Method and primer for on-site rapid detection of parrot Borna virus type 4 RNA (Ribonucleic Acid) through fluorescent isothermal amplification

Abstract

The invention belongs to the technical field of virus detection, and particularly discloses a method and primers for on-site rapid detection of parrot Borna virus type 4 RNA (Ribonucleic Acid) through fluorescent isothermal amplification. The primer and probe combination provided by the invention is used for detecting conservative M gene segments of parrot Borna virus type 4, and has extremely high specificity and sensitivity. The invention further provides a kit for detecting the parrot Borna virus type 4, and the kit can be used for on-site rapid detection of the parrot Borna virus type 4 under non-laboratory conditions.

Description

Technical Field

[0001] The invention relates to the technical field of virus detection, in particular to a method and primers for rapid on-site detection of parrot Borna virus type 4 RNA by fluorescent isothermal amplification. Background Art

[0002] Parrot Bornavirus (PaBV) is a psychotropic RNA virus that can infect a variety of bird species, but is mainly transmitted in parrots and may cause Proventricular Dilatation Disease (PDD). PaBV infection can cause gastrointestinal dysfunction in parrots, diarrhea, and inability to digest food normally. In severe cases, parrots may develop neurological symptoms such as ataxia, blindness, or head tremors, which can lead to large-scale deaths in captive parrots. Due to the characteristics of persistent infection and the lack of antiviral drugs, PaBV is difficult to eradicate once it spreads in aviaries, which has a significant impact on the parrot breeding industry and the ornamental bird trade.

[0003] There is no preventive vaccine or specific treatment for parrot Borna virus infection. Early detection and isolation are effective means to prevent the spread of the virus. At present, there are 8 known genotypes of PaBV, among which parrot Borna virus type 4 (PaBV-4) is the most commonly detected and is the main genotype infected with parrots worldwide. Therefore, it is crucial to establish a highly sensitive, rapid and accurate PaBV-4 detection method. Currently, the commonly used methods for PaBV-4 detection are RT-PCR and RT-qPCR. The detection template of this method is DNA, while PaBV-4 is an RNA virus. Therefore, the detection process not only requires the extraction of viral RNA, but also further reverse transcription into cDNA. This process is more complicated and costly. In addition, the RT-PCR and RT-qPCR methods require professional or expensive equipment in a laboratory environment, and have high professional requirements for testers, and the detection time is longer.

[0004] Therefore, it is urgent to develop new PaBV-4 detection methods to achieve simple, fast and highly sensitive detection of PaBV-4 and meet the needs of rapid detection under non-laboratory conditions. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a primer for fluorescence isothermal amplification detection of parrot Borna virus type 4 (PaBV-4). The primer set provided by the present invention can detect PaBV-4 with extremely high specificity and sensitivity.

[0006] The present invention also provides a probe for detecting PaBV-4.

[0007] The present invention also provides applications of the primer set and probe.

[0008] The present invention also provides a kit for detecting PaBV-4.

[0009] The present invention also provides a method for detecting PaBV-4.

[0010] The present invention also provides application of the above-mentioned kit for detecting PaBV-4.

[0011] In a first aspect of the present invention, a constant temperature fluorescent amplification primer set for detecting parrot Borna virus type 4 is provided, wherein the primer set comprises a specific primer pair of the sequences shown in SEQ ID No: 4 and SEQ ID No: 25, or a specific primer pair of their complementary sequences.

[0012] According to some embodiments of the present invention, the primer set provided by the present invention is a set of primers that have been screened and optimized to have the best specificity and sensitivity for detecting PaBV-4, and the primer set also has high stability and repeatability. Among them, the primer of the sequence shown in SEQ ID No: 25 carries a T7 promoter, which can be transcribed to produce a single-stranded RNA under the catalysis of T7 RNA polymerase.

[0013] In a second aspect of the present invention, a probe for detecting psittacine Borna virus type 4 is provided, wherein the probe comprises the sequence shown in SEQ ID NO: 13 or its complementary sequence.

[0014] According to some embodiments of the present invention, the probe provided by the present invention is an RNA probe, which has been screened and optimized to have the best match with the primer set described in the first aspect of the present invention, and can achieve high specificity and sensitivity for PaBV-4 detection.

[0015] According to some embodiments of the present invention, both ends of the sequence of the probe are labeled with a fluorescent group and a quenching group, respectively.

[0016] According to some embodiments of the present invention, the RNA probe can specifically bind to the single-stranded RNA produced by transcription after amplification, and then be specifically hydrolyzed by the signal amplifier enzyme. The fluorescent groups and quenching groups at both ends of the probe produce fluorescent signals, organically integrating nucleic acid exponential amplification and signal exponential amplification, achieving higher detection sensitivity and specificity, and the detection results are accurate and reliable.

[0017] According to some embodiments of the present invention, the fluorescent group includes a FAM fluorescent group.

[0018] According to some embodiments of the invention, the quencher group comprises a BHQ1 quencher group.

[0019] According to some embodiments of the present invention, the 5' end of the probe is labeled with a fluorescent group.

[0020] According to some embodiments of the present invention, the 3' end of the probe is labeled with a quencher group.

[0021] The third aspect of the present invention provides use of the primer set described in the first aspect of the present invention and the probe described in the second aspect of the present invention in the preparation of a kit for detecting psittacine Borna virus type 4.

[0022] The fourth aspect of the present invention provides a kit for detecting psittacine Borna virus type 4, comprising the constant temperature fluorescent amplification primer set described in the first aspect of the present invention and the probe described in the second aspect of the present invention.

[0023] According to some embodiments of the present invention, the kit further comprises enzyme dry powder, a reverse transcription primer set and an activation solution.

[0024] According to some embodiments of the present invention, the enzyme dry powder includes dNTP, DNA polymerase, T7 RNA polymerase and endonuclease.

[0025] According to some embodiments of the present invention, the enzyme dry powder also includes reverse transcriptase.

[0026] According to some embodiments of the present invention, the reverse transcription primer set includes random primers that specifically bind to the M gene of psittacine Borna virus type 4.

[0027] According to some embodiments of the present invention, the activation solution includes a reaction buffer and magnesium acetate.

[0028] According to some embodiments of the present invention, the kit further comprises a PaBV-4 positive control and a negative control.

[0029] According to some embodiments of the present invention, the PaBV-4 positive control includes PaBV-4 M gene RNA or a recombinant plasmid containing the PaBV-4 M gene sequence, and the negative control is enzyme-free water.

[0030] According to some embodiments of the present invention, the primer set, probe, reverse transcription primer set, enzyme dry powder and activation solution in the kit are freeze-dried to concentrate the materials required for the reaction in one tube. When the kit is used for detection, it only needs to add the sample RNA solution and mix to achieve subsequent reaction and detection.

[0031] A fifth aspect of the present invention provides a method for detecting psittacine Borna virus type 4 for non-disease diagnosis or treatment purposes, comprising the following steps:

[0032] S1, extracting RNA from the sample to be tested;

[0033] S2, using the RNA described in step S1 as a template, using the reagents in the kit described in the fourth aspect of the present invention to perform a fluorescence isothermal amplification reaction, and collecting the fluorescence signal;

[0034] S3. Determine whether the sample to be tested contains psittacine Borna virus type 4 according to the fluorescence amplification result.

[0035] According to some embodiments of the present invention, the method for detecting parrot Borna virus type 4 provided by the present invention has high detection sensitivity and specificity, and the detection results are accurate and reliable; the reaction can be carried out at a constant temperature, without the need for complex temperature cycles and electrophoresis and other steps, the operation is simple and fast, the cost is low, and it is suitable for rapid on-site detection.

[0036] The detection kit and detection method provided by the present invention break through the limitations of ordinary RT-PCR and real-time fluorescence quantitative PCR detection. RNA is used as a template and freeze-drying technology is adopted to concentrate the materials required for the reaction in one tube. During detection, only the sample RNA solution needs to be added to the mixed reagent, and amplification and detection can be achieved under constant temperature conditions. With a portable constant temperature amplification instrument, non-professionals can also achieve rapid detection of parrot Borna virus type 4 in samples under non-laboratory conditions (such as farms).

[0037] This detection method has high sensitivity, strong specificity, good repeatability, low cost, simplicity and rapidity, and no cross-reaction with other avian viruses. It can be used for the detection of clinical samples and epidemic monitoring, providing a scientific and reliable method for the identification of pathogenic microorganisms that cause glandular gastric dilatation in parrots and for reducing economic losses in breeding.

[0038] According to some embodiments of the present invention, in step S2, the fluorescent isothermal amplification reaction is to mix the reagents in the kit described in the fourth aspect of the present invention with the RNA, and set the reaction program as: temperature 39-45°C, 50-70s, 25-35 cycles.

[0039] According to some embodiments of the present invention, the reaction program is set as: temperature 42° C., 60 s, and 30 cycles.

[0040] According to some embodiments of the present invention, the temperature of the reaction procedure is constant.

[0041] According to some embodiments of the present invention, the fluorescent isothermal amplification reaction is carried out in a portable fluorescent quantitative PCR instrument.

[0042] According to some embodiments of the present invention, in step S3, the method for determining whether the sample to be tested contains psittacine bornavirus type 4 comprises:

[0043] If the Ct value of the test result of the sample to be tested is ≤30 and there is an amplification curve, it is judged to be positive for psittacine Borna virus type 4, otherwise it is judged to be negative for psittacine Borna virus type 4.

[0044] According to some embodiments of the present invention, the determination of whether the sample to be tested contains PaBV-4 requires that, on the basis of the establishment of the experiment, the Ct value of the positive control test result is ≤30 and there is an amplification curve, and the negative control test result has no amplification curve.

[0045] In a sixth aspect, the present invention provides use of the kit according to the fourth aspect of the present invention and the method according to the fifth aspect of the present invention in detecting PaBV-4;

[0046] The use is for purposes other than diagnosis or treatment of a disease.

[0047] According to some embodiments of the present invention, the detecting of PaBV-4 includes detecting whether the sample to be tested is infected with PaBV-4, and detecting whether the sample to be tested contains PaBV-4.

[0048] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0050] Figure 1 This is a diagram of the amplification results of RNA probe screening and optimization according to Example 2 of the present invention;

[0051] Figure 2 This is a diagram of the amplification results of the first round of DNA upstream primer screening and optimization in Example 2 of the present invention;

[0052] Figure 3 This is a diagram of the amplification results of the first round of DNA downstream primer screening and optimization in Example 2 of the present invention;

[0053] Figure 4 This is a diagram of the amplification results of the second round of DNA upstream primer screening and optimization in Example 3 of the present invention;

[0054] Figure 5 This is a diagram of the amplification results of the second round of DNA downstream primer screening and optimization in Example 3 of the present invention;

[0055] Figure 6 This is a graph showing the specificity detection results of the detection method of Example 6 of the present invention;

[0056] Figure 7 This is a sensitivity test result diagram of the detection method of Example 7 of the present invention. DETAILED DESCRIPTION

[0057] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0058] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0059] Example 1

[0060] In this example, the detection target of psittacine Borna virus type 4 (PaBV-4) was selected and its plasmid standard was constructed.

[0061] The M gene of the positive pathogenic material of parrot Borna virus type 4 (PaBV-4) whose whole genome has been sequenced and typed in our laboratory was searched on the National Center for Biotechnology Information (NCBI), and all gene sequences with homology of 95%-100% in the NCBI database were downloaded. The conservative sequences were screened using MEGA 11.0, and finally the preferred sub-fragment (42-254) with a length of 213 bp was screened out for the M gene fragment of PaBV-4 as the detection target, and the fragment was cloned into the pMD18-T vector and transformed into DH5α competent cells to prepare the recombinant plasmid standard pMD18-T-PaBV4.

[0062] The copy number of the plasmid standard was calculated according to the formula: Plasmid copy number (copies / μL) = (6.02×10 23 copies / mol)×(plasmid concentration×10 -9 ng / μL) / (MWg / mol), where the average molecular mass of dsDNA (MWg / mol) = (number of bases) × (660 Daltons / base).

[0063] Example 2

[0064] This example designs and optimizes the screening of DNA primers and RNA probes.

[0065] For the preferred sub-fragments screened in Example 1, SnapGene 6.2.2 and Primer Premier 5.0 software were used to design DNA primers and RNA probes, and the BLAST program was used to verify their specificity. The sequences of the primers and probes obtained by the preliminary screening are shown in Table 1.

[0066] Table 1 Sequences of first-round primers and probes

[0067]

[0068]

[0069] The 5' end of the RNA probe is labeled with carboxyfluorescein FAM, and the 3' end is labeled with a quenching group BHQ1.

[0070] 1. Screening and optimization of RNA probes:

[0071] DNA upstream primers F2 and F3 and DNA downstream primers R2 and R3 were selected for cross-combination. The combination arrangement of the primers is shown in Table 2.

[0072] Table 2

[0073]

[0074]

[0075] The plasmid standard of Example 1 was amplified using the four primer combinations in Table 2, and the plasmid concentration in the control sample was 1×10 6 The six RNA probes shown in Table 1 were screened. The reaction system was as follows: 10 μmol / L upstream and downstream primers and 1 μL of RNA probe, 7 μL of plasmid template, and 10 μL of activation solution (containing reaction buffer and magnesium acetate) were added to each enzyme powder tube (containing 50-100 μM dNTPs, 1 μmol endonuclease, 50 nmol DNA polymerase, and 50 nmol T7 RNA polymerase). The mixture was mixed and placed in a portable fluorescence quantitative PCR instrument for on-machine detection. The temperature was set to 42°C for 1 min and 30 cycles. The fluorescence value (RFU) and Ct value of the test samples of each set of primers and RNA probe combinations were tested. The results are shown in the table. Figure 1 shown.

[0076] Among them, the endpoint fluorescence using the primer set F3-R3 and the RNA1 probe combination was the highest and the Ct value was the smallest, which was 6.12. Therefore, the F3-R3-RNA1 combination was selected for subsequent screening.

[0077] 2. Screening and optimization of DNA upstream primers:

[0078] Based on the above optimized combination F3-R3-RNA1, fixed probe RNA1 and DNA downstream primer R3, the concentration of the control plasmid sample was 1 × 10 5 copies / μL, screening DNA upstream primers, the results are as follows Figure 2 shown.

[0079] Among them, the F4-R3-RNA1 combination had the highest endpoint fluorescence value and the smallest Ct value, which was 8.62. Therefore, the F4-R3-RNA1 combination was selected for subsequent screening.

[0080] 3. Screening and optimization of DNA downstream primers:

[0081] Based on the above optimized combination F4-R3-RNA1, fixed probe RNA1 and DNA upstream primer F4, the concentration of the control plasmid sample was 1 × 10 4 copies / μL, screen DNA downstream primers, the results are as follows Figure 3 shown.

[0082] Among them, the F4-R3-RNA1 combination had the highest endpoint fluorescence value and the smallest Ct value, which was 9.46. Therefore, the optimal DNA downstream primer selected in this round was R3.

[0083] Through the above steps, RNA probes and DNA primers were screened and optimized, and finally F4-R3-RNA1 was selected as the optimal combination for subsequent testing and sample detection.

[0084] Example 3

[0085] In this example, a second round of design and optimization screening was performed on DNA primers and RNA probes.

[0086] According to the first round of primer and probe optimization screening results of Example 2, RNA1 was used as the probe, and SnapGene6.2.2 and Primer Premier 5.0 software were used to design the second round of primers, and then the BLAST program was used to verify its specificity. The sequences of the second round primers screened are shown in Table 3.

[0087] Table 3 Second round primer sequences

[0088]

[0089]

[0090] 1. Screening and optimization of the second round of DNA upstream primers:

[0091] Based on the combination F4-R3-RNA1 selected in the first round of optimization in Example 2, the probe RNA1 and the DNA downstream primer R3 were fixed, and the concentration of the plasmid sample was controlled to 1×10 3copies / μL, and the second round of DNA upstream primer optimization was performed. The results were as follows Figure 4 shown.

[0092] Among them, F4-R3-RNA1 had the highest endpoint fluorescence value and the smallest Ct value, which was 13.17, so the F4-R3-RNA1 combination was selected.

[0093] 2. Screening and optimization of the second round of DNA downstream primers:

[0094] Based on the above optimized combination F4-R3-RNA1, fixed probe RNA1 and DNA upstream primer F4, the concentration of the control plasmid sample was 1 × 10 3 copies / μL, and the second round of DNA downstream primer optimization was performed. The results were as follows Figure 5 shown.

[0095] Among them, F4-R7-RNA1 had the highest endpoint fluorescence value and the smallest Ct value, which was 10.69, so the F4-R7-RNA1 combination was selected.

[0096] Through the above steps, the DNA primers were screened and optimized, and finally F4-R7-RNA1 was selected as the optimal combination for subsequent testing and sample detection.

[0097] Example 4

[0098] This example designs and integrates reverse transcription (RT) primers.

[0099] Based on the F4-R7-RNA1 combination screened by the second round of primer and probe optimization in Example 3, a set of specific reverse transcription RT primers was designed using SnapGene 6.2.2 and Primer Premier 5.0 software, and the BLAST program was used to verify its specificity. The RT primer sequences screened were shown in Table 4.

[0100] Table 4 RT primer sequences

[0101]

[0102]

[0103] Example 5

[0104] This example provides a method for rapid on-site detection of PaBV-4 RNA by fluorescence isothermal amplification.

[0105] DNA primer F4 (10 μmol), DNA primer R7 (10 μmol), RNA probe RNA1 (1 μM), RT primer set (1 μmol each), as well as endonuclease (10 μmol), T7 RNA polymerase and DNA polymerase (50 nmol), activation solution (containing reaction buffer, 100 μmol dNTP and magnesium acetate) and reverse transcriptase (50 nmol) were pre-packaged and lyophilized in eight tubes, and a PaBV-4 positive control solution containing the recombinant plasmid pMD18-T-PaBV4 of Example 1 was prepared, and nuclease-free water was used as a negative control.

[0106] The specific steps for rapid on-site detection of PaBV-4 are as follows:

[0107] 1) Obtaining an RNA sample solution to be tested;

[0108] 2) Using the RNA sample to be tested as a template, add 50 μL of RNA sample solution, positive control solution, and negative control solution along the wall of each eight-tube strip, respectively, confirm that the solution contacts the lyophilized powder and the lyophilized powder is completely dissolved, cover the eight-tube strip, pinch both ends of the eight-tube strip, manually mix up and down 20 times, and gently shake the liquid to the bottom of the eight-tube strip 3 times;

[0109] 3) Place the eight-tube strip into a portable fluorescent quantitative PCR instrument for on-machine detection, set the temperature to 42°C, cycle 30 times for 1 min, and collect the fluorescent signal. The result of the detection method is determined as follows: on the basis of the establishment of the experiment, if the test result of the sample is ≤30 in the FAM channel and there is an amplification curve, it is judged as PaBV-4 positive; when the Ct value is >30, it is judged as PaBV-4 negative.

[0110] Example 6

[0111] This example conducts a specificity test on the on-site rapid detection method of RNA fluorescence isothermal amplification of PaBV-4.

[0112] Extract nucleic acid from samples of avian influenza virus (including H5N2, H7N9, and H9N2 subtypes), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), Marek's disease virus (MDV), and infectious bronchitis virus (IBDV). After concentration determination, dilute the sample to 1×10 6 copies / μL, and the detection method of Example 5 was used to detect them respectively, with pMD18-T-PaBV4 plasmid standard (concentration 1×10 6 copies / μL) was used as a positive control, and enzyme-free water was used as a negative control.

[0113] The results are as follows Figure 6As shown, except for the positive control which has a typical amplification curve, the detection results of the other samples are all negative, which shows that the detection method provided by the present invention has good specificity.

[0114] Example 7

[0115] This example conducts a sensitivity test on the on-site rapid detection method of RNA fluorescence isothermal amplification of PaBV-4.

[0116] RNA was extracted from the PaBV-4 virus sample, and the concentration was tested using a QuibitTM 4 fluorometer and diluted to 5×10 7 The RNA solution was diluted to 5 × 10 copies / μL using enzyme-free water. 0 The diluted RNA was used as a template and a sensitivity test was performed using the detection method of Example 5. Enzyme-free water was used as a negative control.

[0117] The results are as follows Figure 7 As shown in the figure, the amplification curve can still be detected when diluted to 5 copies / μL, indicating that the method has good sensitivity.

[0118] Example 8

[0119] This example conducts a repeatability test on the RNA fluorescence isothermal amplification on-site rapid detection method of PaBV-4.

[0120] The plasmid standard was diluted in gradients according to Example 7, and the detection method of Example 5 was used to take 4 concentrations of PaBV-4 RNA for 3 intra-batch and inter-batch repeatability tests, and the mean, standard deviation and coefficient of variation of the cycle number were calculated respectively. The test data are shown in Table 5 below.

[0121] Table 5 Intra-group and inter-group repeatability experimental data

[0122]

[0123] From the experimental data in Table 5, it can be seen that the intra-group coefficient of variation is between 0.72% and 3.80%, and the inter-group coefficient of variation is between 1.80% and 3.45%. Both coefficients of variation are less than 10%. The experimental results show that the detection method of the present invention has high stability and repeatability.

[0124] Example 9

[0125] This example uses clinical samples for testing.

[0126] 108 clinical tissue samples from zoos and wildlife rescue centers were tested using the traditional RT-PCR detection method (Zhai Junqiong et al., 2021) and the detection method of Example 5.

[0127] The results are shown in Table 6. The positive rate of psittacine Borna virus type 4 detected by the detection method of Example 5 was 11.11% (12 / 108), and the positive rate of psittacine Borna virus type 4 detected by the traditional RT-PCR detection method was 8.33% (9 / 108). According to the formula, the positive coincidence rate of clinical samples detected by the two methods was 100% (9 / 9), the negative coincidence rate was 96.97% (9 / 9), and the total rate was 94.44% (9+96 / 108).

[0128] Table 6 Clinical sample test results

[0129]

[0130] Note: Total compliance rate = (true positive + true negative) / (true positive + true negative + false positive + false negative) * 100%; positive compliance rate = true positive / (true positive + false negative) * 100%; negative compliance rate = true negative / (true negative + false positive) * 100%.

[0131] In summary, the method established by the present invention does not require reverse transcription of RNA into cDNA for detection. Compared with the traditional RT-PCR method, it is more sensitive, rapid and accurate, and is suitable for on-site rapid detection under non-laboratory conditions. Non-professionals can also realize on-site rapid detection of parrot Borna virus type 4 nucleic acid in samples under non-laboratory conditions (such as farms), and the entire detection process can be completed in about 30 minutes.

[0132] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A constant temperature fluorescent amplification primer set for detecting parrot Borna virus type 4, characterized in that: The primer set includes a specific primer pair of the sequences shown as SEQ ID No: 4 and SEQ ID No: 25, or a specific primer pair of the complementary sequences thereof.

2. A probe for detecting psittacine Borna virus type 4, characterized in that: The probe includes the sequence shown in SEQ ID NO: 13 or its complementary sequence.

3. The probe according to claim 2, characterized in that: Both ends of the probe sequence are labeled with a fluorescent group and a quenching group respectively.

4. Use of the constant temperature fluorescent amplification primer set according to claim 1, the probe according to claim 2 or the probe according to claim 3 in preparing a kit for detecting parrot Borna virus type 4.

5. A kit for detecting psittacine Borna virus type 4, characterized in that: It comprises the constant temperature fluorescent amplification primer set as claimed in claim 1, and the probe as claimed in claim 2 or 3.

6. The kit according to claim 5, characterized in that The kit also includes enzyme dry powder, reverse transcription primer set and activation solution; Preferably, the enzyme dry powder includes dNTP, DNA polymerase, T7 RNA polymerase and endonuclease; Preferably, the reverse transcription primer set includes random primers that specifically bind to the M gene of psittacine Borna virus type 4.

7. A method for detecting psittacine Borna virus type 4 for non-disease diagnostic or therapeutic purposes, characterized in that: The following steps are involved: S1, extracting RNA from the sample to be tested; S2, using the RNA described in step S1 as a template, using the reagents in the kit described in claim 6 to perform a fluorescence isothermal amplification reaction, and collecting the fluorescence signal; S3. Determine whether the sample to be tested contains psittacine Borna virus type 4 according to the fluorescence amplification result.

8. The method according to claim 7, characterized in that In step S2, the fluorescence isothermal amplification reaction is to mix the reagents in the kit of claim 6 with the RNA, and set the reaction program as follows: temperature 39-45°C, 50-70s, 25-35 cycles.

9. The method according to claim 7, characterized in that: In step S3, the method for determining whether the sample to be tested contains psittacine bornavirus type 4 comprises: If the Ct value of the test result of the sample to be tested is ≤30 and there is an amplification curve, it is judged to be positive for parrot Borna virus type 4, otherwise it is judged to be negative for parrot Borna virus type 4.

10. Use of the kit according to claim 5 or 6 or the method according to any one of claims 7 to 9 in detecting psittacine Borna virus type 4; The use is for purposes other than diagnosis or treatment of a disease.

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