WNV gene detection target sequence and primer combination, kit and detection method thereof

Abstract

The invention discloses a WNV gene detection target sequence and a primer combination, a kit and a detection method thereof, 11 groups of target sequences are screened through WNV whole genome coverage screening, at least one group of five sequences which are more stable relative to other positions is selected as the target sequence for trace WNV gene detection, and the five sequences are nucleotide sequences as shown in SEQ ID No.1-SEQ ID No.5. A nucleic acid sample extracted from faeces of the migrant birds is subjected to PCR / qPCR amplification by utilizing a specially designed primer combination, so that the carrying condition of the WNV pathogens in the migrant birds is quickly judged, and reliable detection data is provided for prevention, control and treatment of diseases; according to the invention, trace detection of WNV in the bird excrement sample can be realized, the detection method is simple and convenient to operate, and the result is reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of trace detection of WNV in special samples, and particularly refers to a WNV gene detection target sequence and a primer combination, a kit and a detection method thereof. Background Art

[0002] West Nile Virus (WNV) was first discovered in Uganda, Africa in 1937. It was isolated from the blood of an adult woman with fever in the West Nile region, hence the name West Nile Virus. West Nile Virus belongs to the Flaviviridae family, the genus Flavivirus. The Flaviviridae family also has more than 70 species, including dengue virus, Japanese encephalitis virus, and yellow fever virus, most of which are arboviruses.

[0003] West Nile virus is mainly transmitted through the bird-mosquito-bird cycle. The virus can infect humans, birds, a variety of mammals, amphibians and reptiles. Humans and birds are the most susceptible. The diseases caused by it are mainly distributed in Europe, Africa and the Middle East countries connected along the bird migration flight routes. Migratory birds play an important role in the global spread and spread of West Nile fever. Its main sources of infection are infected animals in the viremia stage and the natural reservoir host of the virus-migratory birds. The mosquitoes that spread the virus mainly include Culex pipiens, Aedes and Mansonia mosquitoes, especially Culex pipiens, which transmits the virus to humans. Mosquitoes become infected by biting birds infected with WNV, and then the infected mosquitoes transmit West Nile virus to humans and other objects through bites. West Nile virus can cross the blood-brain barrier and interfere with the normal function of the central nervous system. Clinically, it mainly manifests as encephalitis or meningoencephalitis.

[0004] West Nile virus is widely distributed in Europe, America, Africa, the Middle East and West Asia. It mainly causes West Nile fever. Most of the diseases caused by it are zoonotic, causing serious public health problems and lack of effective treatment, prevention and control measures, which poses a severe challenge to disease control. There is also a risk of WNV importation in my country.

[0005] Existing WNV detection technology generally collects human or animal tissue or blood samples for nucleic acid detection, mainly for susceptible hosts for diagnosis. This detection method cannot achieve the purpose of epidemic monitoring and epidemiological analysis; according to the transmission route of WNV, it is mainly transmitted through birds (migratory birds), but migratory birds should not be directly killed, and it is also difficult for birds to collect blood or tissue samples. Therefore, the technical innovation of detecting the WNV carrying status of migratory birds in the present invention has important practical significance, and has practical effects on monitoring the epidemic situation of WNV, import risk analysis, and epidemiological investigation;

[0006] In addition, conventional WNV nucleic acid detection methods all use software to design primers. Ordinary software-designed primers are optimized based on nucleic acid parameters such as the GC content, TM value, and binding free energy of the gene. The designed primers cannot analyze the degradation of viral nucleic acids. The primers designed by the software will not come into contact with the undegraded sequences in actual clinical samples. That is, primers designed based on genomic parameters cannot amplify the WNV sequences actually present in fecal samples. Summary of the invention

[0007] In view of the above technical problems, the purpose of the present invention is to provide a WNV gene detection target sequence and a primer combination, a kit, and a detection method, which can detect the carrying status of WNV through migratory bird feces, so as to analyze the spread of WNV through the carrying status of WNV.

[0008] Since the WNV content in migratory bird feces is low and unstable, it is basically undetectable by existing methods. The present invention has found a relatively stable target for WNV, which can effectively detect migratory bird feces (i.e., WNV trace samples), determine the WNV carrying status in migratory birds, and provide effective prevention and control measures for the spread of WNV.

[0009] According to the first aspect of the present invention, a target sequence for WNV gene detection is provided, and 11 groups of target sequences are screened out through WNV whole genome coverage screening, which are nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.11, and at least one group of sequences is selected for genetic detection of trace WNV.

[0010] In some technical solutions, at least one of five sequences that are more stable than other positions is selected from 11 groups of target sequences as target sequences for trace WNV gene detection, and the five sequences are nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.5.

[0011] In some technical solutions, during the WNV whole genome coverage screening process, the WNV whole genome is divided into fragments of about 200 nt, and there is a 50 nt overlapping sequence between each group of fragments.

[0012] According to a first aspect of the present invention, a primer combination for WNV gene detection is provided, wherein the primer combination is used to amplify the above-mentioned target sequence, wherein:

[0013] The primer combination for amplifying the nucleotide sequence shown in SEQ ID No. 1 is primer combination 1, wherein the primer combination 1 includes primer pair 1, and the primer pair 1 includes upstream primer 1 shown in SEQ ID No. 12 and downstream primer 1 shown in SEQ ID No. 13;

[0014] The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.2 is primer combination 2, and the primer combination 2 includes primer pair 2, and the primer pair 2 includes upstream primer 2 shown in SEQ ID No.14 and downstream primer 2 shown in SEQ ID No.15;

[0015] The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.3 is primer combination 3, and the primer combination 3 includes primer pair 3, and the primer pair 3 includes upstream primer 3 shown in SEQ ID No.16 and downstream primer 3 shown in SEQ ID No.17;

[0016] The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.4 is primer combination 4, and the primer combination 4 includes primer pair 4, and the primer pair 4 includes upstream primer 4 shown in SEQ ID No.18 and downstream primer 4 shown in SEQ ID No.19;

[0017] The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.5 is primer combination 5, and the primer combination 5 includes primer pair 5, and the primer pair 5 includes upstream primer 5 shown in SEQ ID No.20 and downstream primer 5 shown in SEQ ID No.21.

[0018] According to yet another aspect of the present invention, a kit for WNV gene detection is provided, the kit comprising the above primer combination, and reaction reagents for PCR amplification and / or qPCR amplification.

[0019] In some technical solutions, the kit further includes an RNA extraction reagent, a positive control and a negative control; and the reaction reagent includes a DNA polymerase and a DNA polymerase buffer.

[0020] According to another aspect of the present invention, a method for detecting WNV gene is provided, which comprises the steps of performing PCR and / or qPCR using the above-mentioned target sequence or a recombinant plasmid constructed by the target sequence and a plasmid vector as an internal standard gene positive control.

[0021] In some technical solutions, the method comprises the following steps:

[0022] S1. Obtain nucleic acid samples from bird feces;

[0023] S2. Performing PCR and / or qPCR amplification on the nucleic acid sample using the above-mentioned kit;

[0024] S3. Determine whether the bird carries the WNV pathogen based on the amplification results of PCR and / or qPCR.

[0025] In some technical solutions, step S1 specifically includes: collecting fresh viscous or solid migratory bird feces samples and placing them in RNase-free EP tubes, adding RNA preservation solution in a 1:1 ratio and storing them at -70°C to -80°C for a short period of time;

[0026] and / or,

[0027] In step S2, the kit uses a viral RNA extraction kit from QIAGEN;

[0028] and / or,

[0029] The optimized PCR preparation system includes Taq Mix 12.5 μL, guano cDNA 1 μL, upstream primer 20 μM 0.5 μL, downstream primer 20 μM 0.5 μL, and sterile water 9.5 μL; the optimized reaction program is 98° C. for 10 s, 55° C. for 30 s, 72° C. for 20 s, and 30 cycles;

[0030] and / or,

[0031] The optimized qPCR reaction system includes 10 μL 2×Magic SYBR Mixture, 0.4 μL of 10 μM upstream and downstream primers, 2 μL cDNA solution and 7.2 μL water; the optimized reaction conditions are 95°C pre-denaturation for 30 seconds, 95°C 5 seconds denaturation, 60°C 30 seconds extension, and a total of 40 cycles.

[0032] According to another aspect of the present invention, the above target sequence, or the above primer combination, or the above kit is used in the detection of trace amounts of WNV genes.

[0033] The present invention adopts the above technical solution to have at least the following beneficial effects:

[0034] 1. The target sequence for WNV gene detection provided by the present invention is obtained through whole genome coverage screening and is more stable than other positions, so that the qualitative detection of WNV can be achieved in a targeted manner in trace samples of bird droppings, providing a reliable guarantee for determining the carrying status of WNV in bird droppings;

[0035] 2. The target sequence for WNV gene detection provided by the present invention is obtained by screening the whole genome of WNV. Different from the conventional software design of primers, in order to ensure the coverage scan of the whole genome of WNV, the whole genome of WNV is divided into fragments of about 200 nt, and there is a 50 nt repeat between each group of fragments. On the basis of this design, primers are amplified;

[0036] 3. The primer combination for WNV gene detection provided by the present invention is designed to control the above stably existing target sequence and can achieve specific binding;

[0037] 4. The recombinant plasmid provided by the present invention is obtained by connecting the plasmid vector and the target sequence target gene mentioned above through an enzyme, and can be used as a positive control in PCR and qPCR;

[0038] 5. The kit for WNV gene detection provided by the present invention can be used for the extraction and detection of WNV nucleic acid in trace samples of bird droppings. It uses a centrifugal adsorption column that specifically binds to viral RNA and a unique buffer system to quickly extract high-purity nucleic acid samples from bird droppings. After the virus is lysed, the RNA is selectively adsorbed to the silicon matrix membrane in the centrifugal column under a high-order salt state, and then a series of rapid rinsing-centrifugation steps are performed to remove impurities such as salt, cell metabolites, and proteins. Finally, a low-salt elution buffer is used to elute the pure viral RNA from the silicon matrix membrane. The purified viral nucleic acid is free of impurities and PCR inhibitors and can be directly used in PCR / qPCR and other analyses.

[0039] 6. The WNV gene detection method provided by the present invention performs PCR / qPCR amplification on the nucleic acid samples in the feces of migratory birds using the specially designed primer combination of the present invention, so as to quickly determine the carrying status of the WNV pathogen in the migratory birds, and provide reliable detection data for the prevention, control and treatment of the disease; the detection method of the present invention is easy to operate and the results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1a This is the electrophoresis verification pattern of the cDNA of the guano sample of primer combination 1. Figure 1b This is the electrophoresis verification pattern of cDNA of guano samples at different time points (across the year) when the primer combination 1 in the present invention is applied, the target sequence is fragment 1, the product size is 203bp, wherein the first lane M from the left is DNAMarker, the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, 100bp, the second lane 1 from the left is the cDNA of the guano sample, the third lane 2 from the left is the blank control group template is sterile water, the fourth lane 3 from the left is the WNV recombinant plasmid, the fifth lane 4 from the left is the negative guano cDNA, and the sixth lane 5 from the left is the WNV empty plasmid;

[0041] Figure 2a This is the electrophoresis verification pattern of the cDNA of the guano sample of primer combination 2. Figure 2bThis is the electrophoresis verification pattern of cDNA of guano samples at different time points (across the year) when the primer combination 2 in the present invention is applied, the target sequence is fragment 2, the product size is 202bp, wherein the first lane M from the left is DNAMarker, the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, 100bp, the second lane 1 from the left is the cDNA of the guano sample, the third lane 2 from the left is the blank control group template is sterile water, the fourth lane 3 from the left is the WNV recombinant plasmid, and the fifth lane 4 from the left is the negative guano cDNA;

[0042] Figure 3a This is the electrophoresis verification pattern of the cDNA of the guano sample of primer combination 3. Figure 3b This is the electrophoresis verification pattern of cDNA of guano samples at different time points (across the year) when the primer combination 3 in the present invention is applied, the target sequence is fragment 3, the product size is 186bp, wherein the first lane M from the left is DNAMarker, the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, 100bp, the second lane 1 from the left is the cDNA of the guano sample, the third lane 2 from the left is the blank control group template is sterile water, the fourth lane 3 from the left is the WNV recombinant plasmid, the fifth lane 4 from the left is the negative guano cDNA, and the sixth lane 5 from the left is the WNV empty plasmid;

[0043] Figure 4a This is the electrophoresis verification pattern of the cDNA of guano samples of primer combination 4. Figure 4b This is the electrophoresis verification pattern of cDNA of guano samples at different time points (across the year) when the primer combination 4 of the present invention is applied, the target sequence is fragment 4, the product size is 185bp, wherein the first lane M from the left is DNAMarker, the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, 100bp, the second lane 1 from the left is the cDNA of the guano sample, the third lane 2 from the left is the blank control group template is sterile water, the fourth lane 3 from the left is the WNV recombinant plasmid, the fifth lane 4 from the left is the negative guano cDNA, and the sixth lane 5 from the left is the WNV empty plasmid;

[0044] Figure 5a This is the electrophoresis verification pattern of the cDNA of the guano sample of primer combination 5. Figure 5bThis is the electrophoresis verification pattern of cDNA of guano samples at different time points (across the year) when the primer combination 5 of the present invention is applied, the target sequence is fragment 5, the product size is 185bp, wherein the first lane M from the left is DNAMarker, the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, 100bp, the second lane 1 from the left is the cDNA of the guano sample, the third lane 2 from the left is the blank control group template is sterile water, the fourth lane 3 from the left is the WNV recombinant plasmid, the fifth lane 4 from the left is the negative guano cDNA, and the sixth lane 5 from the left is the WNV empty plasmid;

[0045] Figure 6-7 is the melting curve of qPCR amplification using primer combination 1;

[0046] Figure 8 This is the amplification curve of qPCR amplification using primer combination 1. From left to right, the corresponding copy numbers are 5.20×10 9 ~5.20×10 2 Amplification curves of WNV recombinant plasmid and cDNA of the sample to be tested with a dilution ratio of copies / μL, where each dilution ratio is repeated twice and the cDNA of the sample to be tested is repeated three times;

[0047] Figure 9-10 is the melting curve of qPCR amplification using primer combination 2;

[0048] Fig.11 This is the amplification curve of qPCR amplification using primer combination 2. From left to right, the corresponding copy numbers are 5.20×10 9 ~5.20×10 2 Amplification curves of WNV recombinant plasmid and cDNA of the sample to be tested with a dilution ratio of copies / μL, where each dilution ratio is repeated twice and the cDNA of the sample to be tested is repeated three times;

[0049] Figure 12-13 is the melting curve of qPCR amplification using primer combination 3;

[0050] Fig.14 This is the amplification curve of qPCR amplification using primer combination 3. From left to right, the corresponding copy numbers are 1.21×10 9 ~1.21×10 2 Amplification curves of WNV recombinant plasmid and cDNA of the sample to be tested with a dilution ratio of copies / μL, where each dilution ratio is repeated twice and the cDNA of the sample to be tested is repeated three times;

[0051] Figure 15-16 is the melting curve of qPCR amplification using primer combination 4;

[0052] Fig.17 This is the amplification curve of qPCR amplification using primer combination 4. From left to right, the corresponding copy numbers are 5.20×10 9 ~5.20×10 2 Amplification curves of WNV recombinant plasmid and cDNA of the sample to be tested with a dilution ratio of copies / μL, where each dilution ratio is repeated twice and the cDNA of the sample to be tested is repeated three times;

[0053] Figure 18-19 is the melting curve of qPCR amplification using primer combination 5;

[0054] Fig. 20 This is the amplification curve of qPCR amplification using primer combination 5. From left to right, the corresponding copy numbers are 5.20×10 9 ~5.20×10 2 Amplification curves of WNV recombinant plasmid and cDNA of the sample to be tested with a dilution ratio of copies / μL, where each dilution ratio is repeated twice and the cDNA of the sample to be tested is repeated three times;

[0055] Fig.21 This is the design diagram of the WNV amplicon, where red is the primer and pink is the amplified fragment;

[0056] Fig. 22 This is the gene fragment screened from the whole genome of WNV. DETAILED DESCRIPTION

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0058] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0059] 1.1 Collection of migratory bird / bird droppings samples

[0060] Collect samples within two weeks, or at the latest within one month, using a sterile cotton swab to collect fresh viscous or solid migratory bird droppings and place the samples in an RNase-free 2 mL EP tube.

[0061] 1.2 RNA preservation method

[0062] Add RNA preservation solution to the test tube containing the sample at a ratio of 1:1, and transfer it to the laboratory as soon as possible and store it in a -80℃ refrigerator.

[0063] 1.3 Methods for extracting trace RNA from feces and identification of RNA quality standards

[0064] 1.31 Extraction of trace RNA from stool: Sample RNA was extracted using the viral RNA extraction kit from QIAGEN. The specific steps are as follows (the volume of reagents used can be increased or decreased in proportion to the actual sample volume during the specific operation):

[0065] a. Take 560 μL of the prepared Buffer AVL into a 1.5 mL centrifuge tube.

[0066] b. Add 140 μL of sample and vortex mix for about 15 seconds.

[0067] c. Incubate at room temperature for 10 min.

[0068] d. Briefly centrifuge to remove the solution in the tube cap.

[0069] e. Add 560 μL of anhydrous ethanol, vortex mix for about 15 seconds, and then briefly centrifuge to remove the solution in the tube cap.

[0070] f. Gently transfer 630 μL of the solution in step e to the adsorption column (placed in a 2 mL collection tube), centrifuge at 8000 rpm for 1 min, and transfer the adsorption column to a new 2 mL collection tube.

[0071] g. Carefully open the adsorption column and repeat step f until all the solution has been filtered through the adsorption column.

[0072] h. Add 500 μL Buffer AW1 to the adsorption column, centrifuge at 8000 rpm for 1 min, and transfer the adsorption column to a new 2 mL collection tube.

[0073] i. Add 500 μL Buffer AW2 to the adsorption column, centrifuge at the highest speed (14000 rpm) for 3 min, and transfer the adsorption column to a new 2 mL collection tube.

[0074] j. Centrifuge at maximum speed (14000 rpm) for 1 min.

[0075] k. Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 60 μL Buffer AVE, and incubate at room temperature for 1 min.

[0076] 1. Elution of RNA: Centrifuge at 8000 rpm for 1 min. The eluted solution is the solution containing viral RNA.

[0077] 1.32 RNA quality standard identification: The A260 / A280 and A260 / A230 values ​​of RNA were measured using a UV spectrophotometer. The 260 / 280 values ​​of the extracted sample RNA were in the range of 1 to 2.4, and the 260 / 230 values ​​were in the range of 0.35 to 2.4 (the RNA absorbance values ​​here are the range after actual operation. Since the extracted RNA is extracted from the feces of migratory birds, the actual RNA obtained contains impurities, and the absorbance value ratio with that of pure RNA will deviate. This is a normal situation for the successful establishment of this method). The concentration range is 100 to 1000 ng / μL.

[0078] 1.4WNV whole genome scanning: The specific steps are: design amplicon according to the full length of WNV genome ( Fig.21 ), a total of 63 groups, each with 1 pair of primers; each pair of primers amplifies a WNV genomic sequence of about 200 nt in length; between adjacent amplicons, including 50 nt of overlapping sequences, the WNV sequence is covered and amplified to ensure that the genomic fragments within 200 nt of WNV present in the sample can be amplified and no fragments are missed.

[0079] This method ultimately amplified 11 WNV sequences (such as Fig. 22 ), indicating that these 11 WNV sequences have higher stability in fecal samples.

[0080] After whole genome coverage screening, 11 target sequences were screened out, and the information of each target sequence is as follows:

[0081]

[0082]

[0083]

[0084] Five sequences (i.e., SEQ ID No. 1 to SEQ ID No. 5) which are more stable than other positions are selected as target sequences for WNV gene detection.

[0085] The corresponding sequence numbers and SEQ ID No. of the 5 target sequences used for WNV gene detection and the corresponding primer designs are as follows:

[0086]

[0087]

[0088]

[0089] 1.6 PCR method to verify detection efficiency

[0090] This step is a conventional PCR test step. The reagents used in this step include but are not limited to the reagents used when establishing this method. Now take the reagents used in this method as an example: the enzyme used for amplification is TaKaRa Taq Mix, and the preparation system and reaction procedure are as follows:

[0091] System (25 μL):

[0092] Taq Mix: 12.5 μL, guano cDNA: 2 μL, upstream primer (20 μM): 1 μL, downstream primer (20 μM): 1 μL and sterile water: 8.5 μL.

[0093] Reaction procedure:

[0094] ①98℃10s

[0095] ②55℃30s

[0096] ③72℃20s

[0097] ①~③ is a reaction cycle, and a total of 30 cycles are required.

[0098] Illustrations and analysis:

[0099] from Figure 1a and Figure 1b The results show that compared with the control group, the guano sample cDNA did not amplify the target sequence of 203 bp in length. When the primer combination 1 of the present invention was used, the guano sample cDNA and WNV recombinant plasmid at different time points (across the year) were successfully amplified. The target sequence of 203 bp in length.

[0100] from Figure 2a and Figure 2b The results show that compared with the control group, the guano sample cDNA, the guano sample cDNA at different time points (across the year) when the primer combination 2 of the present invention was used, and the WNV recombinant plasmid all successfully amplified a target sequence with a length of 202 bp.

[0101] from Figure 3a and Figure 3b The results show that compared with the control group, both the guano sample cDNA and the WNV recombinant plasmid successfully amplified a target sequence with a length of 186 bp. When the primer combination 3 of the present invention was used, the guano sample cDNA at different time points (across the year) was suspected to amplify a target sequence with a length of 186 bp.

[0102] from Figure 4a and Figure 4b The results show that compared with the control group, the guano sample cDNA, the guano sample cDNA at different time points (across the year) when the primer combination 4 of the present invention was used, and the WNV recombinant plasmid all successfully amplified a target sequence with a length of 185 bp.

[0103] from Figure 5a and Figure 5b The results show that compared with the control group, the guano sample cDNA did not amplify the target sequence of 185 bp in length. When the primer combination 4 of the present invention was used, the guano sample cDNA and WNV recombinant plasmid at different time points (across the year) were successfully amplified. The target sequence of 185 bp in length.

[0104] 1.7 qPCR method to verify detection efficiency

[0105] The WNV recombinant plasmid was diluted 10 times with sterile water for 8 consecutive gradients), and fluorescent quantitative PCR was performed using the fluorescent dye SYBRGreen I: 20μL system: 10μL 2×Magic SYBR Mixture, 0.4μL of upstream and downstream primers (10μM), 2μL cDNA solution and 7.2μL water. Reaction procedure: 95℃ pre-denaturation for 30s, 95℃ 5s denaturation, 60℃ 30s extension, a total of 40 cycles, and then melting curve analysis.

[0106] Illustrations and analysis:

[0107] Figure 6-8 The melting curve is a single peak, indicating that there is no nonspecific amplification and primer dimer. The standard curve obtained by the amplification curve is Y = -3.3079x + 39.743, where x is the logarithmic value of the number of copies of the starting template, y represents the Ct value, and the amplification relation R 2 =0.9964, the lowest detection concentration is 5.20×10 2 copies / μL. According to the sample test results, the effective detection concentration of this primer combination was calculated to be 2.50×10 3 copies / μL.

[0108] Figure 9-11 The melting curve is a single peak, indicating that there is no nonspecific amplification and primer dimer. The standard curve obtained by the amplification curve is Y = -3.2582x + 39.641, where x is the logarithmic value of the number of copies of the starting template, y represents the Ct value, and the amplification relation R 2 =0.9998, the lowest detection concentration is 5.20×10 2 copies / μL. According to the sample test results, the effective detection concentration of this primer combination was calculated to be 1.68×10 3 copies / μL.

[0109] Figure 12-14 The melting curve is a single peak, indicating that there is no nonspecific amplification and primer dimer. The standard curve obtained by the amplification curve is Y = -3.2011x + 38.048, where x is the logarithmic value of the number of copies of the starting template, y represents the Ct value, and the amplification relation R2 =0.9987,1.21×10 2 copies / μL. According to the sample test results, the effective detection concentration of this primer combination was calculated to be 1.56×10 3 copies / μL.

[0110] Figure 15-17 The melting curve is a single peak, indicating that there is no nonspecific amplification and primer dimer. The standard curve obtained by the amplification curve is Y = -3.2243x + 39.484, where x is the logarithmic value of the number of copies of the starting template, y represents the Ct value, and the amplification relation R 2 =0.9994, the lowest detection concentration is 5.20×10 2 copies / μL. According to the sample test results, the effective detection concentration of this primer combination was calculated to be 3.85×10 3 copies / μL.

[0111] Figure 18-20 The melting curve is a single peak, indicating that there is no nonspecific amplification and primer dimer. The standard curve obtained by the amplification curve is Y = -3.0896x + 37.768, where x is the logarithmic value of the number of copies of the starting template, y represents the Ct value, and the amplification relation R 2 =0.9914, the lowest detection concentration is 5.20×10 2 copies / μL. According to the sample test results, the effective detection concentration of this primer combination was calculated to be 7.54×10 3 copies / μL.

[0112] From the above experiments, it can be seen that the target sequences of SEQ ID No. 2 to SEQ ID No. 4 have better application effects in the present invention.

[0113] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A target sequence for WNV gene detection, characterized in that: A total of 11 groups of target sequences were screened out through WNV whole genome coverage screening, which were nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.11, and at least one group of sequences was selected for genetic detection of trace WNV.

2. The target sequence according to claim 1, characterized in that At least one of the five sequences that are more stable than other positions is selected from the 11 target sequences as the target sequence for detecting trace WNV genes. The five sequences are the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.

5.

3. The target sequence according to claim 1, characterized in that During the WNV whole genome coverage screening process, the WNV whole genome was divided into fragments of about 200 nt, and there was a 50 nt overlap sequence between each group of adjacent fragments.

4. A primer combination for WNV gene detection, characterized in that: The primer combination is used to amplify the target sequence according to any one of claims 1 to 3, wherein: The primer combination for amplifying the nucleotide sequence shown in SEQ ID No. 1 is primer combination 1, wherein the primer combination 1 includes primer pair 1, and the primer pair 1 includes upstream primer 1 shown in SEQ ID No. 12 and downstream primer 1 shown in SEQ ID No. 13; The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.2 is primer combination 2, and the primer combination 2 includes primer pair 2, and the primer pair 2 includes upstream primer 2 shown in SEQ ID No.14 and downstream primer 2 shown in SEQ ID No.15; The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.3 is primer combination 3, and the primer combination 3 includes primer pair 3, and the primer pair 3 includes upstream primer 3 shown in SEQ ID No.16 and downstream primer 3 shown in SEQ ID No.17; The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.4 is primer combination 4, and the primer combination 4 includes primer pair 4, and the primer pair 4 includes upstream primer 4 shown in SEQ ID No.18 and downstream primer 4 shown in SEQ ID No.19; The primer combination for amplifying the nucleotide sequence shown in SEQ ID No.5 is primer combination 5, and the primer combination 5 includes primer pair 5, and the primer pair 5 includes upstream primer 5 shown in SEQ ID No.20 and downstream primer 5 shown in SEQ ID No.

21.

5. A kit for WNV gene detection, characterized in that: The kit comprises the primer combination according to claim 4, and reaction reagents for PCR amplification and / or qPCR amplification.

6. The kit according to claim 5, characterized in that The kit also includes an RNA extraction reagent, a positive control and a negative control; and the reaction reagent includes a DNA polymerase and a DNA polymerase buffer.

7. A method for WNV gene detection, characterized in that: The method comprises the steps of performing PCR and / or qPCR using the target sequence described in any one of claims 1 to 3 or a recombinant plasmid constructed by the target sequence and a plasmid vector as an internal standard gene positive control.

8. The method according to claim 7, characterized in that The method comprises the following steps: S1. Obtain nucleic acid samples from bird feces; S2. performing PCR and / or qPCR amplification on the nucleic acid sample using the kit according to claim 5 or 6; S3. Determine whether the bird carries the WNV pathogen based on the amplification results of PCR and / or qPCR.

9. The method according to claim 8, characterized in that Step S1 specifically comprises: collecting fresh viscous or solid migratory bird feces samples and placing them in RNase-free EP tubes, adding RNA preservation solution in a 1:1 ratio and storing them at -70°C to -80°C for a short term; and / or, In step S2, the kit uses a viral RNA extraction kit from QIAGEN; and / or, The optimized PCR preparation system includes Taq Mix 12.5 μL, guano cDNA 1 μL, upstream primer 20 μM 0.5 μL, downstream primer 20 μM 0.5 μL, and sterile water 9.5 μL; the optimized reaction program is 98° C. for 10 s, 55° C. for 30 s, and 72° C. for 20 s, for 30 cycles; and / or, The optimized qPCR reaction system includes 10 μL 2×Magic SYBR Mixture, 0.4 μL of 10 μM upstream and downstream primers, 2 μL cDNA solution and 7.2 μL water; the optimized reaction conditions are 95°C pre-denaturation for 30 seconds, 95°C 5 seconds denaturation, 60°C 30 seconds extension, and a total of 40 cycles.

10. Use of the target sequence according to any one of claims 1 to 3, or the primer combination according to claim 4, or the kit according to claim 5 or 6 in detecting trace amounts of WNV genes.

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