. In summary, this demonstrates that the minimum RNA detection concentration for the established H9 subtype AIV detection method is 1.25 × 10⁻⁶. 1 copies / μL.

[0041] 2.7 Repeatability Test Results of intra-batch and inter-batch repeatability tests for H9 subtype AIV are as follows: Figure 9 As shown, similar and stable fluorescence curves were observed in intra-batch and inter-batch fluorescent RT-RPA amplification reactions, with small differences between the start point and the reaction endpoint. The coefficients of variation for both intra-batch and inter-batch reactions were less than 5%, demonstrating that the established detection method has good repeatability.

[0042] 2.8 Clinical Sample Testing Thirty-two suspected H9 subtype avian influenza samples were simultaneously detected using RT-qPCR and the fluorescent RT-RPA method established in this study. The positive rate of H9 subtype AIV detected by RT-qPCR was 46.88% (15 / 32); correspondingly, the positive rate of H9 subtype AIV detected by the fluorescent RT-RPA method was 43.75% (14 / 32), showing a high degree of consistency with the detection rate of the RT-qPCR method. The results of 31 samples were consistent. The sensitivity of the method established in this study was 93.3%, the specificity was 100%, and the accuracy was 96.88% (see [link to study]. Figure 10 ).

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An RPA primer and probe for detecting H9 subtype avian influenza virus, characterized in that, The RPA primers are divided into upstream primer RPA-F and downstream primer RPA-R, wherein: The nucleotide sequence of the upstream primer RPA-F is any one of those shown in SEQ ID NO. 1~4; The nucleotide sequence of the downstream primer RPA-R is any one of those shown in SEQ ID NO. 5~8; The nucleotide sequence of the probe is shown in SEQ ID NO.

9.

2. The RPA primers and probes according to claim 1, characterized in that, The probe is modified with a fluorescent group at 29 bp at its 5' end; the probe is replaced with tetrahydrofuran (THF) at 31 bp; the probe is modified with a quenching group at 32 bp; and the probe is modified with C3Spacer at its 3' end.

3. The RPA primers and probes according to claim 1, characterized in that, The fluorescent group is one of FAM, HEX, TET, JOE, and VIC; the fluorescence quenching group is one of BHQ1, BHQ2, and BHQ3.

4. The RPA primers and probes according to claim 3, characterized in that, The modified probe sequence is as follows: 5'-ATATGGGCATATAATGCAGAATTGCTAG-i6FAMdT-T-THF-iBHQ1dT-GCTTGAAAACCAGA-C3Spacer-3'.

5. A kit for detecting H9 subtype avian influenza virus, characterized in that, It includes the RPA primers and probes as described in any one of claims 1 to 4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes at least one of the following: reaction buffer, positive control, and negative control.

7. A method for detecting H9 subtype avian influenza virus for non-diagnostic purposes, characterized in that, Includes the following steps: Using H9 subtype avian influenza virus as template DNA, RPA amplification reaction is performed using the RPA primers and probes described in any one of claims 1 to 4. The amplification products are detected by agarose gel electrophoresis. If an amplification band appears at 119-384 bp, it is H9 subtype avian influenza virus; otherwise, it is not.

8. The detection method according to claim 7, characterized in that, The RPA amplification reaction system was as follows: 29.4 μL of buffer A, 10 μmol·L⁻¹ -1 Upstream primer 2 μL, 10 μmol·L -1 Downstream primer 2 μL, 10 μmol·L -1 0.6 μL of fluorescent probe, 3 μL of template RNA, and 10.5 μL of ddH2O were used.

9. The detection method according to claim 7, characterized in that, The RPA amplification reaction was performed at a temperature of 35–45°C for 10–20 minutes.

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