Primer probe group, gene chip, kit and application for detecting seven waterfowl infectious disease pathogens

Abstract

The invention relates to the field of pathogen detection, in particular to a primer probe group, a gene chip and a kit for detecting seven waterfowl infectious disease pathogens and application. The primer probe group provided by the invention can be used for simultaneously detecting the goose parvovirus, the duck viral enteritis virus, the muscovy duck parvovirus, the duck hepatitis A virus type 1, the duck hepatitis A virus type 3, the duck tembusu virus and the novel duck reovirus, and is strong in specificity and high in sensitivity. Furthermore, a gene chip prepared by using the primer probe group provided by the invention not only has strong specificity and high sensitivity (1.0 * 100 copies / [mu] L), but also has good repeatability and stability, can be stably stored within at least 180 days, has sensitivity superior to that of a standard fluorescent quantitative PCR method, can rapidly and efficiently detect main epidemic disease pathogens of waterfowl together, and has good application prospects. The method can be used for epidemiological investigation and disease diagnosis of waterfowls.

Description

Technical Field

[0001] The invention relates to the field of pathogen detection, in particular to a primer probe set, a gene chip, a kit and applications for detecting pathogens of seven waterfowl infectious diseases. 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 Duck Reovirus (NDRV) are all important pathogens affecting the poultry industry. These viruses are widely spread in waterfowl populations, causing certain economic losses to agricultural production. At present, the diagnostic methods for the above-mentioned diseases are mainly based on serological methods and molecular biological methods. The number of pathogens detected in a single test is small, which is difficult to meet the detection needs of the waterfowl industry. In addition, 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 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 new 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: The invention provides a primer-probe group for detecting pathogens of waterfowl infectious diseases, comprising an upstream primer group, a downstream primer group and a probe group; the upstream primer group comprises: upstream primers whose nucleotide sequences are 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 group comprises: downstream primers whose nucleotide sequences are 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 group comprises: probes whose nucleotide sequences are 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.

[0005] Preferably, 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.

[0006] The present invention provides the application of the primer probe group described in the above technical scheme 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.

[0007] Preferably, the product described in 1) includes a gene chip or a kit.

[0008] 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 probes in the primer probe group described in the above technical solution.

[0009] Preferably, the solid phase carrier comprises a nanomembrane.

[0010] 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.

[0011] Preferably, the kit further comprises: horseradish peroxidase-labeled streptavidin and 3,3',5,5'-tetramethylbenzidine.

[0012] The present invention provides a method for detecting pathogens of waterfowl infectious diseases for the direct purpose of non-diagnosis and non-treatment, comprising the following steps: The nucleic acid molecule of the sample to be tested is used as a template, and an upstream primer group and a downstream primer group are used to perform an amplification reaction to obtain an amplification product; the upstream primer group and the downstream primer group are the upstream primer group and the downstream primer group in the primer probe group described in the above technical solution; the amplification product is denatured and hybridized with the probe 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; the first solid phase carrier is reacted with horseradish peroxidase-labeled streptavidin to obtain a second solid phase carrier; the second solid phase carrier is subjected to a color development reaction with 3,3',5,5'-tetramethylbenzidine to obtain a third solid phase carrier; the detection result is determined 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 correspondence between the probe and the pathogen of waterfowl infectious disease includes: the probe with a nucleotide sequence as shown in SEQ ID NO.3 corresponds to goose parvovirus; the probe with a nucleotide sequence as shown in SEQ ID NO.6 corresponds to duck viral enteritis virus; the probe with a nucleotide sequence as shown in SEQ ID NO.9 corresponds to Muscovy duck parvovirus; the probe with a nucleotide sequence as shown in SEQ ID NO.12 corresponds to duck hepatitis A virus type 1; the probe with a nucleotide sequence as shown in SEQ ID NO.15 corresponds to duck hepatitis A virus type 3; the probe with a nucleotide sequence as shown in SEQ ID NO.18 corresponds to duck Tembusu virus; and the probe with a nucleotide sequence as shown in SEQ ID NO.21 corresponds to a new duck reovirus.

[0013] Preferably, the reaction system of the amplification reaction includes: 2×One step Mix 12.5μL, One stepEnzyme Mix 1.25μL, upstream primer set 1μL, downstream primer set 1μL, template 4μL and ddH2O 5.25μL; the reaction procedure of the amplification reaction includes: 50℃ 30 min, 94℃ 4 min; 94℃ 30 sec, 56℃ 30 sec, 72℃ 20sec, 5 cycles; 94℃ 30 sec, 54℃ 30 sec, 72℃ 20 sec, 35 cycles; 72℃ 10 min, 95℃ 5min.

[0014] Beneficial effects: The primer probe set provided by the 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.

[0015] Furthermore, the gene chip prepared by using the primer probe set provided by the present invention is a visualized gene chip, which has high specificity and high sensitivity (1.0×10 0 It has good repeatability and stability, can be stably stored for at least 180 days, and its sensitivity is better than that of the standard fluorescence quantitative PCR method. It can quickly and efficiently detect the above-mentioned major waterfowl disease pathogens and can be used for epidemiological surveys and disease diagnosis of waterfowl. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 This is a layout diagram of the gene chip; Figure 2 This is a graph showing the specificity test results of the gene chip under the seven-layer system; Figure 3 It is the sensitivity test result of single positive plasmid of gene chip; Figure 4 It is the sensitivity test result of the mixed positive plasmid of the gene chip; Figure 5 This is a diagram showing the results of a gene chip shelf life test; Figure 6 This is the specific result diagram of the fluorescent quantitative PCR detection of goose parvovirus; Figure 7 This is the specific result diagram of the detection of duck viral enteritis virus by fluorescent quantitative PCR; Figure 8 This is the specific result diagram of fluorescent quantitative PCR detection of Muscovy duck parvovirus; Fig. 9 This is the specific result diagram of the fluorescent quantitative PCR detection of duck hepatitis A virus type 1; Fig.10 This is the specific result diagram of the fluorescent quantitative PCR detection of duck hepatitis A virus type 3; Fig.11 This is the specific result diagram of the fluorescent quantitative PCR detection of duck Tembusu virus; Fig.12 This is the specific result diagram of the fluorescent quantitative PCR detection of the new duck reovirus; Fig.13 This is the sensitivity result diagram of fluorescent quantitative PCR detection of goose parvovirus; Fig.14 This is the sensitivity result diagram of the fluorescent quantitative PCR detection of duck viral enteritis virus; Fig.15 This is the sensitivity result diagram of fluorescent quantitative PCR detection of Muscovy duck parvovirus; Fig.16 This is the sensitivity result diagram of fluorescent quantitative PCR detection of duck hepatitis A virus type 1; Fig.17 This is the sensitivity result diagram of fluorescent quantitative PCR detection of duck hepatitis A virus type 3; Fig.18 This is the sensitivity result of fluorescent quantitative PCR detection of duck Tembusu virus; Fig.19 This is a graph showing the sensitivity of fluorescent quantitative PCR in detecting the novel duck reovirus. DETAILED DESCRIPTION

[0018] The invention provides a primer-probe group for detecting pathogens of waterfowl infectious diseases, comprising an upstream primer group, a downstream primer group and a probe group; the upstream primer group comprises: upstream primers whose nucleotide sequences are 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 group comprises: downstream primers whose nucleotide sequences are 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 group comprises: probes whose nucleotide sequences are 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.

[0019] The primer probe set provided by the 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] As an embodiment, 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 uses the biotin-SA-HRP / TMB color development system, and adds biotin with high affinity to SA-HRP to the primers. When the amplified target fragment binds to the probe, a blue response point can be displayed, and the detection result can be distinguished by the naked eye, realizing visual detection.

[0021] Based on the above advantages, the present invention provides the application of the primer probe group described in the above technical solution in 1) or 2): 1) preparing products for detecting pathogens of waterfowl infectious diseases; 2) detecting pathogens of waterfowl infectious diseases; the direct purpose of the detection is non-diagnostic and non-therapeutic; the pathogens of waterfowl infectious diseases include: 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 new duck reovirus. As an embodiment, the product described in 1) includes a visualized gene chip or a kit. When the direct purpose of the present invention is non-diagnostic and non-therapeutic detection of pathogens of waterfowl infectious diseases, the sample to be tested can be the food or growth environment of poultry.

[0022] Based on the above advantages, the present invention provides a gene chip for detecting pathogens of waterfowl infectious diseases, comprising: a solid phase carrier with probes fixed thereon; the probes are probes in the primer probe set described in the above technical solution.

[0023] As an embodiment, the solid phase carrier is provided with positive quality control points and negative quality control points; the positive quality control points are fixed with biotin; the negative quality control points are fixed with phosphate buffer. The present invention can avoid false positive or false negative test results by providing positive quality control points and negative quality control points.

[0024] As an embodiment, the solid phase carrier includes a nanomembrane.

[0025] The present invention has no special requirements on the positions of the probes fixed on the solid phase carrier, the positive quality control points and the negative quality control points. Figure 1 The fixing method is only used as a specific implementation method and cannot be understood as the protection scope of the present invention.

[0026] The gene chip provided by the present invention is a visualized gene chip. Through the specificity test of the gene chip, it was found that the seven pathogen probe sites all showed specific binding without cross-reaction, indicating that the pathogen probes used in the visualized gene chip provided by the present invention have good specificity. Through the sensitivity test of the gene chip, the single sample sensitivity of the seven pathogens was 1.0×10 0 copies / μL; the sensitivity of the mixed sample of seven pathogenic plasmids was also 1.0×100 copies / μL, indicating that the visualization gene chip provided by the present invention has high sensitivity. The repeatability test and stability test prove that the visualization 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. The comparative experiment between the gene chip detection method and the standard fluorescence quantitative PCR method proves that the sensitivity of the present invention in detecting seven viral single pathogens and seven mixed pathogens is better than that of the qPCR detection method. The visualization gene chip provided by the present invention can quickly and efficiently jointly detect the above-mentioned major waterfowl disease pathogens, and can be used for epidemiological surveys and disease diagnosis of waterfowl.

[0027] The present invention also provides a method for preparing the gene chip described in the above technical solution, comprising the following steps: spotting each probe on a solid phase carrier according to the chip arrangement order, placing the spotted solid phase carrier in a humidity of 40% to 60% and a temperature of 22 to 25°C to dry overnight to obtain the gene chip.

[0028] 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 a gene chip as 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 as described in the above technical solution. As an embodiment, the kit also includes: horseradish peroxidase-labeled streptavidin and 3,3',5,5'-tetramethylbenzidine.

[0029] Based on the above advantages, the present invention provides a method for directly detecting pathogens of waterfowl infectious diseases for non-diagnostic and non-therapeutic purposes, comprising the following steps: The nucleic acid molecule of the sample to be tested is used as a template, and an upstream primer group and a downstream primer group are used to perform an amplification reaction to obtain an amplification product; the upstream primer group and the downstream primer group are the upstream primer group and the downstream primer group in the primer probe group described in the above technical solution; the amplification product is denatured and hybridized with the probe 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; the first solid phase carrier is reacted with horseradish peroxidase-labeled streptavidin to obtain a second solid phase carrier; the second solid phase carrier is subjected to a color development reaction with 3,3',5,5'-tetramethylbenzidine 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 correspondence between the probe and the pathogen of waterfowl infectious disease includes: the probe with a nucleotide sequence as shown in SEQ ID NO.3 corresponds to goose parvovirus; the probe with a nucleotide sequence as shown in SEQ ID NO.6 corresponds to duck viral enteritis virus; the probe with a nucleotide sequence as shown in SEQ ID NO.9 corresponds to Muscovy duck parvovirus; the probe with a nucleotide sequence as shown in SEQ ID NO.12 corresponds to duck hepatitis A virus type 1; the probe with a nucleotide sequence as shown in SEQ ID NO.15 corresponds to duck hepatitis A virus type 3; the probe with a nucleotide sequence as shown in SEQ ID NO.18 corresponds to duck Tembusu virus; and the probe with a nucleotide sequence as shown in SEQ ID NO.21 corresponds to a new duck reovirus.

[0030] As an embodiment, the reaction system of the amplification reaction includes: 2×One step Mix 12.5μL, One step Enzyme Mix 1.25μL, upstream primer set 1μL, downstream primer set 1μL, template 4μL and ddH2O 5.25μL; the reaction procedure of the amplification reaction includes: 50℃ 30 min, 94℃ 4 min; 94℃ 30 sec, 56℃ 30 sec, 72℃ 20 sec, 5 cycles; 94℃ 30 sec, 54℃ 30 sec, 72℃ 20 sec, 35 cycles; 72℃ 10 min, 95℃ 5 min.

[0031] As an embodiment, the sample to be tested may be food or growth environment of poultry.

[0032] The method provided by the present invention can quickly and efficiently detect the above-mentioned major waterfowl disease pathogens, and can be used for the detection of samples waiting to be tested in poultry food or growth environment, thereby reducing the risk of poultry infection.

[0033] 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 are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 Establishment of a visualized gene chip and virus detection method 1. The pathogens used in the present invention: goose parvovirus (GPV, CVCC AV239) and duck viral enteritis virus (DEV, CVCC AV18) are supplied by the National Veterinary Microbiology and Virus 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), and duck adenovirus type 3 (DAdV-3) are all pathogens isolated and identified by conventional methods.

[0035] 2. Design of primers and probes: In order to ensure the detection effect of 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. NS Gene, DEV UL6 Gene, MDPV VP Gene, DHAV-1 VP1 Gene, DHAV-3 VP1 Gene, DTMUV E Gene, NDRV S4 Genes, primers and probes for GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV and NDRV were designed, and the results are shown in Table 1.

[0036] Table 1 Primers and probes for seven viral gene chips Note: Forward in Table 1 means the upstream primer; Reverse means the downstream primer, and the 5' end of the downstream primer is modified with a Biotin group; Probe means the probe, and the 5' end of the probe is modified with NH2C6 (C6 amino modification).

[0037] 3. Preparation of seven virus recombinant positive plasmids: 1) According to the instructions of the TAKARA virus genome RNA / DNA extraction kit, nucleic acids of 7 virus samples were extracted respectively, and 7 pairs of upstream and downstream primers in Table 1 were mixed with equal mass to obtain upstream and downstream primer mixtures. The extracted nucleic acids were used as templates, and the upstream and downstream primer mixtures were used to amplify the target fragments, and purified to obtain purified PCR products; the reaction system was: 2×One step Mix 12.5μL, One step Enzyme Mix 1.25μL, upstream primer mixture 1μL, downstream primer mixture 1μL, template 4μL and ddH2O 5.25μL; the reaction program was: 50℃ reverse transcription for 30 min, 94℃ 3 min; 94℃ 30 sec, 54℃ 30 sec, 72℃ 30 sec, 35 cycles; 72℃ 7 min, 4℃ storage.

[0038] 2) According to the instructions of TARAKA's pMD™19-T Vector Cloning Kit, add 1 μL of pMD19-T Vector, 1 μL of purified PCR product, 3 μL of double distilled water, and 5 μL of Solution I to a centrifuge tube. React at 16℃ for 30 minutes, add the entire amount (10 μL) to 100 μL of JM109 competent cells, place on ice for 30 minutes, heat shock in a 42℃ water bath for 45~60 seconds, quickly transfer to an ice bath, and let stand for 2 minutes. Add 890 μL of non-resistant LB culture medium to the centrifuge tube, and culture at 37℃, 200rpm for 60 minutes. Pipette 80 μL of culture medium and evenly spread it on the Amp-resistant culture medium, and culture it upside down in a 37℃ incubator overnight.

[0039] 3) Pick a single colony and add it to 5 mL of Amp-resistant liquid culture medium. After culturing at 37°C and 200 rpm for 12 h, select PCR-positive bacterial samples for sequencing, use NCBI Nucleotide Blast to align the sequencing results, and extract the recombinant positive plasmids of each virus according to the instructions of Tiangen's rapid plasmid extraction kit.

[0040] 4. Preparation of chip: The probe was diluted with phosphate buffer to a final concentration of 6 μmol / L, and biotin was diluted with phosphate buffer to a final concentration of 6 μmol / L as a positive quality control, and phosphate buffer was used as a negative quality control. The spotter parameter was set to 100 drops, and each probe was spotted on the nanomembrane according to the chip arrangement sequence. The chip arrangement sequence is shown in Figure 1 , where Biotin is the positive quality control of the chip; Buffer is the negative quality control of the chip. After spotting, place the membrane substrate in a humidity of 40% to 60% and a temperature of 22 to 25°C to dry overnight and store at 2 to 8°C.

[0041] 5. Hybridization, washing and color development of the chip: Extract the nucleic acid of the sample to be tested according to the instructions of the TAKARA virus genome RNA / DNA extraction kit, and perform PCR amplification using the reaction system described in step 3 1) to obtain the amplified product; the reaction procedure of PCR amplification is: 50℃ 30 min, 94℃ 4 min; 94℃ 30 sec, 56℃ 30 sec, 72℃ 20 sec, 5 cycles; 94℃ 30 sec, 54℃ 30 sec, 72℃ 20 sec, 35 cycles; 72℃ 10 min, 95℃ 5 min. Denature the amplified product at 95℃ for 5 min and quickly place it on ice to obtain a denatured amplified product. The chip was preheated in a microplate constant temperature oscillator at 47°C, 200 rpm for 10 min; 100 μL of solution A and 20 μL of the denatured amplification product were added to each well of the chip, hybridized at 47°C, 200 rpm for 20 min, and the hybridization solution was aspirated; 150 μL of solution B preheated to 47°C was added to each well for washing twice, and then 150 μL of solution B preheated to 47°C was added, and incubated at 47°C, 200 rpm in a constant temperature oscillator for 10 min; the solution A and solution B were purchased from Nanjing Sen Microbiology Technology Co., Ltd., item number ABT-Z-1006. After the vacuum pump dried the liquid, 150 μL of horseradish peroxidase-labeled streptavidin (SA-HRP) working solution was added to each well, and incubated at 47°C, 200 rpm in a constant temperature oscillator for 10 min. Aspirate the SA-HRP working solution, add 150 μL of solution A to each well and wash three times, then add 150 μL of solution C to each well and wash three times, then aspirate the liquid. Add 90 μL of 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution to each well and let stand at room temperature in the dark for 5-10 min. Aspirate the TMB colorimetric solution, wash each well of the chip with pure water, remove the liquid on the surface of the nanomembrane, and observe the results with the naked eye or using a chip imager.

[0042] 6. Test result determination: The negative control point does not show color, the color of the positive control point is obviously stronger than the blue-purple color of the negative control point, and at least one of the three positive control points shows color for a valid experimental result. If the probe point where the sample is located does not show color, it is negative; if the probe point where the sample is located is blue-purple, it is positive.

[0043] Example 2 Specificity test of gene chip The recombinant positive plasmids of GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV, and NDRV were constructed by the method of step 3 in Example 1 as templates, and the recombinant positive plasmids, viral nucleic acid molecules (DAstv and DAdV-3), and healthy duck serum were PCR amplified by the method of step 5 in Example 1, and the specificity of the chip was verified after the PCR amplification products were hybridized with the chip for color development. The results are shown in FIG. Figure 2 As shown, 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; and J is healthy duck serum.

[0044] The results show that the negative quality control point has no response, the positive quality control point responds, and the test results are established. All seven pathogen probe sites show specific binding without cross-reaction, indicating that the pathogen probes used in the chip have good specificity.

[0045] Example 3 Sensitivity test of gene chip 1. Sensitivity test of single positive plasmid on gene chip The concentration of each positive plasmid constructed in step 3 of Example 1 was measured using an ultra-micro UV 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 The primers in Table 1 were used to perform PCR amplification respectively at four concentrations of 100 copies / μL. After the PCR amplification products were hybridized with the chip for color development, the sensitivity of the chip for single positive plasmid was determined. The results are shown in Figure 3 As 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.

[0046] The results show that the negative quality control point has no response, the positive quality control point responds, and the test results are valid. The single sample sensitivity of GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV, and NDRV is 1.0×10 0 copies / μL.

[0047] 2. Sensitivity test of mixed positive plasmids on gene chip Each positive plasmid constructed in step 3 of Example 1 was diluted to 1.0×10 8 After the mixture was diluted 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 After five concentrations of 100 copies / μL, PCR amplification was performed with the primers in Table 1. After the PCR amplification products were hybridized with the chip for color development, the sensitivity of the mixed positive plasmid of the chip was measured. The results are shown in Figure 4 As 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.

[0048] The results show that the negative quality control point has no response, the positive quality control point responds, and the test result is established. The sensitivity of the mixed sample of seven plasmids, GPV, DEV, MDPV, DHAV-1, DHAV-3, DTMUV, and NDRV, is 1.0×10 0 copies / μL.

[0049] Example 4 Repeatability test of gene chip In order to verify the repeatability of the chip, different experimenters used the amplified products of the same quality control sample (positive recombinant plasmid) to perform chip experiments at different time points. In each experiment, the PCR reaction conditions were the same as the chip hybridization and color development operations to ensure the consistency of the conditions. The experiment was divided into repeatability verification within the same batch and repeatability verification between different batches. By analyzing and comparing the results, the repeatability of the chip within the same batch and under different batches of chips was verified.

[0050] To verify the repeatability of the gene chip detection method, different experimenters used the same quality control sample (positive recombinant plasmid) with different dilutions of amplified products to perform chip experiments at different time points. Repeatability was verified for batch CH240828001 and batch CH240620001 and CH240718001. In each experiment, the PCR reaction conditions were the same as the chip hybridization color development operation to ensure the consistency of the conditions. The results are shown in Table 2. The consistency of the test results within and between batches reached 100%, indicating that the method has good repeatability.

[0051] Table 2 Intra-batch and inter-batch reproducibility of gene chips Example 5 Shelf Life Test of Gene Chip In order to test the stability of gene chip detection, seven virus mixed positive samples were used for detection on the 0th day, 30th day, 60th day, 90th day, 120th day and 180th day after chip production. The stability of the chip at different storage times was verified by analyzing and comparing the results.

[0052] Using seven mixed positive plasmids as templates, stability tests were performed at 0 days, 30 days, 60 days, 90 days, 120 days, and 180 days after chip production. Figure 5 As shown, A is 0 days, B is 30 days, C is 60 days, D is 90 days, E is 120 days, and F is 180 days.

[0053] The results show that under the storage condition of 2℃~8℃, the gene chip can be stably stored for at least 180 days, indicating that this method has good long-term storage stability.

[0054] Example 6 Comparative study on specificity and sensitivity of gene chip method and fluorescence quantitative PCR detection method 1. Reconstruction of standard fluorescence quantitative PCR method According to the industry standards of each virus diagnostic 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 method of each virus was re-established and its specificity and sensitivity were measured. The primers and probes are shown in Table 3.

[0055] 2. Standard fluorescent quantitative PCR method The method for constructing the recombinant positive plasmid is similar to step 3 in Example 1, except that the primers are the primers in Table 3.

[0056] Table 3 Primers and probes for seven viral gene chips Note: Forward in Table 3 indicates the upstream primer; Reverse indicates the downstream primer, and the 5' end of the downstream primer is modified with a Biotin group; Probe indicates the probe, and the 5' end of the probe is modified with NH2C6 (C6 amino modification).

[0057] 3. qPCR reaction program: 25℃ for 10 min; 95℃ for 30 s; 95℃ for 5 s, 48℃ for 30 s, 72℃ for 20 s, 40 cycles.

[0058] 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.

[0059] The results are as follows Figures 6 to 12 As shown, in the qPCR detection methods of the respective pathogens, after three replicate verifications, the amplification curve was only observed when the corresponding pathogen nucleic acid was used as a template, and no amplification signals were observed for other pathogens, indicating that the qPCR detection method has good specificity.

[0060] Each pathogen-positive plasmid was diluted tenfold to 1.0×10 8 copies / μL~1.0×10 0 copies / μL, and qPCR sensitivity tests were performed on each pathogen. Figures 13 to 19 As shown in the figure, the detection sensitivity of GPV, DEV, MDPV, and DTMUV is 1.0×10 1 copies / μL, and the detection sensitivity of DHAV-1, DHAV-3, and NDRV was 1.0×10 2 copies / μL.

[0061] The results show that the sensitivity of the visualized gene chip of the present invention in detecting seven viral single pathogens and seven combined pathogens is slightly better than that of the qPCR detection method.

[0062] Example 7 Comparative test of clinical sample compliance rate Throat swabs, anal swabs, livers, lungs and hearts of animals suspected of being positive for each pathogen were collected in Shandong Province, Jiangxi Province, Fujian Province and this laboratory as clinical samples, and the chip in Example 1 and the qPCR method in Example 6 were used for detection, respectively, and the compliance rate was calculated. Compliance rate = (total number of clinical samples tested - number of different results) / total number of clinical samples tested × 100%.

[0063] A total of 210 clinical samples suspected of being positive for pathogens were used for detection by chip and qPCR detection methods, respectively. The consistency rate of the results of the two detection methods was 97.6%-100%, and the Kappa value was 0.935, indicating that the two detection methods had good consistency.

[0064] Table 4 Clinical sample test results

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, 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 includes 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 group includes probes having 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.

2. The primer probe set according to claim 1, characterized in that: 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.

3. Use of the primer probe set according to claim 1 or 2 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 non-diagnostic and non-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.

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

5. A gene chip for detecting pathogens of waterfowl infectious diseases, characterized in that: It comprises a solid phase carrier having a probe fixed thereon; the probe is a probe in the primer probe set according to claim 1 or 2.

6. The visualized gene chip according to claim 5, characterized in that: The solid phase carrier includes a nanomembrane.

7. A kit for detecting pathogens of waterfowl infectious diseases, characterized in that: It comprises an independently packaged upstream primer set, a downstream primer set and the gene chip according to claim 5 or 6; 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 or 2.

8. The kit according to claim 7, characterized in that The kit also includes: horseradish peroxidase-labeled streptavidin and 3,3',5,5'-tetramethylbenzidine.

9. 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 2; 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 according to claim 5 or 6, or the gene chip in the kit according to claim 8; 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 development 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.

10. The method according to claim 9, 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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