Primer-probe sets, gene chips, kits and applications for detecting seven waterfowl infectious disease pathogens

Abstract

The present invention relates to the field of pathogen detection, in particular to a primer-probe set, gene chip, kit and application for detecting seven kinds of waterfowl infectious disease pathogens. The primer-probe set provided by the present invention can simultaneously detect goose parvovirus, duck viral enteritis virus, Muscovy duck parvovirus, duck hepatitis A virus type 1, duck hepatitis A virus type 3, duck Tembusu virus and novel duck reovirus, and has strong specificity and high sensitivity. Further, the gene chip prepared by using the primer-probe set provided by the present invention not only has strong specificity and high sensitivity (1.0×10<supgt;0< / supgt> copies / μL), but also has good repeatability and stability, can be stably stored for at least 180 days, and its sensitivity is also better than that of the standard fluorescence quantitative PCR method. It can quickly and efficiently co-detect the above-mentioned main pathogens of waterfowl diseases and can be used for the epidemiological investigation and disease diagnosis of waterfowl.

Description

Technical Field

[0001] The present invention relates to the field of pathogen detection, in particular to a primer-probe set, a gene chip, a kit and an application for detecting seven kinds of waterfowl infectious disease pathogens. Background Art

[0002] Goose Parvovirus (GPV), Duck Enteritis Virus (DEV), Muscovy duck parvovirus (MDPV), Duck Hepatitis A Virus Type 1 (DHAV-1), Duck Hepatitis A Virus Type 3 (DHAV-3), Duck Tembusu virus (DTMUV) and Novel Duck Reovirus (NDRV) are all important pathogens affecting the poultry breeding industry. These viruses are widely spread in the waterfowl population, causing certain economic losses to agricultural production. At present, the diagnostic methods for the above diseases mainly rely on serological methods and molecular biological methods, with fewer pathogen types detected in a single test, which is difficult to meet the detection needs of the waterfowl industry. Moreover, the existing multiplex PCR detection methods have poor specificity and sensitivity. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a primer-probe set, a gene chip, a kit and an application for detecting seven kinds of waterfowl infectious disease pathogens. The primer-probe set provided by the present invention can simultaneously detect Goose Parvovirus, Duck Enteritis Virus, Muscovy duck parvovirus, Duck Hepatitis A Virus Type 1, Duck Hepatitis A Virus Type 3, Duck Tembusu virus and Novel Duck Reovirus, and has strong specificity and high sensitivity.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a primer-probe set for detecting pathogens of waterfowl infectious diseases, comprising an upstream primer set, a downstream primer set and a probe set; the upstream primer set includes upstream primers with nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16 and SEQ ID NO.19; the downstream primer set includes downstream primers with nucleotide sequences as shown in SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.17 and SEQ ID NO.20; the probe set includes probes with nucleotide sequences as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18 and SEQ ID NO.21.

[0006] Preferably, biotin is modified on the primers in the upstream primer set and / or the downstream primer set, and amino groups including NH2C6 are modified on the probes in the probe set.

[0007] The present invention provides the application of the primer-probe set described in the above technical solution in 1) or 2): 1) preparing a product for detecting pathogens of waterfowl infectious diseases; 2) detecting pathogens of waterfowl infectious diseases; the direct purpose of the detection is non-diagnosis and non-treatment; the pathogens of waterfowl infectious diseases include one or more of goose parvovirus, duck viral enteritis virus, Muscovy duck parvovirus, duck hepatitis A virus type 1, duck hepatitis A virus type 3, duck Tembusu virus and novel duck reovirus.

[0008] Preferably, 1) the product includes a gene chip or a kit.

[0009] The present invention provides a gene chip for detecting pathogens of waterfowl infectious diseases, comprising a solid-phase carrier fixed with probes; the probes are the probes in the primer-probe set described in the above technical solution.

[0010] Preferably, the solid-phase carrier includes a nano-film.

[0011] The present invention provides a kit for detecting pathogens of waterfowl infectious diseases, comprising an independently packaged upstream primer set, a downstream primer set and the gene chip described in the above technical solution; the upstream primer set and the downstream primer set are the upstream primer set and the downstream primer set in the primer-probe set described in the above technical solution.

[0012] Preferably, the kit further includes: horseradish peroxidase-labeled streptavidin and 3,3',5,5'-tetramethylbenzidine.

[0013] The present invention provides a method for non-diagnostic and non-therapeutic detection of pathogens of waterfowl infectious diseases, comprising the following steps:

[0014] Using the nucleic acid molecule of the sample to be tested as a template, performing an amplification reaction with an upstream primer set and a downstream primer set to obtain an amplification product; the upstream primer set and the downstream primer set are the upstream primer set and the downstream primer set in the primer-probe set described in the above technical solution; denaturing the amplification product and hybridizing it with the probes on the gene chip to obtain a first solid-phase carrier; the gene chip is the gene chip described in the above technical solution or the gene chip in the kit described in the above technical solution; reacting the first solid-phase carrier with horseradish peroxidase-labeled streptavidin to obtain a second solid-phase carrier; performing a color reaction on the second solid-phase carrier with 3,3',5,5'-tetramethylbenzidine to obtain a third solid-phase carrier; determining the detection result according to the color reaction result of the third solid-phase carrier:

[0015] When the third solid-phase carrier shows color at the site where the probe is immobilized, the sample to be tested contains the pathogen of waterfowl infectious disease corresponding to the probe; when the third solid-phase carrier does not show color at the site where the probe is immobilized, the sample to be tested does not contain the pathogen of waterfowl infectious disease corresponding to the probe;

[0016] The corresponding relationship between the probe and the pathogen of waterfowl infectious disease includes: the probe with the nucleotide sequence shown in SEQ ID NO.3 corresponds to goose parvovirus; the probe with the nucleotide sequence shown in SEQ ID NO.6 corresponds to duck viral enteritis virus; the probe with the nucleotide sequence shown in SEQ ID NO.9 corresponds to Muscovy duck parvovirus; the probe with the nucleotide sequence shown in SEQ ID NO.12 corresponds to duck hepatitis A virus type 1; the probe with the nucleotide sequence shown in SEQ ID NO.15 corresponds to duck hepatitis A virus type 3; the probe with the nucleotide sequence shown in SEQ ID NO.18 corresponds to duck Tembusu virus; the probe with the nucleotide sequence shown in SEQ ID NO.21 corresponds to novel duck reovirus.

[0017] Preferably, the reaction system of the amplification reaction includes: 2×One step Mix 12.5 μL, One step Enzyme Mix 1.25 μL, upstream primer set 1 μL, downstream primer set 1 μL, template 4 μL and ddH2O 5.25 μL; the reaction program of the amplification reaction includes: 50°C for 30 min, 94°C for 4 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 20 sec, 5 cycles; 94°C for 30 sec, 54°C for 30 sec, 72°C for 20 sec, 35 cycles; 72°C for 10 min, 95°C for 5 min.

[0018] Beneficial effects:

[0019] The primer-probe set provided by the present invention can simultaneously detect goose parvovirus, duck viral enteritis virus, Muscovy duck parvovirus, duck hepatitis A virus type 1, duck hepatitis A virus type 3, duck Tembusu virus and novel duck reovirus, and has strong specificity and high sensitivity.

[0020] Furthermore, the gene chip prepared by using the primer-probe set provided by the present invention is a visual gene chip, which not only has strong specificity and high sensitivity (1.0×10 0 copies / μL), but also has good repeatability and stability, can be stably stored for at least 180 days, and its sensitivity is also better than that of the standard fluorescence quantitative PCR method. It can quickly and efficiently co-detect the main pathogenic agents of the above-mentioned waterfowl diseases and can be used for the epidemiological investigation and disease diagnosis of waterfowl. Brief description of the drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0022] Figure 1 It is the layout diagram of the gene chip;

[0023] Figure 2 It is the specific test result diagram of the gene chip under the seven-plex system;

[0024] Figure 3 It is the sensitivity test result of the single positive plasmid of the gene chip;

[0025] Figure 4 It is the sensitivity test result of the mixed positive plasmids of the gene chip;

[0026] Figure 5 It is the test result diagram of the storage period of the gene chip;

[0027] Figure 6 It is the specific test result diagram of detecting goose parvovirus by fluorescence quantitative PCR;

[0028] Figure 7 It is the specific test result diagram of detecting duck viral enteritis virus by fluorescence quantitative PCR;

[0029] Figure 8 It is the specific test result diagram of detecting Muscovy duck parvovirus by fluorescence quantitative PCR;

[0030] Figure 9 It is the specific test result diagram of detecting duck hepatitis A virus type 1 by fluorescence quantitative PCR;

[0031] Figure 10Specific result diagram for detecting duck hepatitis A virus type 3 by fluorescence quantitative PCR;

[0032] Figure 11 Specific result diagram for detecting duck Tembusu virus by fluorescence quantitative PCR;

[0033] Figure 12 Specific result diagram for detecting novel duck reovirus by fluorescence quantitative PCR;

[0034] Figure 13 Sensitivity result diagram for detecting goose parvovirus by fluorescence quantitative PCR;

[0035] Figure 14 Sensitivity result diagram for detecting duck viral enteritis virus by fluorescence quantitative PCR;

[0036] Figure 15 Sensitivity result diagram for detecting Muscovy duck parvovirus by fluorescence quantitative PCR;

[0037] Figure 16 Sensitivity result diagram for detecting duck hepatitis A virus type 1 by fluorescence quantitative PCR;

[0038] Figure 17 Sensitivity result diagram for detecting duck hepatitis A virus type 3 by fluorescence quantitative PCR;

[0039] Figure 18 Sensitivity result diagram for detecting duck Tembusu virus by fluorescence quantitative PCR;

[0040] Figure 19 Sensitivity result diagram for detecting novel duck reovirus by fluorescence quantitative PCR. Specific implementation manners

[0041] The present invention provides a primer-probe set for detecting pathogens of waterfowl infectious diseases, including an upstream primer set, a downstream primer set, and a probe set; the upstream primer set includes: upstream primers with nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16, and SEQ ID NO.19; the downstream primer set includes: downstream primers with nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.17, and SEQ ID NO.20; the probe set includes: probes with nucleotide sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18, and SEQ ID NO.21.

[0042] The primer-probe set provided by the present invention can simultaneously detect goose parvovirus, duck viral enteritis virus, Muscovy duck parvovirus, duck hepatitis A virus type 1, duck hepatitis A virus type 3, duck Tembusu virus, and novel duck reovirus, and has strong specificity and high sensitivity.

[0043] As an implementation manner, the primers in the upstream primer set and / or the downstream primer set are modified with biotin, and the probes in the probe set are modified with amino groups, and the amino groups include NH2C6. The present invention utilizes the biotin-SA-HRP / TMB color development system, adds biotin with high affinity for SA-HRP to the primers. When the amplified target fragment binds to the probe, a blue response point can be shown, and the detection result can be distinguished by the naked eye, realizing visual detection.

[0044] Based on the above advantages, the present invention provides the application of the primer-probe set described in the above technical solution in 1) or 2): 1) preparing a product for detecting pathogens of waterfowl infectious diseases; 2) detecting pathogens of waterfowl infectious diseases; the direct purpose of the detection is non-diagnosis and non-treatment; the pathogens of waterfowl infectious diseases include: one or more of goose parvovirus, duck viral enteritis virus, Muscovy duck parvovirus, duck hepatitis A virus type 1, duck hepatitis A virus type 3, duck Tembusu virus, and novel duck reovirus. As an implementation manner, 1) the product includes a visual gene chip or a kit. When the direct purpose of the present invention is non-diagnosis and non-treatment detection of pathogens of waterfowl infectious diseases, the test sample can be the food or growth environment of poultry.

[0045] Based on the above advantages, the present invention provides a gene chip for detecting pathogens of waterfowl infectious diseases, comprising: a solid-phase carrier fixed with probes; the probes are the probes in the primer-probe group described in the above technical solution.

[0046] As an implementation manner, positive control points and negative control points are provided on the solid-phase carrier; biotin is fixed on the positive control points; and phosphate buffer solution is fixed on the negative control points. By setting the positive control points and negative control points, the present invention can avoid false positive or false negative test results.

[0047] As an implementation manner, the solid-phase carrier comprises a nano-membrane.

[0048] The present invention has no special requirements on the positions of the probes, positive control points and negative control points fixed on the solid-phase carrier. In the embodiments Figure 1 the fixing method is only taken as a specific implementation manner, and it should not be construed as the protection scope of the present invention.

[0049] The gene chip provided by the present invention is a visual gene chip. Through the specificity test of the gene chip, it is measured that specific binding occurs at the probe sites of seven pathogens, and there is no cross-reaction phenomenon, indicating that the specificities of the various pathogen probes used in the visual gene chip provided by the present invention are good. Through the sensitivity test of the gene chip, the sensitivity of a single sample of seven pathogens is measured to be 1.0×10 0 copies / μL; the sensitivity of the mixed sample of seven pathogen plasmids is also 1.0×10 0 copies / μL, indicating that the visual gene chip provided by the present invention has high sensitivity. Through the repeatability test and stability test, it is proved that the visual gene chip provided by the present invention not only has good repeatability, but also has good stability and can be stably stored for at least 180 days. Through the comparative experiment between the gene chip detection method and the standard fluorescence quantitative PCR method, it is proved that the sensitivity of the present invention for detecting single pathogens of seven viruses and seven-fold mixed pathogens is better than that of the qPCR detection method. The visual gene chip provided by the present invention can quickly and efficiently co-detect the above-mentioned main pathogen of waterfowl diseases and can be used for the epidemiological investigation and disease diagnosis of waterfowl.

[0050] The present invention also provides a preparation method of the gene chip described in the above technical solution, comprising the following steps: spotting each probe on the solid-phase carrier according to the chip layout order, and placing the spotted solid-phase carrier in an environment with a humidity of 40% - 60% and a temperature of 22 - 25°C to dry overnight to obtain the gene chip.

[0051] Based on the above advantages, the present invention provides a kit for detecting pathogens of waterfowl infectious diseases, comprising an independently packaged upstream primer set, a downstream primer set, and the gene chip described in the above technical solution; the upstream primer set and the downstream primer set are the upstream primer set and the downstream primer set in the primer-probe set described in the above technical solution. As an implementation manner, the kit further comprises: horseradish peroxidase-labeled streptavidin and 3,3',5,5'-tetramethylbenzidine.

[0052] Based on the above advantages, the present invention provides a method for non-diagnostic and non-therapeutic detection of pathogens of waterfowl infectious diseases, comprising the following steps:

[0053] Using the nucleic acid molecule of the sample to be tested as a template, performing an amplification reaction with the upstream primer set and the downstream primer set to obtain an amplification product; the upstream primer set and the downstream primer set are the upstream primer set and the downstream primer set in the primer-probe set described in the above technical solution; after denaturing the amplification product, performing a hybridization reaction with the probes on the gene chip to obtain a first solid-phase carrier; the gene chip is the gene chip described in the above technical solution, or the gene chip in the kit described in the above technical solution; reacting the first solid-phase carrier with horseradish peroxidase-labeled streptavidin to obtain a second solid-phase carrier; performing a color reaction on the second solid-phase carrier with 3,3',5,5'-tetramethylbenzidine to obtain a third solid-phase carrier;

[0054] Determining the detection result according to the color development result of the third solid-phase carrier:

[0055] When the third solid-phase carrier develops color at the site where the probe is immobilized, the sample to be tested contains the waterfowl infectious disease pathogen corresponding to the probe; when the third solid-phase carrier does not develop color at the site where the probe is immobilized, the sample to be tested does not contain the waterfowl infectious disease pathogen corresponding to the probe;

[0056] The corresponding relationship between the probe and the waterfowl infectious disease pathogen includes: the probe with the nucleotide sequence shown in SEQ ID NO.3 corresponds to goose parvovirus; the probe with the nucleotide sequence shown in SEQ ID NO.6 corresponds to duck viral enteritis virus; the probe with the nucleotide sequence shown in SEQ ID NO.9 corresponds to Muscovy duck parvovirus; the probe with the nucleotide sequence shown in SEQ ID NO.12 corresponds to duck hepatitis A virus type 1; the probe with the nucleotide sequence shown in SEQ ID NO.15 corresponds to duck hepatitis A virus type 3; the probe with the nucleotide sequence shown in SEQ ID NO.18 corresponds to duck Tembusu virus; the probe with the nucleotide sequence shown in SEQ ID NO.21 corresponds to novel duck reovirus.

[0057] As an implementation manner, the reaction system of the amplification reaction includes: 2×One step Mix 12.5 μL, One step Enzyme Mix 1.25 μL, upstream primer set 1 μL, downstream primer set 1 μL, template 4 μL, and ddH2O 5.25 μL; the reaction procedure of the amplification reaction includes: 50°C for 30 min, 94°C for 4 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 20 sec, for 5 cycles; 94°C for 30 sec, 54°C for 30 sec, 72°C for 20 sec, for 35 cycles; 72°C for 10 min, 95°C for 5 min.

[0058] As an implementation manner, the sample to be tested can be the food or growth environment of poultry.

[0059] The method provided by the present invention can quickly and efficiently co-detect the main pathogenic agents of the above-mentioned waterfowl diseases, and can be used for the detection of samples to be tested such as poultry food or growth environment, reducing the risk of poultry infection.

[0060] In order to further illustrate the present invention, the primer-probe set, gene chip, kit and application for detecting seven waterfowl infectious disease pathogens provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1 Establishment of a visual gene chip and virus detection method

[0062] 1. Pathogenic agents used in the present invention: Goose parvovirus (GPV, CVCC AV239), Duck viral enteritis virus (DEV, CVCCAV18) were supplied by the National Veterinary Microbial Culture Collection Center; Muscovy duck parvovirus (MDPV), Duck hepatitis A virus type 1 (DHAV-1), Duck hepatitis A virus type 3 (DHAV-3), Duck Tembusu virus (DTMUV), Novel duck reovirus (NDRV), Duck astrovirus (DAstv), Duck adenovirus type 3 (DAdV-3) were all pathogens isolated and identified by conventional methods.

[0063] 2. Design of primers and probes: In order to ensure the detection effect of the primers and probes used in the gene chip detection method, all available sequences of GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV and NDRV were obtained from the GenBank database and analyzed. According to the NS gene of GPV, the UL6 gene of DEV, the VP gene of MDPV, the VP1 gene of DHAV-1, the VP1Genes of DTMUV E Genes of NDRV S4 Genes, primers and probes of GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV and NDRV were designed, and the results are shown in Table 1.

[0064] Table 1 Primers and Probes of Gene Chip for Seven Viruses

[0065]

[0066] Note: Forward in Table 1 represents the upstream primer; Reverse represents the downstream primer, and the 5'-end of the downstream primer is modified with a Biotin group; Probe represents the probe, and the 5'-end of the probe is modified with NH2C6 (C6 amino modification).

[0067] 3. Preparation of Recombinant Positive Plasmids of Seven Viruses:

[0068] 1) According to the instructions of the TAKARA Viral Genome RNA / DNA Extraction Kit, nucleic acids of 7 virus samples were extracted respectively. The 7 pairs of upstream and downstream primers in Table 1 were mixed with equal mass to obtain upstream and downstream primer mixtures. Using the extracted nucleic acids as templates respectively, the target fragments were amplified with the upstream and downstream primer mixtures, and the purified PCR products were obtained after purification; the reaction system was: 2×One step Mix 12.5 μL, One step Enzyme Mix 1.25 μL, upstream primer mixture 1 μL, downstream primer mixture 1 μL, template 4 μL and ddH2O 5.25 μL; the reaction program was: reverse transcription at 50°C for 30 min, 94°C for 3 min; 94°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec, 35 cycles; 72°C for 7 min, stored at 4°C.

[0069] 2) According to the instructions of the TARAKA pMD™19-T Vector Cloning Kit, 1 μL of pMD19-T Vector, 1 μL of the purified PCR product, 3 μL of double-distilled water and 5 μL of Solution I were added to a centrifuge tube. React at 16°C for 30 min, add the whole volume (10 μL) to 100 μL of JM109 competent cells, place on ice for 30 min, heat shock in a 42°C water bath for 45 - 60 sec, quickly transfer to an ice bath, and let stand for 2 min. Add 890 μL of LB culture medium without resistance to the centrifuge tube, shake culture at 37°C and 200 rpm for 60 min. Pipette 80 μL of the culture medium and spread it evenly on the Amp-resistant medium, and culture it upside down in a 37°C incubator overnight.

[0070] 3) Pick a single colony and add it to 5 mL of liquid medium with Amp resistance. After culturing at 37 °C and 200 rpm for 12 h, select the PCR-positive bacterial liquid sample for sequencing. Use NCBI Nucleotide Blast to compare the sequencing results, and extract the recombinant positive plasmids of each virus according to the instructions of the Tiangen Quick Plasmid Mini Kit.

[0071] 4. Chip preparation: Dilute the probe with phosphate buffer to a final concentration of 6 μmol / L, and dilute biotin with phosphate buffer to a final concentration of 6 μmol / L as the positive control, and phosphate buffer as the negative control. Set the parameters of the spotter to 100 drop, and spot each probe on the nanofilm according to the chip layout order. The chip layout order is shown in Figure 1 , where Biotin is the positive control of the chip; Buffer is the negative control of the chip. After spotting, place the membrane substrate in an environment with a humidity of 40% - 60% and a temperature of 22 - 25 °C to dry overnight, and store it at 2 - 8 °C.

[0072] 5. Hybridization, Washing and Color Development of the Chip: Extract the nucleic acid of the test sample according to the instructions of the TAKARA Viral Genome RNA / DNA Extraction Kit, and perform PCR amplification using the reaction system described in 1) of step 3 to obtain the amplification product. The reaction program for PCR amplification is: 50°C for 30 min, 94°C for 4 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 20 sec, for 5 cycles; 94°C for 30 sec, 54°C for 30 sec, 72°C for 20 sec, for 35 cycles; 72°C for 10 min, 95°C for 5 min. After denaturing the amplification product at 95°C for 5 min, quickly place it on ice to obtain the denatured amplification product. Preheat the chip in a microplate constant temperature oscillator at 47°C and 200 rpm for 10 min; add 100 μL of solution A and 20 μL of the denatured amplification product to each well of the chip, hybridize at 47°C and 200 rpm for 20 min, and aspirate the hybridization solution; after washing 2 times with 150 μL of solution B preheated to 47°C in each well, add 150 μL of solution B preheated to 47°C, and incubate in a constant temperature oscillator at 47°C and 200 rpm for 10 min; Solution A and Solution B are purchased from Nanjing Senwei Biotechnology Co., Ltd., product number ABT-Z-1006. After sucking dry the liquid with a vacuum pump, add 150 μL of streptavidin-horseradish peroxidase (SA-HRP) working solution to each well, and incubate in a constant temperature oscillator at 47°C and 200 rpm for 10 min. Aspirate the SA-HRP working solution, wash 3 times with 150 μL of solution A in each well, and then wash 3 times with 150 μL of solution C in each well and aspirate the liquid. Add 90 μL of 3,3',5,5'-tetramethylbenzidine (TMB) color development solution to each well, and let it stand at room temperature in the dark for 5 - 10 min. Aspirate the TMB color development solution, wash each well of the chip with pure water to remove the liquid on the surface of the nanofilm, and observe the results with the naked eye or using a chip imager.

[0073] 6. Judgment of the Detection Results: The negative control point does not show color, the color of the positive control point is significantly stronger than the blue-violet color of the negative control point, and at least one of the three positive control points showing color is a valid experimental result. If the probe point where the test sample is located does not show color, it is negative; if the probe point where the test sample is located is blue-violet, it is positive.

[0074] Example 2 Specificity Test of the Gene Chip

[0075] Using the recombinant positive plasmids of GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV, and NDRV constructed by the method in step 3 of Example 1 as templates, the recombinant positive plasmids, viral nucleic acid molecules (DAstv and DAdV-3), and healthy duck sera were respectively subjected to PCR amplification by the method in step 5 of Example 1. After the PCR amplification products were hybridized and color-developed with the chip, the specificity of the chip was verified. The results are as Figure 2 shown, where A is GPV; B is DEV; C is MDPV; D is DHAV-1; E is DHAV-3; F is DTMUV; G is NDRV; H is DAstv; I is DAdV-3; J is healthy duck serum.

[0076] It can be seen from the results that the negative control points did not respond, and the positive control points responded, indicating that the test results were valid. Specific binding occurred at the probe sites of all seven pathogens, and there was no cross-reaction phenomenon, indicating that the specificity of each pathogen probe used in the chip was good.

[0077] Example 3 Sensitivity Test of Gene Chip

[0078] 1. Sensitivity Test of Single Positive Plasmid of Gene Chip

[0079] The concentrations of each positive plasmid constructed in step 3 of Example 1 were measured using an ultra-micro ultraviolet spectrophotometer, and each positive plasmid was diluted to 1.0×10 3 copies / μL, 1.0×10 2 copies / μL, 1.0×10 1 copies / μL, and 1.0×10 0 copies / μL at four concentrations. PCR amplification was performed using the primers in Table 1 respectively. After the PCR amplification products were hybridized and color-developed with the chip, the sensitivity of the single positive plasmid of the chip was measured. The results are as Figure 3 shown, where A: 1.0×10 3 copies / μL; B: 1.0×10 2 copies / μL; C: 1.0×10 1 copies / μL; D: 1.0×10 0 copies / μL; E: negative control.

[0080] It can be seen from the results that the negative control points did not respond, and the positive control points responded, indicating that the test results were valid. The sensitivity of a single sample of GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV, and NDRV was all 1.0×10 0 copies / μL.

[0081] 2. Sensitivity Test of Gene Chip Mixed Positive Plasmids

[0082] Dilute each positive plasmid constructed in step 3 of Example 1 to 1.0×10 8 copies / μL and then mix them in equal volumes. Dilute the mixed plasmids to 1.0×10 4 copies / μL, 1.0×10 3 copies / μL, 1.0×10 2 copies / μL, 1.0×10 1 copies / μL and 1.0×10 0 copies / μL at five concentrations. Then perform PCR amplification with the primers in Table 1 respectively. After the PCR amplification products are hybridized and color-developed with the chip, measure the sensitivity of the mixed positive plasmids on the chip. The results are as Figure 4 shown, where A: 1.0×10 4 copies / μL; B: 1.0×10 3 copies / μL; C: 1.0×10 2 copies / μL; D: 1.0×10 1 copies / μL; E: 1.0×10 0 copies / μL.

[0083] It can be seen from the results that the negative control points have no response and the positive control points have a response, indicating that the test results are valid. The sensitivity of the mixed sample of seven plasmids, namely GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV, and NDRV, is 1.0×10 0 copies / μL.

[0084] Example 4 Repeatability Test of Gene Chip

[0085] To verify the repeatability of the chip, the amplification products of the same quality control sample (positive recombinant plasmid) were used for chip experiments by different experimenters at different time points. In each experiment, the PCR reaction conditions were the same as those for chip hybridization and color development to ensure the consistency of conditions. The experiment was divided into repeatability verification within the same batch and repeatability verification between different batches. By analyzing and comparing the results, verify the repeatability of the chip within the same batch and under different batches of chips.

[0086] To verify the repeatability of the gene chip detection method, chip experiments were carried out by different experimenters at different time points using amplification products of the same quality control sample (positive recombinant plasmid) at different dilution degrees. Repeatability verification was performed for batch CH240828001 and batches CH240620001 and CH240718001. In each experiment, the PCR reaction conditions were the same as those for the chip hybridization and color development operations to ensure the consistency of conditions. The results are shown in Table 2. The consistency of the detection results within and between batches reached 100%, indicating that this method has good repeatability.

[0087] Table 2 Repeatability within and between batches of gene chips

[0088]

[0089] Example 5 Shelf life test of gene chips

[0090] To test the stability of gene chip detection, seven-virus mixed positive samples were used for detection on the 0th, 30th, 60th, 90th, 120th, and 180th days after chip fabrication. By analyzing and comparing the results, the stability of the chip under different storage times was verified.

[0091] Using the seven-fold mixed positive plasmid as a template, stability tests were carried out on the 0th, 30th, 60th, 90th, 120th, and 180th days after chip fabrication. The results are as Figure 5 shown, where A is the 0th day, B is the 30th day, C is the 60th day, D is the 90th day, E is the 120th day, and F is the 180th day.

[0092] It can be seen from the results that under the storage conditions of 2°C - 8°C, the gene chip can be stably stored for at least 180 days, indicating that this method has good long-term storage stability.

[0093] Example 6

[0094] Comparison test on specificity and sensitivity between gene chip method and fluorescence quantitative PCR detection method

[0095] 1. Reconstruction of the standard fluorescence quantitative PCR method

[0096] According to the industry standards of each virus diagnosis technology and the primers, probes, and qPCR reaction conditions in the literature, in order to ensure the reliability of the detection method, the qPCR detection methods for each virus were re-established, and their specificity and sensitivity were determined. The primers and probes are shown in Table 3.

[0097] 2. The method for constructing the recombinant positive plasmid of the standard fluorescence quantitative PCR method is similar to step 3 in Example 1, except that the primers are the primers in Table 3.

[0098] Table 3 Primers and Probes for Gene Chip of Seven Viruses

[0099]

[0100] Note: In Table 3, Forward represents the upstream primer; Reverse represents the downstream primer, and the 5'-end of the downstream primer is modified with a Biotin group; Probe represents the probe, and the 5'-end of the probe is modified with NH2C6 (C6 amino modification).

[0101] 3. qPCR reaction procedure: 25°C for 10 min; 95°C for 30 s; 95°C for 5 s, 48°C for 30 s, 72°C for 20 s, 40 cycles.

[0102] qPCR system: 2 × Animal Detection U + Probe qPCR Super PreMix 12.5 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, probe 0.5 μl, ddH2O 6 μl, and template 5 μl.

[0103] The results are as Figures 6 - 12 shown. In the qPCR detection methods for their respective pathogens, after three replicates verification, amplification curves were observed only when using the corresponding pathogen nucleic acid as the template, and no amplification signals were observed for other pathogens, indicating that the qPCR detection method has good specificity.

[0104] The positive plasmids of each pathogen were ten-fold serially diluted to 1.0×10 8 copies / μL~1.0×10 0 copies / μL, and the qPCR sensitivity test was performed on each pathogen. The results are as Figures 13 - 19 shown. The detection sensitivities of GPV, DEV, MDPV, and DTMUV were 1.0×10 1 copies / μL, and the detection sensitivities of DHAV-1, DHAV-3, and NDRV were 1.0×10 2 copies / μL.

[0105] The results show that the visualization gene chip of the present invention has slightly better sensitivity than the qPCR detection method when detecting single pathogens and seven-fold pathogens of seven viruses.

[0106] Example 7 Comparative Test of Clinical Sample Compliance

[0107] In Shandong Province, Jiangxi Province, and Fujian Province, throat swabs, anal swabs, livers, lungs, and hearts of suspected positive diseased animals of various pathogens were collected as clinical samples in this laboratory, and were respectively detected using the chip in Example 1 and the qPCR method in Example 6, and the coincidence rate was calculated. Coincidence rate = (total number of detected clinical samples - number of different results) / total number of detected clinical samples × 100%.

[0108] A total of 210 clinical samples suspected of being positive for pathogens were used in this invention and detected respectively by the chip and the qPCR detection method. The coincidence rate of the results of the two detection methods was 97.6% - 100%, and the Kappa value was 0.935, indicating that the consistency of the two detection methods was good.

[0109] Table 4 Detection Results of Clinical Samples

[0110]

[0111] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer probe set for detecting pathogens of waterfowl infectious diseases, characterized in that: It consists of an upstream primer set, a downstream primer set and a probe set; The upstream primer set consists of the following upstream primers: upstream primers with nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16 and SEQ ID NO.19; The downstream primer set consists of the following downstream primers: downstream primers with nucleotide sequences as shown in SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.17 and SEQ ID NO.20; The probe set consists of the following probes: probes with nucleotide sequences as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18 and SEQ ID NO.21; The primers in the upstream primer set and / or the downstream primer set are modified with biotin, and the probes in the probe set are modified with NH2C6.

2. Use of the primer probe set according to claim 1 in 1) or 2): 1) Preparation of products for detecting pathogens of waterfowl infectious diseases; 2) Detection of pathogens of waterfowl infectious diseases; the direct purpose of such detection is neither diagnostic nor therapeutic; The pathogens of waterfowl infectious diseases include: One or more of goose parvovirus, duck viral enteritis virus, Muscovy duck parvovirus, duck hepatitis A virus type 1, duck hepatitis A virus type 3, duck Tembusu virus and novel duck reovirus.

3. The use according to claim 2, characterized in that: 1) The products include gene chips or kits.

4. A kit for detecting pathogens of waterfowl infectious diseases, characterized in that: It comprises independently packaged upstream primer sets, downstream primer sets and gene chips; the upstream primer sets and downstream primer sets are the upstream primer sets and downstream primer sets in the primer-probe set described in claim 1; the gene chip comprises a solid phase carrier fixed with probes; the probes are the probes in the primer-probe set described in claim 1.

5. The kit according to claim 4, characterized in that The kit also includes: horseradish peroxidase-labeled streptavidin and 3,3',5,5'-tetramethylbenzidine.

6. The kit according to claim 4, characterized in that The solid phase carrier includes a nanomembrane.

7. A method for the direct purpose of non-diagnostic and non-therapeutic detection of pathogens of waterfowl infectious diseases, characterized in that: The following steps are involved: Using the nucleic acid molecule of the sample to be tested as a template, an upstream primer set and a downstream primer set are used to perform an amplification reaction to obtain an amplification product; the upstream primer set and the downstream primer set are the upstream primer set and the downstream primer set in the primer probe set according to claim 1; Denaturing the amplified product and performing hybridization reaction with the probe on the gene chip to obtain a first solid phase carrier; the gene chip is the gene chip in the kit according to any one of claims 4 to 6; reacting the first solid phase carrier with horseradish peroxidase-labeled streptavidin to obtain a second solid phase carrier; The second solid phase carrier and 3,3',5,5'-tetramethylbenzidine are subjected to a color reaction to obtain a third solid phase carrier; Determine the detection result according to the color development result of the third solid phase carrier: When the third solid phase carrier develops color at the site where the probe is fixed, the sample to be tested contains the waterfowl infectious disease pathogen corresponding to the probe; when the third solid phase carrier does not develop color at the site where the probe is fixed, the sample to be tested does not contain the waterfowl infectious disease pathogen corresponding to the probe; The corresponding relationship between the probe and the pathogen of waterfowl infectious disease includes: The probe whose nucleotide sequence is shown in SEQ ID NO. 3 corresponds to goose parvovirus; The probe whose nucleotide sequence is shown in SEQ ID NO.6 corresponds to duck viral enteritis virus; The probe whose nucleotide sequence is shown in SEQ ID NO.9 corresponds to Muscovy duck parvovirus; The probe whose nucleotide sequence is shown in SEQ ID NO.12 corresponds to duck hepatitis A virus type 1; The probe whose nucleotide sequence is shown in SEQ ID NO.15 corresponds to duck hepatitis A virus type 3; The probe whose nucleotide sequence is shown in SEQ ID NO. 18 corresponds to duck Tembusu virus; The probe whose nucleotide sequence is shown in SEQ ID NO.21 corresponds to a novel duck reovirus.

8. The method according to claim 7, characterized in that The reaction system of the amplification reaction includes: 2×Onestep Mix 12.5 μL, One step Enzyme Mix 1.25 μL, upstream primer set 1 μL, downstream primer set 1 μL, template 4 μL and ddH2O 5.25 μL; The reaction procedure of the amplification reaction includes: 50°C 30 min, 94°C 4 min; 94°C 30 sec, 56°C 30 sec, 72°C 20 sec, 5 cycles; 94°C 30 sec, 54°C 30 sec, 72°C 20 sec, 35 cycles; 72°C 10 min, 95°C 5 min.

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