Compositions for detecting paBV types 2, 4 and 5, kits and uses thereof

Abstract

The application belongs to the technical field of virus detection, and discloses a composition for detecting PaBV type 2, type 4 and type 5, a kit and application thereof. Specifically disclosed is a reagent comprising a primer set and / or a probe set. The reagent provided by the application can be used for detecting a P gene fragment of PaBV which is highly conservative, can simultaneously and rapidly detect and identify three genotypes of the borna disease virus type 2, type 4 and type 5 causing psittacine proventricular dilatation syndrome, and has very high detection sensitivity and specificity, and the detection result is accurate and reliable. The three genotypes of the pathogen of the psittacine borna disease virus can be detected by only performing one PCR amplification in one reaction.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to compositions, kits and applications for detecting PaBV types 2, 4 and 5. Background Technology

[0002] Parrot Bornavirus (PaBV) is a member of the Bornaviridae family that causes proventricular dilatation disease (PDD) in parrots. PDD is a deadly infectious disease that can cause mass mortality in captive parrots. Due to its persistent nature and the lack of antiviral drugs, PaBV is difficult to eradicate once it spreads in aviaries, posing a significant threat to the global parrot breeding industry. On the one hand, due to the bright plumage and mimicry abilities of parrots, many countries keep them as pets and ornamental birds, leading to a year-on-year increase in the international trade of parrots. On the other hand, because PaBV infection can be observed in asymptomatic parrots and clinical symptoms may not appear for at least two months after detection, it is essential to establish highly sensitive detection methods for PaBV infection to isolate infected birds before the virus spreads. Researchers have developed RT-PCR and RT-qPCR methods for detecting PaBV RNA, with the latter showing higher sensitivity.

[0003] There are seven genotypes of PaBV, among which PaBV-4 is the dominant genotype in parrots worldwide. In recent years, there have been increasing reports on PaBV-2 in cockatiels and PaBV-5 in cockatoos, but research on PaBV-5 is still limited. Different genotypes cause very similar clinical symptoms and epidemiological characteristics in parrots. Without differentiation, cross-infection and viral mutations can easily occur in captive parrot populations, leading to greater economic losses and public safety hazards. Therefore, it is necessary to develop a rapid detection method for clinical prevention and differentiation of the three PaBV genotypes (PaBV-2, PaBV-4, and PaBV-5). Summary of the Invention

[0004] The first aspect of the present invention is to provide a reagent.

[0005] A second aspect of the present invention is to provide a reagent kit.

[0006] The object of a third aspect of the present invention is to provide the application of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention.

[0007] The fourth aspect of this invention aims to provide a triple fluorescent PCR detection method for simultaneously detecting PaBV-2, PaBV-4 and / or PaBV-5.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a reagent comprising a primer set and / or a probe set, the nucleotide sequences of which are shown below:

[0010] The primer set is used to detect PaBV-2, PaBV-4 and / or PaBV-5.

[0011] The primers for detecting PaBV-2 are:

[0012] PaBV-2-F1: 5'-GAACGAAACTATGAAGATGA-3', or the complementary sequence of this sequence;

[0013] PaBV-2-R1: 5'-GTTGGGTAAATTCTAGAAGG-3', or the complementary sequence of this sequence;

[0014] The primers for detecting PaBV-4 are:

[0015] PaBV-4-F1: 5'-GAGCAAGTCAAGAAGAAC-3', or the complementary sequence of this sequence;

[0016] PaBV-4-R1: 5'-CCGAATTAGGTCATCATTC-3', or the complementary sequence of this sequence;

[0017] The primers for detecting PaBV-5 are:

[0018] PaBV-5-F1: 5'-AGACCATCAAGAAGAACC-3', or the complementary sequence of this sequence;

[0019] PaBV-5-R1: 5'-CCTCTTAATCCTTCATTCTC-3', or the complementary sequence of this sequence;

[0020] The probe set is used to detect PaBV-2, PaBV-4 and / or PaBV-5.

[0021] The probe for detecting PaBV-2 is:

[0022] PaBV-2-P1: 5'-TCTTCTCTATTCGGCGATGGCAA-3', or the complementary sequence of this sequence;

[0023] The probe used to detect PaBV-4 is:

[0024] PaBV-4-P1: 5'-TCCATGATCTCAGACCAAGAGCC-3', or the complementary sequence of this sequence;

[0025] The probe used to detect PaBV-5 is:

[0026] PaBV-5-P1: 5'-CTCCGCCAATTCAGTTACGAGC-3', or the complementary sequence of this sequence.

[0027] In some embodiments of the present invention, both ends of the probe sequence are labeled with a fluorescent group and a quenching group, respectively, and the fluorescent groups labeled with each probe sequence are different.

[0028] In some embodiments of the present invention, the fluorescent group is at least one of FAM, Hex, VIC, TAMRA, ROX, Texas-Red and CY5.

[0029] In some embodiments of the present invention, the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

[0030] In some embodiments of the present invention, the fluorescent group is attached to the 5' end of the probe.

[0031] In some embodiments of the present invention, the quenching group is attached to the 3' end of the probe.

[0032] In some preferred embodiments of the present invention, the 5' end of PaBV-2-P is connected to the fluorescent group FAM, and the 3' end is connected to the quenching group BHQ1.

[0033] In some preferred embodiments of the present invention, the 5' end of the PaBV-4-P is connected to the fluorescent group Hex, and the 3' end is connected to the quenching group BHQ1.

[0034] In some preferred embodiments of the present invention, the 5' end of PaBV-5-P is connected to the fluorescent group Texas-Red, and the 3' end is connected to the quenching group BHQ1.

[0035] A second aspect of the present invention provides a reagent kit comprising the reagent of the first aspect of the present invention.

[0036] In some embodiments of the present invention, the kit further includes PCR reaction solution and positive reference.

[0037] A third aspect of the present invention provides the use of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention in any one of (1) to (6):

[0038] (1) Identify PaBV-2, PaBV-4 and / or PaBV-5 types;

[0039] (2) Prepare products for detecting or assisting in the detection of PaBV-2, PaBV-4 and / or PaBV-5;

[0040] (3) Detect whether the sample to be tested is PaBV-2, PaBV-4 and / or PaBV-5;

[0041] (4) Prepare products for testing or auxiliary testing to determine whether the sample to be tested is of PaBV-2, PaBV-4 and / or PaBV-5 type;

[0042] (5) Detect whether the sample to be tested is infected with PaBV-2, PaBV-4 and / or PaBV-5;

[0043] (6) Prepare products for detecting or assisting in the detection of whether the sample to be tested is infected with PaBV-2, PaBV-4 and / or PaBV-5;

[0044] The above applications are used for the diagnosis and treatment of non-disease conditions.

[0045] A fourth aspect of the present invention provides a triple fluorescent PCR detection method for simultaneously detecting PaBV-2, PaBV-4, and / or PaBV-5, comprising the steps of using the reagents of the first aspect of the present invention and / or the kit of the second aspect of the present invention for detection, said method being used for non-disease diagnosis and treatment.

[0046] In some embodiments of the present invention, the detection method includes the following steps:

[0047] 1) Extract nucleic acid from the sample to be tested;

[0048] 2) Using the nucleic acid from step 1) as a template, perform triple fluorescent PCR amplification reaction with the reagent or the kit described above, and collect the fluorescence signal;

[0049] 3) Determine whether the sample contains PaBV-2, PaBV-4 and / or PaBV-5 based on the fluorescence signal.

[0050] In some embodiments of the present invention, the multiplex fluorescent PCR amplification reaction system in step 2) is as follows:

[0051]

[0052] In some embodiments of the present invention, the triple fluorescent PCR amplification reaction program in step 2) is as follows: 35–40°C for 2–3 min; 94–96°C for 4–6 min; 90–95°C for 8–10 s; 50–62°C for 30–35 s; 40–45 cycles.

[0053] In some embodiments of the present invention, the triple fluorescent PCR amplification reaction program in step 2) is as follows: digestion at 37°C for 2 min, pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, for 45 cycles.

[0054] In some embodiments of the present invention, the PCR reaction solution is AceQ Universal U+Probe Master MixV2.

[0055] In some embodiments of the present invention, the result determination method of the detection method is as follows: based on the experimental validity, if the test sample's detection result is ≤37.01 in the FAM channel and has an amplification curve, it is judged to be positive for PaBV2 type; if the Ct value is ≤36.64 in the Hex channel and has an amplification curve, it is judged to be positive for PaBV4 type; if the Ct value is ≤35.29 in the Texas-Red channel and has an amplification curve, it is judged to be positive for PaBV5 type; if there is no Ct value or the Ct value is >37.01 in all three channels, it is judged to be negative for PaBV2, PaBV4 and PaBV5 types; if the test sample has a Ct value <37.01 in any channel (35.29 < Ct value ≤37.01), the sample should be re-extracted for nucleic acid and tested again. If the repeated test result has no Ct value, it is negative; otherwise, it is positive.

[0056] The beneficial effects of this invention are:

[0057] The reagents provided by this invention can be used to detect the highly conserved P gene fragment of PaBV, and can simultaneously and rapidly detect and identify three genotypes of Bornavirus (BBV) – types 2, 4, and 5 – that cause parrot proventricular dilatation. It exhibits extremely high detection sensitivity and specificity, and the detection results are accurate and reliable. This invention enables the detection of the pathogens of three genotypes of Parrot Bornavirus with only one PCR amplification in a single reaction tube.

[0058] The detection kit provided by this invention breaks through the limitations of single fluorescent PCR detection, and can simultaneously detect the content of three genotypes of parrot Borna virus (PNP) types 2, 4 and 5 in samples in real time. It has high sensitivity, strong specificity, good repeatability, low cost, and can detect a large number of samples at the same time. Moreover, it has no cross-reactivity with other avian viruses, making it very suitable for the detection of large numbers of clinical samples and epidemic monitoring. It provides a scientific and reliable method for the identification of pathogenic microorganisms causing parrot proventricular ectasia and for reducing economic losses in poultry farming. Attached Figure Description

[0059] Figure 1 The amplification curves are shown for the detection of plasmid standards using the detection method in Example 3.

[0060] Figure 2 The standard curve for detecting plasmid standard (PaBV-2) using the detection method in Example 3 is shown.

[0061] Figure 3 The standard curve for detecting plasmid standard (PaBV-4) using the detection method in Example 3 is shown.

[0062] Figure 4 The standard curve for detecting plasmid standard (PaBV-5) using the detection method in Example 3 is shown.

[0063] Figure 5 The results show the sensitivity test results of the detection method in Example 5.

[0064] Figure 6 The results are specific detection results of the detection method in Example 7. Detailed Implementation

[0065] The present invention will now be described in detail with reference to specific embodiments, but this does not limit the scope of the invention.

[0066] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0067] Example 1: Target selection, primer and probe screening and optimization

[0068] The full genome sequences of PaBV-2, PaBV-4, and PaBV-5 were downloaded from the National Center for Biotechnology Information (NCBI). Conserved sequences were screened using MEGA 11.0. Primers and probes for the P gene fragment of PaBV were designed and screened using Primer Premier 6.0 and Oliogo 7. The primers obtained from the preliminary screening are shown in Table 1. After further BLAST comparison and verification, the specificity of the primers and probes was determined. The nucleic acid sequences of the final selected primer-probe combinations were PaBV-2-F1 (SEQ ID NO:1), PaBV-2-R1 (SEQ ID NO:2), PaBV-2-P1 (SEQ ID NO:3); PaBV-4-F1 (SEQ ID NO:6), PaBV-4-R1 (SEQ ID NO:7), PaBV-4-P1 (SEQ ID NO:8); PaBV-5-F1 (SEQ ID NO:11), PaBV-5-R1 (SEQ ID NO:12), PaBV-5-P1 (SEQ ID NO:13).

[0069] Table 1 Primer and probe sequences

[0070]

[0071] Among them, the 5' end of the PaBV2 detection probe is labeled with carboxyfluorescein FAM, and the 3' end is labeled with quencher group BHQ1;

[0072] The 5' end of the PaBV4 detection probe is labeled with carboxyfluorescein Hex, and the 3' end is labeled with the quencher group BHQ1.

[0073] The 5' end of the PaBV5 detection probe is labeled with carboxyfluorescein Texas-Red, and the 3' end is labeled with the quencher group BHQ2.

[0074] Example 2: Construction of plasmid standards

[0075] Homology alignment and phylogenetic analysis of the three PaBV genotypes were performed using MEGA 11.0 to screen for conserved sequences. To ensure uniform plasmid concentration during subsequent optimization of the three viruses, pET-23a-PaBV-based plasmid standards were synthesized from the conserved sequences of PaBV-2, PaBV-4, and PaBV-5. The plasmids were synthesized by Shanghai Sangon Biotech Co., Ltd. The concentration was calculated according to the formula (concentration ng / μL × 6.02 × 10²³ × 10⁻⁶).-9 Calculate the copy number of each plasmid standard using (DNA length × 660) and dilute it to the appropriate copy number.

[0076] Example 3: Establishment and optimization of a triple fluorescence PCR detection method

[0077] A triplet real-time quantitative PCR detection method for detecting three PaBV genotypes includes the following steps:

[0078] (1) Obtain the nucleic acid from the sample to be tested;

[0079] (2) Using the nucleic acid in step (1) as a template, triple real-time quantitative PCR was performed using the primer and probe combination designed and screened in Example 1. The triplet real-time quantitative PCR amplification conditions were as follows: 10 μL of 2×AceQ Universal U+Probe Master Mix V2; 0.4 μL each of 10 μM upstream and downstream primers for Psittacovirus type 2 (PaBV-2), and 0.2 μL of 10 μM probe for Psittacovirus type 2 (PaBV-2); 0.6 μL each of 10 μM upstream and downstream primers for Psittacovirus type 4 (PaBV-4), and 0.2 μL of 10 μM probe for Psittacovirus type 4 (PaBV-4); 0.3 μL each of 10 μM upstream and downstream primers for Psittacovirus type 5 (PaBV-5), and 0.2 μL of 10 μM probe for Psittacovirus type 5 (PaBV-5); 2 μL of template; and sterile deionized water to a final volume of 20 μL. The reaction procedure was as follows: digestion at 37℃ for 2 min, pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, for 45 cycles.

[0080] (3) Amplification was carried out using the above reaction conditions and system to obtain amplification kinetic curves. The standard linear regression equation (standard curve) was derived by plotting the common logarithm (lgC) of the initial copy number of the standard (prepared in Example 2) on the x-axis and the cycle number threshold (Ct value) on the y-axis, and its sensitivity test data were obtained.

[0081] Result Interpretation: Based on the established experimental results, if the test sample's Ct value in the FAM channel is ≤37.01 and an amplification curve is present, it is judged as positive for PaBV2 type; if the Ct value in the Hex channel is ≤36.64 and an amplification curve is present, it is judged as positive for PaBV4 type; if the Ct value in the Texas-Red channel is ≤35.29 and an amplification curve is present, it is judged as positive for PaBV5 type; if no Ct value or a Ct value >37.01 is detected in any of the three channels, it is judged as negative for PaBV2, PaBV4, and PaBV5 types; if the test sample's Ct value is ≤37.01 and <35.29 in any channel, the sample should be re-extracted for nucleic acid and retested. If the repeated test result has no Ct value, it is negative; otherwise, it is positive.

[0082] The TaqMan triple qPCR reaction system was optimized using the matrix method. The optimization of PaBV-2, PaBV-4 and PaBV-5 used a single recombinant plasmid pET-23a-PaBV as a template, as shown in Table 2 below.

[0083] Table 2 Taqman reaction system

[0084]

[0085]

[0086] Annealing temperature optimization: The system was optimized at three temperature gradients: 50℃, 55℃, and 60℃. By calculating the amplification efficiency, the optimal annealing temperature was determined to be 60℃. The optimization results are shown in Table 3.

[0087] Table 3 Optimization results of annealing temperature

[0088]

[0089] Optimization of primer and probe concentrations: The qPCR primers screened in Table 1 were diluted to 20 μmol / L. Each upstream and downstream primer was mixed in equal portions, and the mixture was aliquoted into 50 μL tubes. qPCR amplification was performed at seven gradients, from 0.2 μL to 0.8 μL, to determine the optimal primer concentration. The qPCR probes were diluted to 10 μmol / L, aliquoted, and qPCR amplification was performed at four gradients, from 0.1 μL to 0.4 μL, to determine the optimal probe addition volume for all values ​​of 0.2 μL. The optimization results are shown in Tables 4-6.

[0090] Table 4. Optimization results of PaBV-2 primers

[0091]

[0092]

[0093] Table 5. Optimization results of PaBV-4 primers

[0094]

[0095] Table 6. Optimization results of PaBV-5 primers

[0096]

[0097]

[0098] After optimization, the triplet real-time quantitative PCR amplification conditions were as follows: 10 μL of 2×AceQ Universal U+Probe Master Mix V2; 0.4 μL each of 10 μM upstream and downstream primers for Psittacovirus type 2 (PaBV-2), and 0.2 μL of 10 μM probe for Psittacovirus type 2 (PaBV-2); 0.6 μL each of 10 μM upstream and downstream primers for Psittacovirus type 4 (PaBV-4), and 0.2 μL of 10 μM probe for Psittacovirus type 4 (PaBV-4); 0.3 μL each of 10 μM upstream and downstream primers for Psittacovirus type 5 (PaBV-5), and 0.2 μL of 10 μM probe for Psittacovirus type 5 (PaBV-5); template was 2 μL. Sterile deionized water was added to a final volume of 20 μL.

[0099] The reaction procedure was as follows: digestion at 37℃ for 2 min, pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, for 45 cycles.

[0100] Example 4: Establishment of Standard Curve

[0101] Using the plasmid standard (Example 2) as a template, it was serially diluted 10-fold to obtain: 3.483 × 10⁻⁶. 6 copies / μL, 3.483×10 5 copies / μL, 3.483×10 4 copies / μL, 3.483×10 3 copies / μL, 3.483×10 2 copies / μL, 3.483×10 1 The diluted plasmid was then aliquoted into copies / μL and stored at -20°C; the diluted plasmid was then amplified and detected using the reaction system described in Example 3. The amplification results are as follows: Figure 1 As shown, the template copy number and cycle number (Ct value) were obtained. After the detection was completed, a standard curve was plotted by comparing the concentration lg value (X-axis) of each standard with its corresponding Ct value (Y-axis). The results are shown in [Figure 1]. Figures 2-4 .

[0102] According to the formula: Amplification efficiency E = 10 -1 / 斜率 -1, by Figures 2-4 It can be seen that the slopes of PaBV-2, PaBV-4, and PaBV-5 are -3.250, -3.200, and -3.310, respectively. The amplification efficiencies of PaBV-2, PaBV-4, and PaBV-5 are calculated to be 103.09%, 105.35%, and 100.50%, respectively, proving that the triplet real-time PCR constructed in Example 3 has high amplification efficiency.

[0103] Example 5 Sensitivity Test

[0104] The recombinant standard plasmid (pET-23a-PaBV, Example 2) powder synthesized by Shanghai Sangon Biotech was diluted with appropriate amount of sterile water to 100 ng / μL, according to the formula (concentration ng / μL × 6.02 × 10). 23 ×10 -9 Calculate the copy number of each plasmid by () / (plasmid length × 660) and perform serial dilutions (10-fold serial dilution). Use the diluted standard plasmids as templates for sensitivity testing.

[0105] Triple real-time quantitative PCR was performed using the detection method described in Example 3. Results are shown below. Figure 5 The detection limits for PaBV-2, PaBV-4, and PaBV-5 were all 34.8 copies / μL, corresponding to cycle numbers of 37.01, 36.64, and 35.29, respectively, demonstrating that the established method has good sensitivity.

[0106] Example 6 Repeatability Test

[0107] Using a 10-fold serially diluted quantitative PCR plasmid standard (Example 2) as a template, the following 10-fold serial dilutions were performed to obtain a plasmid standard of 3.483 × 10⁻⁶. 6 copies / μL, 3.483×10 5 copies / μL, 3.483×10 4 copies / μL, 3.483×10 3 The samples were collected in copies / μL and detected using the triplet real-time quantitative PCR method described in Example 3. Each test was performed in triplicate for three cycles. The mean cycle number, standard deviation, and coefficient of variation were calculated for each cycle. The results are shown in Table 7. The coefficients of variation within and between groups were all less than 0.76%, demonstrating that the established method has good reproducibility.

[0108] Table 7. Repeatability test results of the triple real-time PCR detection method

[0109]

[0110]

[0111] Example 7 Specificity Detection

[0112] The detection method described in Example 3 was used to detect common avian viruses and other PaBV genotypes. Commercially available recombinant avian influenza virus (H5+H7) bivalent inactivated vaccine (H5N2 rFJ56 strain + H7N9 rGD76 strain), Newcastle disease live vaccine (La Sota strain), and avian infectious bronchitis H120 strain vaccine were used as positive controls for avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus. Positive plasmids for PaBV-1, PaBV-3, and PaBV-7 were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0113] The experimental results are shown in Figure 6 (The amplification curves shown in the figure are amplification curves for plasmid standards). The detection results indicate that the detection results for common avian viruses such as avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus, as well as other genotypes of PaBV, were all negative, and the plasmid standards were detected normally, indicating that the detection method in Example 3 has good specificity.

[0114] Example 8: Detection of Clinical Samples

[0115] Three hundred and eighty clinical samples from zoos and wildlife rescue centers were tested. Both conventional singlet RT-PCR (Zhai et al., 2021) and the method described in Example 3 were used. Results showed that the positive rates for PaBV-2 detected by the method in Example 3 were 3.16% (12 / 380), PaBV-4 11.05% (42 / 380), and PaBV-5 24 / 380. For singlet RT-PCR, the positive rates for PaBV-2 were 3.42% (13 / 380), PaBV-4 11.84% (45 / 380), and PaBV-5 6.31% (25 / 380). The specific number of positive and negative samples determined by both methods is shown in Table 8. According to the calculation results, the positive concordance rate of the samples using the detection method of Example 3 is 84%–88.89%, the negative concordance rate is 99.15%–99.73%, and the overall concordance rate is 98.06%–99.21%. This indicates that the detection method of Example 3 can rapidly, accurately, and reliably detect and identify psittac Borna virus types 2, 4, and 5 in clinical samples.

[0116] Table 8 Clinical Sample Comparison Tests

[0117]

[0118]

[0119] Note: Overall concordance rate = (true positive + true negative) / (true positive + true negative + false positive + false negative) * 100%; Positive concordance rate = true positive / (true positive + false negative) * 100%; Negative concordance rate = true negative / (true negative + false positive) * 100%.

[0120] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A reagent comprising a primer set and a probe set, the nucleotide sequences of said primer set and probe set being shown below: The primer set is used to detect PaBV-2, PaBV-4 and PaBV-5 types; in, The primers for detecting PaBV-2 are: PaBV-2-F1: 5'-GAACGAAACTATGAAGATGA-3'; PaBV-2-R1: 5'-GTTGGGTAAATTCTAGAAGG-3'; The primers for detecting PaBV-4 are: PaBV-4-F1: 5'-GAGCAAGTCAAGAAGAAC-3'; PaBV-4-R1: 5'-CCGAATTAGGTCATCATTC-3'; The primers for detecting PaBV-5 are: PaBV-5-F1: 5'-AGACCATCAAGAAGAACC-3'; PaBV-5-R1: 5'-CCTCTTAATCCTTCATTCTC-3'; The probe set is used to detect PaBV-2, PaBV-4 and PaBV-5 types; The probe for detecting PaBV-2 is: PaBV-2-P: 5'-TCTTCTCTATTCGGCGATGGCAA-3'; The probe used to detect PaBV-4 is: PaBV-4-P: 5'-TCCATGATCTCAGACCAAGAGCC-3'; The probe used to detect PaBV-5 is: PaBV-5-P: 5'-CTCCGCCAATTCAGTTACGAGC-3'; The probes are labeled with a fluorescent group and a quenching group at both ends of their sequences, and the fluorescent groups labeled with each probe sequence are different.

2. The reagent according to claim 1, characterized in that, The fluorescent group is three of the following: FAM, Hex, VIC, TAMRA, ROX, Texas-Red, and CY5; and / or, the quenching group is three of the following: TAMRA, MGB, BHQ1, BHQ2, and BHQ3.

3. A kit for detecting PaBV-2, PaBV-4, and PaBV-5, characterized in that, The kit contains the reagent as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, The kit also includes PCR reaction solution, enzymes, and a positive reference.

5. The use of the reagent according to claim 1 or 2 or the kit according to claim 3 or 4 in any one of (1) to (6); (1) Identify PaBV-2, PaBV-4 and PaBV-5 types; (2) Prepare products for the detection or auxiliary detection of PaBV-2, PaBV-4 and PaBV-5 types; (3) Detect whether the sample to be tested is of PaBV-2, PaBV-4 or PaBV-5 type; (4) Prepare products for testing or auxiliary testing to determine whether the sample to be tested is of PaBV-2, PaBV-4 or PaBV-5 type; (5) Detect whether the sample to be tested is infected with PaBV-2, PaBV-4 and PaBV-5; (6) Prepare products for detecting or assisting in the detection of whether the sample to be tested is infected with PaBV-2, PaBV-4 and PaBV-5; The above applications are used for the diagnosis and treatment of non-disease conditions.

6. A triple fluorescent PCR detection method for simultaneously detecting PaBV-2, PaBV-4 and PaBV-5, comprising the step of using the reagent of claim 1 or 2 or the kit of claim 3 or 4 for detection, the method being used for non-disease diagnosis and treatment.

7. The detection method according to claim 6, characterized in that, The detection method includes the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Using the nucleic acid from step 1) as a template, perform triple fluorescent PCR amplification reaction with the reagent or the kit described above, and collect the fluorescence signal; 3) Determine whether the sample contains PaBV-2, PaBV-4 and PaBV-5 based on the fluorescence signal.

8. The detection method according to claim 7, characterized in that, The triple fluorescence PCR amplification reaction program in step 2) is as follows: 35~40℃, 2~3min; 94~96℃, 4~6min; 90~95℃, 8~10s; 50~62℃, 30~35s; 40~45 cycles.

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