Fluorescence quantitative PCR primer probe set and kit for detecting avian influenza virus H3, H4 and H5 subtypes

Abstract

The present invention discloses a fluorescent quantitative PCR primer probe set and a kit for detecting H3, H4 and H5 subtypes of avian influenza virus, as well as a fluorescent quantitative PCR kit containing the primer probe set. The kit also contains fluorescent quantitative PCR reagents, ddH2O and positive plasmid standards, and can simultaneously detect whether the sample to be tested contains H3, H4 and H5 subtypes of avian influenza virus. The kit provided by the present invention has good specificity for H3, H4 and H5 subtypes of avian influenza virus; compared with the common PCR detection method, the kit provided by the present invention has higher sensitivity, and the minimum detection limit for the three subtypes of avian influenza virus is 2.1×10 2 The kit provided by the present invention also has the advantages of high stability and good repeatability. Therefore, the present invention can be used for the rapid diagnosis and detection of avian influenza virus H3, H4 and H5 subtypes, which is of great significance for the prevention and control of avian influenza virus H3, H4 and H5 subtypes.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to a fluorescent quantitative PCR primer probe set and a kit for detecting H3, H4 and H5 subtypes of avian influenza virus. Background Art

[0002] Avian influenza virus is a respiratory disease of poultry that not only causes huge economic losses to the poultry industry, but also poses a constant threat to human health. The influenza A virus gene pool in waterfowl provides all the genetic diversity required for the emergence of pandemic influenza viruses in humans, lower animals and birds. In recent years, H3, H4 and H5 subtypes of avian influenza viruses have been widely circulated in waterfowl, and infections with H3 and H5 subtypes of avian influenza viruses have been observed in humans.

[0003] Common detection methods for avian influenza virus include ELISA antibody detection, PCR, recombinase-mediated detection (RAA) and loop-mediated isothermal amplification (LAMP) detection technologies. ELISA antibody detection has poor repeatability and is interfered by autoantibodies, heterophilic antibodies, etc., and is prone to false positives; although ordinary PCR is simple and fast, it takes longer than fluorescent quantitative PCR; recombinase street detection can be rapidly amplified at a constant temperature under enzyme mediation, reducing the requirements for equipment, but there are requirements for both ends of the primer, and the primers need to be redesigned; loop-mediated Dengwen amplification is prone to false positives due to aerosol contamination caused by opening the lid during the experiment. The Chinese invention patent with application number CN104498629A discloses a dual real-time fluorescent quantitative PCR detection kit for H3N2 subtype avian influenza virus. The disadvantage of this invention is that it can only detect H3N2 subtype avian influenza virus, and cannot detect multiple avian influenza subtype viruses that may appear in waterfowl. Summary of the invention

[0004] Based on this, the present invention provides a fluorescent quantitative PCR primer probe set and a kit for detecting H3, H4 and H5 subtype avian influenza viruses, so as to achieve simultaneous detection of H3, H4 and H5 subtype avian influenza viruses.

[0005] The first aspect of the present invention provides a fluorescent quantitative PCR primer probe set for detecting H3, H4 and H5 subtypes of avian influenza virus, characterized in that it includes the following primers and probes:

[0006] H3 subtype upstream primer H3-F, which has the nucleotide sequence shown in SEQ ID NO.1;

[0007] H3 subtype downstream primer H3-R, which has the nucleotide sequence shown in SEQ ID No. 2;

[0008] H4 subtype upstream primer H4-F, which has the nucleotide sequence shown in SEQ ID NO.3;

[0009] H4 subtype downstream primer H4-R, which has the nucleotide sequence shown in SEQ ID NO.4;

[0010] H5 subtype upstream primer H5-F, which has the nucleotide sequence shown in SEQ ID NO.5;

[0011] H5 subtype downstream primer H5-R, which has the nucleotide sequence shown in SEQ ID NO.6;

[0012] H3 subtype probe H3-Probe, which has the nucleotide sequence shown in SEQ ID NO.7;

[0013] H4 subtype probe H4-Probe, which has the nucleotide sequence shown in SEQ ID NO.8;

[0014] The H5 subtype probe H5-Probe has a nucleotide sequence as shown in SEQ ID NO.9.

[0015] According to the AIV sequence published on NCBI, the present invention selects the HA genes of the domestic H3, H4 and H5 subtype avian influenza viruses in the past five years, designs three pairs of primers and three probes respectively, and uses the fluorescent quantitative Taqman probe PCR to detect related pathogens. The H3 standard curve equation is Y=-3.343lgX+39.94, and the correlation coefficient R 2 =0.997, amplification efficiency E = 99.14%; H4 standard curve equation is Y = -3.42lgX + 40.19, correlation coefficient R 2 =0.999, amplification efficiency E = 96.06%; H5 standard curve equation is Y = -3.405lgX + 39.78, correlation coefficient R 2 =0.998, amplification efficiency E = 96.65%. The minimum detection amount is 2.1×10 2 copies / μL, with high sensitivity and good specificity, and the coefficient of variation within and between batches is less than 1.5%. Therefore, the present invention can be used as a rapid, sensitive and accurate detection method in actual production and epidemic monitoring, and provide a reference for the identification and detection of H3, H4 and H5 subtype avian influenza viruses in the future.

[0016] Furthermore, the 5' ends of the H3-Probe, H4-Probe and H5-Probe are respectively labeled with fluorescent genes, and the 3' ends are respectively labeled with quenching genes.

[0017] Furthermore, the H3-Probe is labeled with the fluorescent gene FAM at the 5' end and the quenching gene BHQ1 at the 3' end; the H4-Probe is labeled with the fluorescent gene HEX at the 5' end and the quenching gene BHQ1 at the 3' end; the H5-Probe is labeled with the fluorescent gene CY5 at the 5' end and the quenching gene BHQ2 at the 3' end.

[0018] The second aspect of the present invention also claims the use of the above primer probe set in the preparation of a fluorescent quantitative PCR kit for detecting H3, H4 and H5 subtypes of avian influenza virus.

[0019] The third aspect of the present invention provides a fluorescent quantitative PCR kit for detecting H3, H4 and H5 subtypes of avian influenza virus. Specifically, the kit contains any of the primer probe sets described above.

[0020] Furthermore, the kit contains H3-F 0.4 μL, H3-R 0.4 μL, H3-Probe 0.2 μL, H4-F 0.4 μL, H4-R 0.4 μL, H4-Probe 0.2 μL, H5-F 0.2 μL, H5-R 0.2 μL and H5-Probe 0.1 μL, and the concentration of each primer and probe is 10 μM.

[0021] Furthermore, the kit also contains 10 μL of fluorescent quantitative PCR reagent, 5.5 μL of ddH2O and 2 μL of positive plasmid standard.

[0022] Furthermore, the fluorescent quantitative PCR reagent is AceQ Universal U+Probe Master Mix V2.

[0023] In one embodiment, the positive plasmid standard is prepared by the following steps:

[0024] S1: PCR amplification and purification of target fragments: Use upstream primers and downstream primers to perform conventional PCR amplification of the target fragments with H3, H4 and H5 gene templates, and purify the PCR products by gel electrophoresis; the reaction program of conventional PCR amplification is: ① 95℃ for 3min; ② 95℃ for 15s, ③ 55℃ for 20s, ④ 72℃ for 2min, ②-④35 cycles; ⑤ 72℃ for 10min.

[0025] S2: Target fragment connection: Use Tarkara's pMD18-T Vector to connect the purified product to the vector;

[0026] S3: Introduce the recombinant plasmid into the recipient bacterial cells;

[0027] S4: Screening and identification of recombinant plasmids: Select single colonies with good growth for culture, and take suspected positive bacterial liquid as a template for PCR identification;

[0028] S5: Extraction of positive plasmids: Expand the bacterial solution with the correct sequencing results in step (4) and extract the plasmid using a plasmid extraction kit.

[0029] In one embodiment, the steps of using the fluorescent quantitative PCR kit for detecting H3, H4 and H5 subtypes of avian influenza virus provided by the present invention are as follows: extracting total RNA from the sample to be tested, using the obtained total RNA as a template, using the kit provided by the present invention to perform fluorescent quantitative PCR detection, and judging whether the sample to be tested is an avian influenza virus H3, H4 or H5 subtype according to the fluorescent CT value, if the CT value is greater than 35, it is negative, and if the CT value is less than or equal to 35, it is positive.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The present invention has good specificity for H3, H4 and H5 subtype avian influenza viruses, and can simultaneously detect H3, H4 and H5 subtype avian influenza viruses widely spread in waterfowl; the kit provided by the present invention contains positive plasmid standard products, and the standard products are gradiently diluted and amplified. The results show that there is a good linear relationship between the standards of different concentrations, which is in line with the expected results; the standard products are diluted to 2.1×10 9 copies / μL-2.1×10 0 The reaction system provided by the present invention was used to detect the virus and determine the minimum detection limit. The minimum detection limits of H3, H4 and H5 subtype avian influenza viruses were all 2.1×10 2 The sensitivity of the primer probe set and the kit provided by the present invention is about 1 order of magnitude higher than that of the common PCR detection method. The real-time fluorescence quantitative PCR amplification within and between batches of the standard products was performed, and it was found that the coefficient of variation within the batch was less than 1%, and the coefficient of variation between batches was less than 1.5%. Therefore, the primer probe set and the kit provided by the present invention have high stability and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a standard curve diagram of the H3 subtype avian influenza virus fluorescence quantitative PCR in Example 4 of the present invention;

[0033] Figure 2 The standard curve of the fluorescence quantitative PCR of H4 subtype avian influenza virus in Example 4 of the present invention;

[0034] Figure 3 This is a standard curve diagram of the H5 subtype avian influenza virus fluorescence quantitative PCR in Example 4 of the present invention;

[0035] Figure 4 This is a sensitivity detection curve of fluorescent quantitative PCR of H3 subtype avian influenza virus in Example 5 of the present invention;

[0036] Figure 5 This is a sensitivity detection curve of fluorescent quantitative PCR of H4 subtype avian influenza virus in Example 5 of the present invention;

[0037] Figure 6 This is a sensitivity detection curve of fluorescent quantitative PCR of H5 subtype avian influenza virus in Example 5 of the present invention;

[0038] Figure 7 This is the common PCR electrophoresis diagram of Example 5 of the present invention;

[0039] Figure 8 This is a fluorescence quantitative PCR specific detection curve diagram of Example 6 of the present invention. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the technical field of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0042] In the present invention, "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "the first", "the second", "the third", "the fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features. In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between the minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0044] The percentage contents involved in the present invention, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing. The percentage concentrations involved in the present invention, unless otherwise specified, refer to final concentrations. The final concentration refers to the proportion of the added component in the system after the addition of the component. The temperature parameters in the present invention, unless otherwise specified, are allowed to be either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0045] Example 1, synthesis and design of primers:

[0046] According to the AIV sequence published on NCBI, the HA genes of H3, H4 and H5 subtype avian influenza viruses in China in the past five years were selected, and three pairs of primers and three probes were designed respectively. The HA gene sequences of the three subtypes of avian influenza viruses and the designed primer pairs and probe sequences are shown in Table 1, wherein the 5' end of H3-Probe is labeled with the fluorescent gene FAM, and the 3' end is labeled with the quenching gene BHQ1; the 5' end of H4-Probe is labeled with the fluorescent gene HEX, and the 3' end is labeled with the quenching gene BHQ1; the 5' end of H5-Probe is labeled with the fluorescent gene CY5, and the 3' end is labeled with the quenching gene BHQ2.

[0047] Table 1. Designed primer pairs and probes and primer pairs for HA genes of H3, H4 and H5 subtype avian influenza viruses

[0048]

[0049] Example 2, Preparation of plasmid standards:

[0050] (1) PCR amplification and purification of target fragments

[0051] Use upstream primers and downstream primers (10 μM) to amplify the target fragment with H3, H4 and H5 gene templates. The reaction procedure is: ① 95°C for 3 min; ② 95°C for 15 s, ③ 55°C for 20 s, ④ 72°C for 2 min, ②-④35 cycles; ⑤ 72°C for 10 min.

[0052] Table 2. Conventional PCR reaction system

[0053]

[0054] After the PCR program is completed, all PCR products are added to a 2% agarose gel containing Goldview and electrophoresed for 30 minutes. Under ultraviolet light, the target band is cut out and gel recovery is performed using the Thermo Fisher gel recovery kit. The Binding Buffer and Wash Buffer in step (1) are both from the kit. The specific steps are as follows:

[0055] (1-1) Use a clean scalpel or blade to cut out the gel slice containing the DNA fragment, cutting as close to the DNA as possible to minimize the gel volume. Place the gel slice in a pre-weighed 1.5 mL tube and weigh it. Record the weight of the gel slice.

[0056] (1-2) Add 1:1 volume of Binding Buffer (volume:weight, e.g., add 100 μL Binding Buffer to every 100 mg of agarose gel) to the gel slice.

[0057] (1-3) Incubate the gel mixture at 55°C for 10 minutes or until the gel slice is completely dissolved. Invert the mixing tube every few minutes to facilitate the melting process. Make sure the gel is completely dissolved. Vortex the gel mixture briefly before loading. Check the color of the solution. Yellow indicates the optimal pH for DNA binding.

[0058] (1-4) Transfer up to 800 μL of the dissolved gel solution from the above step to the GeneJET purification column. Centrifuge at 12000 rpm for 1 min. Discard the filtrate and return the column to the same collection tube.

[0059] (1-5) Add 100 μL Binding Buffer, centrifuge for 1 min, discard the filtrate, and return the column to the same collection tube. (1-6) Add 700 μL Wash Buffer to the GeneJET purification column. Centrifuge at 12000 rpm for 1 min. Discard the filtrate, and return the column to the same collection tube.

[0060] (1-7) Centrifuge the empty GeneJET purification column at 12,000 rpm for 1 minute to completely remove the residual wash buffer. (1-8) Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube. Add 40 μL of DEPC water to the center of the purification column membrane. Centrifuge at 12,000 rpm for 1 minute.

[0061] (1-9) Discard the GeneJET purification column, check its concentration, and store the purified DNA at -20°C.

[0062] (2) Connection of target fragments

[0063] The purified PCR product obtained in step (1-9) was connected to the vector with reference to the instruction manual of pMD18-T Vector of Tarkara Company. The reaction system is shown in Table 3. After the reactants shown in Table 3 are mixed, they are placed in a PCR instrument at 16°C for 4 hours and at 4°C overnight to obtain the connection product.

[0064] Table 3. Vector ligation reaction system

[0065]

[0066] (3) Introducing the recombinant plasmid into recipient bacterial cells (3-1) Add the ligation products obtained in step (2) into 50 μL top10 competent cells respectively.

[0067] (3-2) Gently mix the ligation product and competent cells, immediately place on ice for 30 min, then heat shock at 42°C for 45 s, and then immediately place on ice for 2 min.

[0068] (3-3) Add 400 μL of fresh LB liquid culture medium to each tube and incubate with slow shaking in a 37°C incubator for 1 h.

[0069] (3-4) Spread 100 μL of bacterial solution on a 1.5% (W / V) LB agar plate containing Amp (100 μg / mL) and incubate at 37°C for 12-16 h.

[0070] (4) Screening and identification of recombinant plasmids.

[0071] A single colony with good growth was picked and placed in 500 μL of LB liquid culture medium containing ampicillin (AMP), and placed in a 37°C constant temperature shaker for shaking culture for 4-6 h. The suspected positive bacterial solution was used as a template for PCR identification. The PCR identification reaction system is shown in Table 4.

[0072] Table 4. PCR identification reaction system

[0073]

[0074] PCR reaction program: ① 95℃ for 3min; ② 95℃ for 15s, ③ 55℃ for 20s, ④ 72℃ for 2min, ②-④ 35 cycles; ⑤ 72℃ for 10min. The screened positive bacterial solution was sent to Shenggong Biotechnology Co., Ltd. for sequencing.

[0075] (5) Extraction of positive plasmid

[0076] The bacterial cultures with correct sequencing results in step (4) were named 18T-H3, 18T-H4 and 18T-H5 and expanded for culture. Plasmids were extracted using the Novezan plasmid extraction kit. The specific steps are as follows:

[0077] (5-1) Cultivation of Escherichia coli: A single colony was selected from the plate culture medium and inoculated into 5 ml of LB liquid culture medium containing ampicillin (AMP) and cultured at 37°C overnight (12-16 hours).

[0078] (5-2) Take 5 ml of overnight culture solution, centrifuge at 12000 rpm for 5 minutes, and discard the supernatant.

[0079] (5-3) Add 250 μL of Buffer P1 to the centrifuge tube containing the bacterial pellet and mix well using a pipette or vortex.

[0080] (5-4) Add 250 μL of Buffer P2 to step (5-3) and mix gently by inverting up and down 8-10 times to fully lyse the bacteria.

[0081] (5-5) Add 350 μL of Buffer P3 to step (5-4) and immediately gently invert the solution 8-10 times to completely neutralize Buffer P2. A white flocculent precipitate should appear. Centrifuge at 13,000×g for 10 min.

[0082] (5-6) Place the FastPure DNA Mini Columns adsorption column in a 2 ml collection tube. Carefully transfer the supernatant from step (5-5) to the adsorption column using a pipette, taking care not to aspirate the precipitate, and centrifuge at 13,000 × g for 1 min. Pour off the waste liquid in the collection tube and place the adsorption column back into the collection tube.

[0083] (5-7) Add 500 μl Buffer PW1 to the adsorption column. Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds. Discard the waste liquid and return the adsorption column to the collection tube.

[0084] (5-8) Add 600 μL Buffer PW2 to the adsorption column. Centrifuge at 13,000×g for 1 min. Discard the waste liquid and return the adsorption column to the collection tube.

[0085] (5-9) Repeat step (5-8).

[0086] (5-10) Place the adsorption column in a new sterilized 1.5 ml centrifuge tube. Add 30-100 μL Elution Buffer to the center of the column membrane. Let stand at room temperature for 2 minutes, and centrifuge at 13,000 × g for 1 minute to elute the DNA.

[0087] (5-11) Discard the adsorption column and store the DNA product at -20°C to prevent DNA degradation.

[0088] The plasmid concentration was measured using an ultra-micro fluorescence spectrophotometer and the gene copy number (copies / μL) was 6.02×10 23 ×Plasmid concentration (ng / μL)×10 -9 / [plasmid size (bp) × 660], and calculate its gene copy number.

[0089] Example 3: Detection of H3, H4 and H5 influenza viruses using primers and probes of the present invention

[0090] (1) RNA extraction (AxyPrepTM Body Fluid Viral DNA / RNA Miniprep kit)

[0091] (1-1) Centrifuge at 12000×g for 5 min, collect 200 μL of sample and transfer it into a 1.5 ml centrifuge tube.

[0092] (1-2) Add 200 μL of Buffer VL, vortex to mix well, and let stand at room temperature for 5 min.

[0093] (1-3) Add 75 μL of Buffer VN, vortex to mix, and centrifuge at 12,000 × g for 5 min.

[0094] (1-4) Transfer the supernatant to a new 2 ml centrifuge tube (provided in the kit), add 300 μL of isopropanol (1% glacial acetic acid), invert upside down 6-8 times, and mix gently.

[0095] (1-5) Place the preparation tube in a 2 ml centrifuge tube (provided in the kit, can hold up to 800 μL), transfer the mixed solution in step 4 into the preparation tube in two batches, add 800 μL in the first batch, centrifuge at 6000×g for 1 min, and discard the filtrate. Transfer the remaining 750 ul and centrifuge at 6000×g for 1 min.

[0096] (1-6) Discard the filtrate, place the preparation tube back into a 2 ml centrifuge tube, add 500 μL Buffer W1A, let stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min.

[0097] (1-7) Discard the filtrate, place the preparation tube back into a 2 ml centrifuge tube, add 800 μL Buffer W2, and centrifuge at 12,000 × g for 1 min. (1-8) Place the preparation tube back into a 2 ml centrifuge tube, and centrifuge at 12,000 × g for 1 min.

[0098] (1-9) Place the preparation tube in a clean 1.5 ml centrifuge tube (provided in the kit), add 30 μL of DEPC water to the center of the preparation tube membrane, and let it stand at room temperature for 1 minute. Centrifuge at 12000×g for 1 minute to elute the RNA.

[0099] (2) Reverse transcription III 1stStrand cDNA synthesis Kit)

[0100] (2-1) RNA template denaturation:

[0101] The RNA solution obtained in step (1-9) was placed in a 65°C water bath in a PCR instrument for 5 min, and then quickly placed on ice for 2 min.

[0102] (2-2) Genome removal

[0103] Carefully mix the following solutions, centrifuge briefly, and incubate in a PCR machine at 42°C for 2 min.

[0104] 10 μL of the solution obtained in step (1-9) 5×gDNA wiper Mix 5.5μL

[0105] (2-3) Use a pipette to gently mix the following solution.

[0106]

[0107] (2-4) The above mixed solution is placed in a PCR instrument for PCR amplification. The reaction procedure is as follows:

[0108]

[0109] The amplified products were stored in a -80°C refrigerator.

[0110] (3) Fluorescence real-time PCR of H3, H4 and H5 subtype avian influenza viruses

[0111] The reaction system is as follows:

[0112]

[0113]

[0114] The reaction procedure was: ① 37°C for 2 min; ② 95°C for 5 min, ③ 95°C for 10 s, ④ 60°C for 30 s, and steps ③-④ were repeated for 40 cycles.

[0115] (4) Result determination

[0116] Negative: If the Ct value of the test sample is greater than 35, it is negative.

[0117] Positive: The sample is positive if the Ct value is less than or equal to 35.

[0118] Example 4: Construction of Fluorescence Quantitative PCR Reaction System and Standard Curve

[0119] The 18T-H3, 18T-H4 and 18T-H5 plasmids were mixed at a ratio of 1:1:1 and serially diluted to obtain 2.1×10 9 copies / μL-2,1×10 0 copies / μL 10 dilutions of the standard, 2.1×10 8 copies / μL-2.1×10 2 1000 copies / μL was used as the reaction template, and each dilution was repeated 3 times and a negative control was established. The reaction system is shown in Table 5, and the results are shown in Table 5. Figure 1 As shown, the H3 standard curve equation is Y = -3.343lgX + 39.94; the correlation coefficient R 2 =0.997; amplification efficiency E = 99.14%, H4 standard curve equation is Y = -3.42lgX + 40.19; correlation coefficient R 2 =0.999; amplification efficiency E = 96.06%, H5 standard curve equation is Y = -3.405lgX + 39.78; correlation coefficient R 2 =0.998; amplification efficiency E=96.65%, and the results showed that there was a good linear relationship between standards of different concentrations, where the X-axis was the copy number of the plasmid standard and the Y-axis was the cycle threshold, which was consistent with the expected results.

[0120] Table 5. Fluorescence quantitative PCR reaction system

[0121]

[0122]

[0123] Reaction procedure: ① 37°C for 2 min; ② 95°C for 5 min, ③ 95°C for 10 s, ④ 60°C for 30 s, steps ③-④ were repeated for 40 cycles.

[0124] Example 5: Sensitivity experiment

[0125] 2.1×10 7 copies / μL-2.1×10 0 The plasmid standard with 8 dilutions of copies / μL was used as a template, and amplification was performed according to the fluorescent quantitative PCR reaction system provided in Example 3. At the same time, the same template was amplified by ordinary PCR, and the lowest detection limits of the real-time fluorescent quantitative PCR and ordinary PCR were determined and the sensitivity of the two methods was compared. Figure 2 As shown, the fluorescence quantitative PCR reaction system provided in Example 3 can detect the minimum detection amount of positive standard products of 2.1×10 2 copies / μL. Figure 3 As shown in the figure, lane M is DL500 DNA Marker; lanes 2-10 are 2.1×10 9 copies / μL-2.1×10 0 The sample has 200 copies / μL; Lane 1 is negative control. It can be seen that the minimum detection amount of ordinary PCR is 2.1×10 3 In comparison, the real-time fluorescence quantitative PCR detection method established in Example 3 is about one order of magnitude higher in sensitivity than the common PCR detection method.

[0126] Example 6: Specificity Experiment

[0127] The established real-time fluorescence quantitative PCR method was used to amplify the H1N1, H3N8, H4N6, H5N6, H5N8, H6N6, H7N9, H9N2 subtype avian influenza viruses, MDRV (Muscovy duck reovirus), NDRV (novel duck reovirus), MDV (Marek's disease virus), AILT (infectious laryngotracheitis), CIAV (infectious anemia virus), IBV (infectious bronchitis virus) and NDV (Newcastle disease virus) pathogens to verify the specificity of the fluorescence quantitative PCR reaction system provided in Example 3. The results are as follows. Figure 4 As shown, it is shown that the fluorescent quantitative PCR reaction system provided in Example 3 has specific amplification for H3, H4 and H5 subtype avian influenza viruses, and has no fluorescent signals for H1N1, H6N6, H7N9, H9N2 subtype AIV, AILT, CIAV, IBV and NDV, but the Ct values ​​of MDRV, NDRV and MDV are all after 35. Therefore, the method is positive when the Ct value is less than or equal to 35, and negative when it is greater than 35.

[0128] Example 7, Repeatability Test

[0129] The real-time fluorescence quantitative PCR amplification was performed on three batches of plasmids, and three batches were repeated. The changes in Ct values ​​were compared to verify the stability of the method. The stability of the method was evaluated by the intra-batch and inter-batch coefficients of variation. The results are shown in Table 6. The intra-batch coefficient of variation was less than 1%, and the inter-batch coefficient of variation was less than 1.5%, indicating good repeatability.

[0130] Table 6. Results of fluorescence quantitative PCR repeatability test

[0131]

[0132] The above-mentioned embodiments only express the preferred implementation of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A fluorescent quantitative PCR primer probe set for detecting H3, H4 and H5 subtypes of avian influenza virus, characterized in that: Includes the following primer pairs and probes: H3 subtype upstream primer H3-F, the nucleotide sequence of which is shown in SEQ ID NO.1; H3 subtype downstream primer H3-R, the nucleotide sequence of which is shown in SEQ ID No. 2; H3 subtype probe H3-Probe, whose nucleotide sequence is shown in SEQ ID NO.3; H4 subtype upstream primer H4-F, the nucleotide sequence of which is shown in SEQ ID NO.4; H4 subtype downstream primer H4-R, the nucleotide sequence of which is shown in SEQ ID NO.5; H4 subtype probe H4-Probe, whose nucleotide sequence is shown in SEQ ID NO.6; H5 subtype upstream primer H5-F, the nucleotide sequence of which is shown in SEQ ID NO.7; H5 subtype downstream primer H5-R, the nucleotide sequence of which is shown in SEQ ID NO.8; The nucleotide sequence of H5 subtype probe H5-Probe is shown in SEQ ID NO.

9.

2. A fluorescent quantitative PCR primer probe set for detecting H3, H4 and H5 subtypes of avian influenza virus according to claim 1, characterized in that: The 5' ends of the H3-Probe, H4-Probe and H5-Probe are respectively labeled with fluorescent genes, and the 3' ends are respectively labeled with quenching genes.

3. A fluorescent quantitative PCR primer probe set for detecting H3, H4 and H5 subtypes of avian influenza virus according to claim 2, characterized in that: The H3-Probe 5' end is labeled with the fluorescent gene FAM, and the 3' end is labeled with the quenching gene BHQ1; the H4-Probe 5' end is labeled with the fluorescent gene HEX, and the 3' end is labeled with the quenching gene BHQ1; the H5-Probe 5' end is labeled with the fluorescent gene CY5, and the 3' end is labeled with the quenching gene BHQ2.

4. Use of the primer probe set according to any one of claims 1 to 3 in preparing a fluorescent quantitative PCR kit for detecting H3, H4 and H5 subtypes of avian influenza virus.

5. A fluorescent quantitative PCR kit for detecting H3, H4 and H5 subtypes of avian influenza virus, characterized in that: A primer probe set comprising any one of claims 1 to 3.

6. The fluorescent quantitative PCR kit according to claim 5, characterized in that: It contains H3-F 0.4μL, H3-R 0.4μL, H3-Probe 0.2μL, H4-F 0.4μL, H4-R 0.4μL, H4-Probe 0.2μL, H5-F 0.2μL, H5-R 0.2μL and H5-Probe 0.1μL, and the concentration of each primer and probe is 10μM.

7. The fluorescent quantitative PCR kit according to claim 6, characterized in that: It also contains 10 μL of fluorescent quantitative PCR reagent, 5.5 μL of ddH2O and 2 μL of positive plasmid standard; the fluorescent quantitative PCR reagent is AceQ Universal U+Probe Master Mix V2.

8. The fluorescent quantitative PCR kit according to claim 7, characterized in that: The positive plasmid standard is prepared by the following steps: S1: PCR amplification and purification of target fragments: Use upstream primers and downstream primers to perform conventional PCR amplification of the target fragments with H3, H4 and H5 gene templates, and purify the PCR products by gel electrophoresis; S2: Target fragment connection: Use pMD18-T Vector to connect the purified product to the vector; S3: Introduce the recombinant plasmid into the recipient bacterial cells; S4: Screening and identification of recombinant plasmids: Select single colonies with good growth for culture, and take suspected positive bacterial liquid as a template for PCR identification; S5: Extraction of positive plasmids: Expand the bacterial solution with the correct sequencing results in step S4 and use a plasmid extraction kit to extract the plasmids.

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