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

Abstract

The application discloses a WNV gene detection target sequence, a primer combination, a kit and a detection method, 11 groups of target sequences are screened out through WNV whole genome coverage screening, and at least one group of five sequences which are relatively stable compared with other positions is selected as a target sequence for micro WNV gene detection, the five sequences are nucleotide sequences shown in SEQ ID No. 1-SEQ ID No. 5. The nucleic acid sample extracted from the droppings of migratory birds is amplified by PCR / qPCR using the special primer combination of the application, so that the carrying condition of the WNV pathogen in the migratory birds is rapidly judged, and reliable detection data for the prevention, control and treatment of diseases is provided. The application can realize the micro detection of WNV in the droppings of birds, the detection method is simple to operate, and the result is reliable.

Description

Technical Field

[0001] This invention belongs to the field of trace detection technology of WNV in special samples, especially the target sequence of WNV gene detection and its primer combination, kit and detection method. Background Technology

[0002] West Nile virus (WNV) was first discovered in Uganda, Africa in 1937, isolated from the blood of a feverish adult woman in the West Nile region, hence its name. West Nile virus belongs to the genus Flaviviridae in the family Flaviviridae. The Flaviviridae family also includes more than 70 other species, such as dengue virus, Japanese encephalitis virus, and yellow fever virus, most of which are arboviruses.

[0003] West Nile virus (WNV) is primarily transmitted through a bird-mosquito-bird cycle. This virus can infect humans, birds, various mammals, amphibians, and reptiles, with humans and birds being the most susceptible. The disease is mainly distributed in Europe, Africa, and the Middle East along bird migration routes. Migratory birds play a crucial role in the global prevalence and spread of West Nile fever. Its main sources of infection are infected animals in the viremic phase and migratory birds, the virus's natural reservoir hosts. Mosquitoes that transmit the virus mainly include Culex, Aedes, and Mansonia, with Culex pipiens being particularly effective in transmitting the virus to humans. Mosquitoes become infected with WNV by biting birds, and then transmit the virus to humans and other objects through bites. West Nile virus can cross the blood-brain barrier, interfering with normal central nervous system function, clinically manifesting primarily as encephalitis or meningoencephalitis.

[0004] West Nile virus (WNV) is widely distributed in Europe, America, Africa, the Middle East, and West Asia, primarily causing West Nile fever. Most of the diseases it causes are zoonotic, posing serious public health problems, and effective treatment, prevention, and control measures are often lacking, presenting a severe challenge to disease control. There is also a risk of WNV importation into my country.

[0005] Current WNV detection technologies generally rely on collecting human or animal tissue or blood samples for nucleic acid testing, primarily targeting susceptible hosts for diagnosis. This method fails to achieve the goals of epidemic monitoring and epidemiological analysis. Given that WNV primarily spreads through birds (migratory birds), and that migratory birds are not suitable for direct capture, collecting blood or tissue samples from them is also difficult. Therefore, the technological innovation in this invention for detecting WNV carrying status in migratory birds has significant practical implications, playing a role in WNV epidemic monitoring, input risk analysis, and epidemiological investigations.

[0006] Furthermore, conventional WNV nucleic acid detection methods all use software-designed primers. These software-designed primers are optimized based on nucleic acid parameters such as GC content, TM value, and binding free energy, and therefore cannot analyze viral nucleic acid degradation. Software-designed primers do not access undegraded sequences in actual clinical samples. In other words, primers designed based on genomic parameters cannot amplify actual WNV sequences present in fecal samples. Summary of the Invention

[0007] To address the above technical problems, the present invention aims to provide WNV gene detection target sequences and their primer combinations, kits, and detection methods, which can detect WNV carrying status through migratory bird droppings, so as to analyze the spread and prevalence of WNV through WNV carrying status.

[0008] Because the content of WNV in migratory bird droppings is low and unstable, existing methods can hardly detect it. However, this invention has found a relatively stable target for WNV, which can effectively detect migratory bird droppings (i.e., trace samples of WNV), determine the carrying status of WNV in migratory birds, and provide effective prevention and control measures for the spread of WNV.

[0009] According to a first aspect of the present invention, target sequences for WNV gene detection are provided. A total of 11 target sequences are screened through WNV whole genome coverage screening, which are the nucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 11. At least one of these sequences is selected for gene detection of trace amounts of WNV.

[0010] In some technical solutions, at least one set of five sequences that are relatively more stable than other positions are selected from 11 sets of target sequences as the target sequences for the detection of trace WNV genes. These five sequences are the nucleotide sequences shown in 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 approximately 200 nt each, with 50 nt of overlapping sequences between each group of fragments.

[0012] According to a first aspect of the invention, a primer set for WNV gene detection is provided, the primer set being used to amplify the aforementioned target sequence, wherein,

[0013] The primer combination for amplifying the nucleotide sequence shown in SEQ ID No. 1 is primer combination 1, which includes primer pair 1, which 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, which includes primer pair 2, which 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, which includes primer pair 3, which 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, which includes primer pair 4, including 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, which includes primer pair 5, including upstream primer 5 shown in SEQ ID No. 20 and downstream primer 5 shown in SEQ ID No. 21.

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

[0019] In some technical solutions, the kit also includes RNA extraction reagents, positive controls, and negative controls; the reaction reagents include DNA polymerase and DNA polymerase buffer.

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

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

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

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

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

[0025] In some technical solutions, step S1 specifically involves: collecting fresh viscous or solid migratory bird droppings samples and placing them in RNase-free EP tubes, adding RNA preservation solution at a 1:1 ratio, and storing them briefly at -70℃ to -80℃.

[0026] And / or,

[0027] In step S2, the kit used is the viral RNA extraction kit from QIAGEN.

[0028] And / or,

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

[0030] And / or,

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

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

[0033] The present invention, by employing the above technical solution, has at least the following beneficial effects:

[0034] 1. The target sequence for WNV gene detection provided by this invention is obtained through whole-genome coverage screening and is more stable than other positions, thus enabling targeted qualitative identification of WNV in trace samples of bird droppings, providing a reliable guarantee for determining the WNV carrying status in bird droppings;

[0035] 2. The target sequence for WNV gene detection provided by this invention is obtained through screening the entire WNV genome. Unlike conventional software-designed primers, in order to ensure a comprehensive scan of the entire WNV genome, the entire WNV genome is divided into fragments of approximately 200 nt, with 50 nt repeats between each group of fragments. Primers are then used for amplification based on this design.

[0036] 3. The primer combination for WNV gene detection provided by this invention is designed based on the stable target sequences mentioned above, and can achieve specific binding;

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

[0038] 5. The kit for WNV gene detection provided by this invention can be used for WNV nucleic acid extraction and detection in trace samples of bird droppings. It employs a centrifugal adsorption column that specifically binds to viral RNA and a unique buffer system, enabling rapid extraction of high-purity nucleic acid samples from bird droppings. After viral lysis, the RNA selectively adsorbs onto the silica matrix membrane within the centrifugal column under high-salt conditions. A series of rapid rinsing-centrifugation steps remove impurities such as salt, cell metabolites, and proteins. Finally, a low-salt elution buffer elutes the pure viral RNA from the silica matrix membrane. The purified viral nucleic acid is free of impurities and PCR inhibitors and can be directly used in PCR / qPCR analyses.

[0039] 6. The WNV gene detection method provided by this invention rapidly determines the WNV pathogen carrying status in migratory birds by performing PCR / qPCR amplification on nucleic acid samples from migratory bird feces using primer combinations specially designed by this invention, providing reliable detection data for disease prevention, control and treatment; the detection method of this invention is simple to operate and the results are reliable. Attached Figure Description

[0040] Figure 1a This is an electrophoretic verification pattern of cDNA from bird droppings samples using primer combination 1. Figure 1b The electrophoretic verification pattern of cDNA from bird droppings samples at different time points (across years) using primer combination 1 in this invention is shown. The target sequence is fragment 1, and the product size is 203bp. In the image, lane M from the left is the DNA marker, and the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, and 100bp, respectively. Lane 1 from the left is the cDNA from bird droppings sample. Lane 2 from the left is the blank control group with sterile water as the template. Lane 3 from the left is the WNV recombinant plasmid. Lane 4 from the left is the negative bird droppings cDNA. Lane 5 from the left is the WNV empty vector plasmid.

[0041] Figure 2a The electrophoretic verification pattern of cDNA from bird droppings sample using primer combination 2. Figure 2bThe electrophoretic verification pattern of cDNA from bird droppings samples at different time points (across years) using primer combination 2 in this invention is shown. The target sequence is fragment 2, and the product size is 202bp. In the image, lane M from the left is the DNA marker, and the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, and 100bp, respectively. Lane 1 from the left is the cDNA from bird droppings samples. Lane 2 from the left is the blank control group with sterile water as the template. Lane 3 from the left is the WNV recombinant plasmid. Lane 4 from the left is the negative bird droppings cDNA.

[0042] Figure 3a Electrophoretic verification pattern of cDNA from bird droppings sample using primer combination 3. Figure 3b The electrophoretic verification pattern of cDNA from bird droppings samples at different time points (across years) using primer combination 3 in this invention is shown. The target sequence is fragment 3, and the product size is 186bp. In the image, lane M from the left is the DNA marker, and the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, and 100bp, respectively. Lane 1 from the left is the cDNA from bird droppings sample. Lane 2 from the left is the blank control group with sterile water as the template. Lane 3 from the left is the WNV recombinant plasmid. Lane 4 from the left is the negative bird droppings cDNA. Lane 5 from the left is the WNV empty vector plasmid.

[0043] Figure 4a Electrophoretic verification pattern of cDNA from bird droppings sample using primer combination 4. Figure 4b The electrophoretic verification pattern of cDNA from bird droppings samples at different time points (across years) using primer combination 4 in this invention is shown. The target sequence is fragment 4, and the product size is 185bp. In the diagram, lane M from the left is the DNA marker, and the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, and 100bp, respectively. Lane 1 from the left is the cDNA from bird droppings sample. Lane 2 from the left is the blank control group with sterile water as the template. Lane 3 from the left is the WNV recombinant plasmid. Lane 4 from the left is the negative bird droppings cDNA. Lane 5 from the left is the WNV empty vector plasmid.

[0044] Figure 5a Electrophoretic verification pattern of cDNA from bird droppings sample using primer combination 5. Figure 5bThe electrophoretic verification pattern of cDNA from bird droppings samples at different time points (across years) using primer combination 5 in this invention is shown. The target sequence is fragment 5, and the product size is 185bp. In the diagram, lane M from the left is the DNA marker, and the fragment sizes from top to bottom are 600bp, 500bp, 400bp, 300bp, 200bp, and 100bp, respectively. Lane 1 from the left is the cDNA from bird droppings sample. Lane 2 from the left is the blank control group with sterile water as the template. Lane 3 from the left is the WNV recombinant plasmid. Lane 4 from the left is the negative bird droppings cDNA. Lane 5 from the left is the WNV empty vector plasmid.

[0045] Figure 6-7 Melting curves for qPCR amplification using primer combination 1;

[0046] Figure 8 The image shows the amplification curve for qPCR using primer combination 1, where the copy numbers from left to right correspond to 5.20 × 10⁻⁶ copies. 9 ~5.20×10 2 Amplification curves of serially diluted WNV recombinant plasmid (copy / μL) and cDNA from test samples, where each dilution is a duplicate and the cDNA from test samples is a triplet;

[0047] Figure 9-10 Melting curves for qPCR amplification using primer combination 2;

[0048] Figure 11 The image shows the amplification curve for qPCR using primer combination 2, where the copy numbers from left to right correspond to 5.20 × 10⁻⁶ copies. 9 ~5.20×10 2 Amplification curves of serially diluted WNV recombinant plasmid (copy / μL) and cDNA from test samples, where each dilution is a duplicate and the cDNA from test samples is a triplet;

[0049] Figure 12-13 Melting curves for qPCR amplification using primer combination 3;

[0050] Figure 14 The image shows the amplification curves for qPCR using primer combination 3, where the copy numbers from left to right correspond to 1.21 × 10⁻⁶ copies. 9 ~1.21×10 2 Amplification curves of serially diluted WNV recombinant plasmid (copy / μL) and cDNA from test samples, where each dilution is a duplicate and the cDNA from test samples is a triplet;

[0051] Figure 15-16 Melting curves for qPCR amplification using primer combination 4;

[0052] Figure 17 The image shows the amplification curve for qPCR using primer combination 4, where the copy numbers from left to right correspond to 5.20 × 10⁻⁶ copies. 9 ~5.20×10 2 Amplification curves of serially diluted WNV recombinant plasmid (copy / μL) and cDNA from test samples, where each dilution is a duplicate and the cDNA from test samples is a triplet;

[0053] Figure 18-19 Melting curves for qPCR amplification using primer combination 5;

[0054] Figure 20 The image shows the amplification curves for qPCR using primer combination 5, where the copy numbers from left to right correspond to 5.20 × 10⁻⁶ copies. 9 ~5.20×10 2 Amplification curves of serially diluted WNV recombinant plasmid (copy / μL) and cDNA from test samples, where each dilution is a duplicate and the cDNA from test samples is a triplet;

[0055] Figure 21 This is a design diagram of the WNV amplicon, where red indicates primers and pink indicates amplification fragments;

[0056] Figure 22 Gene fragments screened from the entire WNV genome. Detailed Implementation

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

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

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

[0060] Collect samples within two weeks, or at the latest within one month, using sterile cotton swabs to collect fresh, viscous or solid migratory bird droppings. Place the samples in 2 mL EP tubes without RNase.

[0061] 1.2 RNA preservation methods

[0062] Add RNA preservation solution to the test tube containing the sample at a 1:1 ratio, and transfer it to the laboratory as soon as possible for storage at -80°C.

[0063] 1.3 Methods for extracting trace RNA from feces, and RNA quality standard identification.

[0064] 1.31 Fecal microRNA extraction: RNA was extracted from the samples using the QIAGEN viral RNA extraction kit. The specific steps are as follows (the volume of reagents used can be increased or decreased proportionally according to the actual sample volume):

[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 for about 15 seconds.

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

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

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

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

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

[0072] h. Add 500 μL of BufferAW1 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 of BufferAW2 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 the highest speed (14000 rpm) for 1 min.

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

[0076] 1. Eluting 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 measured range of the extracted sample RNA 260 / 280 value was 1–2.4, and the range of the 260 / 230 value was 0.35–2.4 (these RNA absorbance values ​​are the actual ranges after actual operation. Since the extracted RNA was obtained from migratory bird droppings, the actual obtained RNA contains impurities, and the absorbance ratio will deviate from that of pure RNA. This is a normal situation for the successful establishment of this method). The concentration range was 100–1000 ng / μL.

[0078] 1.4 WNV whole genome scanning: The specific steps are as follows: design amplicon assays based on the full length of the WNV genome. Figure 21 There are 63 groups in total, with one pair of primers in each group; each pair of primers amplifies a WNV genome sequence of about 200 nt in length; between adjacent amplicon, including a 50 nt overlap sequence, the WNV sequence is covered and amplified to ensure that the WNV genome fragments within 200 nt present in the sample can be amplified and that no fragments are missed.

[0079] This method ultimately amplified 11 WNV sequences (e.g. Figure 22 This indicates that these 11 WNV sequences have higher stability in fecal samples.

[0080] A total of 11 target sequences were identified through genome-wide screening. The information for each target sequence is as follows:

[0081]

[0082]

[0083]

[0084] Five sequences (SEQ ID No. 1 to SEQ ID No. 5) that are relatively more stable than those in other positions were selected as target sequences for WNV gene detection.

[0085] The five target sequences used for WNV gene detection, their corresponding sequence numbers, SEQ ID Nos, and primer designs are as follows:

[0086]

[0087]

[0088]

[0089] 1.6 PCR method to verify detection efficiency

[0090] This step is a standard PCR procedure. The reagents used in this step include, but are not limited to, those used in establishing this method. Taking the reagents used in this method as an example: The enzyme used to amplify the fragment is TaKaRa Taq Mix, and the preparation system and reaction procedure are as follows:

[0091] System (25 μL):

[0092] Taq Mix: 12.5 μL, bird droppings 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℃ for 10 seconds

[0095] ②55℃ for 30 seconds

[0096] ③72℃ for 20 seconds

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

[0098] Attached Figure Descriptions and Analysis:

[0099] from Figure 1a and Figure 1b The results showed that, compared with the control group, the cDNA of bird droppings samples did not amplify the target sequence of 203 bp. However, when primer combination 1 of the present invention was used, the cDNA of bird droppings samples and WNV recombinant plasmids at different time points (across years) successfully amplified the target sequence of 203 bp.

[0100] from Figure 2a and Figure 2b The results showed that, compared with the control group, the cDNA of bird droppings samples, the cDNA of bird droppings samples at different time points (across years) when primer combination 2 of the present invention was used, and the WNV recombinant plasmid were all successfully amplified to a target sequence of 202 bp in length.

[0101] from Figure 3a and Figure 3b The results showed that, compared with the control group, the cDNA and WNV recombinant plasmid of bird droppings samples successfully amplified the target sequence of 186 bp. When using primer combination 3 in this invention, the cDNA of bird droppings samples at different time points (across years) was suspected to amplify the target sequence of 186 bp.

[0102] from Figure 4a and Figure 4b The results showed that, compared with the control group, the cDNA of bird droppings samples, the cDNA of bird droppings samples at different time points (across years) using primer combination 4 in this invention, and the WNV recombinant plasmid all successfully amplified the target sequence with a length of 185 bp.

[0103] from Figure 5a and Figure 5b The results showed that, compared with the control group, the cDNA of bird droppings samples did not amplify the target sequence of 185 bp. However, when using primer combination 4 in this invention, the cDNA and WNV recombinant plasmid of bird droppings samples at different time points (across years) successfully amplified the target sequence of 185 bp.

[0104] 1.7 Validation of detection efficiency using the qPCR method

[0105] The WNV recombinant plasmid was serially diluted 10-fold with sterile water (eight consecutive gradients). Quantitative real-time PCR was performed using the fluorescent dye SYBR Green I. The 20 μL system consisted of 10 μL 2×Magic SYBR Mixture, 0.4 μL each of forward and reverse primers (10 μM), 2 μL cDNA solution, and 7.2 μL water. The reaction program was: 95℃ pre-denaturation for 30 s, followed by 95℃ denaturation for 5 s, and 60℃ extension for 30 s, for a total of 40 cycles. Melting curve analysis was then performed.

[0106] Attached Figure Descriptions and Analysis:

[0107] Figure 6-8 The melting curve showed a single peak, indicating the absence of nonspecific amplification and primer dimers. The standard curve obtained from the amplification curve was Y = -3.3079x + 39.743, where x is the logarithm of the initial template copy number, y represents the Ct value, and the amplification relation Rt is the coefficient of variation. 2 =0.9964, the lowest detectable concentration is 5.20×10 2 Based on the sample detection results, the effective detection concentration of this primer combination was calculated to be 2.50 × 10⁻⁶ copies / μL. 3 Copy / μL.

[0108] Figure 9-11 The melting curve showed a single peak, indicating the absence of nonspecific amplification and primer dimers. The standard curve obtained from the amplification curve was Y = -3.2582x + 39.641, where x is the logarithm of the initial template copy number, y represents the Ct value, and the amplification relation R is... 2 =0.9998, the lowest detectable concentration is 5.20×10 2 Based on the sample detection results, the effective detection concentration of this primer combination was calculated to be 1.68 × 10⁻⁶ copies / μL. 3 Copy / μL.

[0109] Figure 12-14 The melting curve showed a single peak, indicating the absence of nonspecific amplification and primer dimers. The standard curve obtained from the amplification curve was Y = -3.2011x + 38.048, where x is the logarithm of the initial template copy number, y represents the Ct value, and the amplification relation Rt is the coefficient of variation.2 =0.9987, 1.21 × 10 2 Based on the sample detection results, the effective detection concentration of this primer combination was calculated to be 1.56 × 10⁻⁶ copies / μL. 3 Copy / μL.

[0110] Figure 15-17 The melting curve showed a single peak, indicating the absence of nonspecific amplification and primer dimers. The standard curve obtained from the amplification curve was Y = -3.2243x + 39.484, where x is the logarithm of the initial template copy number, y represents the Ct value, and the amplification relation Rt is the coefficient of variation. 2 =0.9994, the lowest detectable concentration is 5.20×10 2 Based on the sample detection results, the effective detection concentration of this primer combination was calculated to be 3.85 × 10⁻⁶ copies / μL. 3 Copy / μL.

[0111] Figure 18-20 The melting curve showed a single peak, indicating the absence of nonspecific amplification and primer dimers. The standard curve obtained from the amplification curve was Y = -3.0896x + 37.768, where x is the logarithm of the initial template copy number, y represents the Ct value, and the amplification relation Rt is the coefficient of variation. 2 =0.9914, the lowest detectable concentration is 5.20×10 2 Based on the sample detection results, the effective detection concentration of this primer combination was calculated to be 7.54 × 10⁻⁶ copies / μL. 3 Copy / μL.

[0112] As can be seen from the above experiments, the target sequences of SEQ ID No. 2 to SEQ ID No. 4 are the most effective for application of this invention.

[0113] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A nucleic acid molecule for WNV gene detection, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID No.

4.

2. A primer combination for WNV gene detection, characterized in that, The primer combination is used to amplify the nucleic acid molecule of claim 1. The primer combination includes the upstream primer shown in SEQ ID No. 18 and the downstream primer shown in SEQ ID No.

19.

3. A kit for WNV gene detection, characterized in that, The kit includes the primer combination as described in claim 2, and PCR amplification reaction reagents.

4. The reagent kit according to claim 3, characterized in that, The kit also includes RNA extraction reagents, a positive control, and a negative control; the reaction reagents include DNA polymerase and DNA polymerase buffer.

5. The use of the nucleic acid molecule of claim 1 or the primer combination of claim 2 in the preparation of a WNV gene detection kit.

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