A rapid detection method for avian influenza virus

Abstract

The application belongs to the technical field of nucleic acid detection methods, and particularly relates to a rapid detection method of avian influenza virus, which comprises using a primer pair and a probe designed for a conserved region of M gene of AIV, and amplifying amplification reagents containing a sample to be detected based on super-fast qPCR technology, wherein the sequence of a forward primer in the primer pair is 5'-GGCCCCCTCAAAGCCGA-3'; the sequence of a reverse primer is 5'-CGTCTACGCTGCAGTCC-3'; and the sequence of the probe is ROX-CACTGGGCACGGTGAGCGT-BHQ2. The method of the application can rapidly and accurately detect whether the avian influenza virus exists in the sample to be detected and the content of the avian influenza virus, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection methods, and particularly relates to a rapid detection method for avian influenza virus. Background Technology

[0002] Avian influenza virus ( Avian Influenza Virus AIV is a virus belonging to the family Orthomyxoviridae (AIV). Orthomyxoviridae Influenza A virus (AIV) is an RNA virus that primarily infects birds, but it also has the potential to spread across species to humans and other mammals. Statistics from the World Health Organization (WHO) show that the mortality rate for human infection with H5N1 is as high as 53%, and that of H7N9 is 39%. Although only about 50 mild cases of the H9 subtype have been reported, serological studies indicate that the antibody positivity rate in occupationally exposed populations is 3%-7%, posing a serious threat to global public health and the poultry farming industry. Furthermore, the lag in monitoring viral mutations using traditional surveillance technologies may delay the golden window for prevention and control. Therefore, developing rapid, accurate, and on-site detection-suitable AIV diagnostic technologies has become a core requirement for breaking the virus transmission chain and preventing the escalation of the epidemic.

[0003] Current rapid on-site detection technologies for AIV mainly include immunochromatographic strips (such as antigen detection), loop-mediated isothermal amplification (LAMP), and portable real-time quantitative PCR (qPCR). Although these technologies have shortened the detection time (to approximately 30-60 minutes), significant bottlenecks remain: immunochromatography has low sensitivity (>10). 3 Virus copies / μL are insufficient to identify early or low-viral-load infections; LAMP technology relies on complex primer design and is susceptible to non-specific amplification interference; while conventional qPCR has high sensitivity, it requires cold-chain transport of liquid reagents, time-consuming nucleic acid extraction steps, and bulky temperature control equipment, making it difficult to meet the needs of point-of-care testing (POCT) in remote areas or emergency scenarios. Furthermore, the low-temperature storage and transportation requirements of nucleic acid amplification reagents further increase the application cost in resource-scarce areas. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid detection method for avian influenza virus. This method can quickly and accurately detect whether avian influenza virus is present in a sample and the amount of avian influenza virus, and has good application prospects.

[0005] This invention provides a rapid detection method for avian influenza virus. The method includes using primer pairs and probes designed for the conserved region of the M gene of AIV, and amplifying amplification reagent containing the sample to be tested based on ultra-rapid qPCR technology. The sequence of the forward primer in the primer pair is: 5'-GGCCCCCTCAAAGCCGA-3'; the sequence of the reverse primer is: 5'-CGTCTACGCTGCAGTCC-3'; and the sequence of the probe is ROX-CACTGGGCACGGTGAGCGT-BHQ2.

[0006] In some embodiments of the present invention, the concentration of the primer pair in the amplification reagent is 0.2 to 0.8 μmol / L; preferably 0.6 to 0.8 μmol / L; more preferably 0.8 μmol / L.

[0007] In some embodiments of the present invention, the concentration of the probe in the amplification reagent is 0.3 to 0.5 μmol / L; preferably 0.4 to 0.5 μmol / L; more preferably 0.5 μmol / L.

[0008] In some embodiments of the present invention, the amplification reagent further includes a PCR buffer; preferably, the PCR buffer includes Tris, KCl, MgCl2, dNTPs, dUTPs, etc.; more preferably, the PCR buffer includes 100 mmol / L Tris, 50 mmol / L KCl, 5 mmol / L MgCl2, 25 mmol / L dNTPs, and 100 mmol / L dUTPs; even more preferably, the PCR buffer accounts for 48% to 50% of the volume of the amplification reagent, preferably 49%.

[0009] In some embodiments of the present invention, the amplification reagent further includes a DNA polymerase; preferably, the DNA polymerase is Taq DNA polymerase, and / or, the concentration of the DNA polymerase in the amplification reagent is 0.18–0.22 U / µL, preferably 0.2006 U / µL.

[0010] In some embodiments of the present invention, the amplification reagent further includes reverse transcriptase; preferably, the concentration of the reverse transcriptase in the amplification reagent is 8-8.5 U / µL, more preferably 8.264 U / µL.

[0011] In some embodiments of the present invention, the amplification reagent further includes UDG enzyme; preferably, the concentration of UDG enzyme in the amplification reagent is 0.4 to 0.45 U / µL, more preferably 0.4132 U / µL.

[0012] In some embodiments of the present invention, the amplification reagent further includes an RNase inhibitor; preferably, the concentration of the RNase inhibitor in the amplification reagent is 16-17 U / µL, more preferably 16.528 U / µL.

[0013] In some embodiments of the present invention, the amplification reagent consists of a sample to be tested and a PCR detection premix.

[0014] In some embodiments of the present invention, the PCR detection premix includes the primer pairs, probes, DNA polymerase, reverse transcriptase, PCR buffer, UDG enzyme, and RNase inhibitor described above.

[0015] In some embodiments of the present invention, the rapid detection method includes reconstituted a lyophilized PCR detection premix with ultrapure water and then mixing it with the sample to be tested to form an amplification reagent; preferably, the ultrapure water is DEPC water.

[0016] In some embodiments of the present invention, the lyophilized PCR detection premix is ​​prepared by a method comprising the following steps: adding a lyophilization protectant to the PCR detection premix; pre-freezing the PCR detection premix containing the lyophilization protectant at -80°C for 2 hours, and then performing vacuum freeze-drying: first stage drying at -40°C / 0.1 mbar for 12 hours, and second stage drying at 25°C / 0.01 mbar for 3 hours.

[0017] In some embodiments of the present invention, the lyophilization protectant includes trehalose, PEG20000 and mannitol; preferably, the content of trehalose in the PCR detection premix containing the lyophilization protectant is 8 wt%, and / or the content of PEG20000 in the PCR detection premix containing the lyophilization protectant is 3 wt%, and / or the content of mannitol in the PCR detection premix containing the lyophilization protectant is 1.5 wt%.

[0018] In some embodiments of the present invention, the annealing temperature for amplification is 58°C to 62°C; preferably 58°C to 60°C; more preferably 58°C.

[0019] In some embodiments of the present invention, the amplification program is as follows: 50℃ for 2 min; 95℃ for 30 s, 95℃ for 10 s, 58~62℃ for 30 s; for a total of 40 cycles.

[0020] In some embodiments of the present invention, the amplification program is as follows: 50℃ for 2 min; 95℃ for 30 s, 95℃ for 10 s, 58℃ for 30 s; for a total of 40 cycles.

[0021] In some embodiments of the present invention, the amplification is performed using an ultra-fast qPCR amplification instrument.

[0022] In some embodiments of the present invention, the rapid detection method further includes extracting AIV nucleic acid from the sample to be tested using a direct-amplification nucleic acid release agent before amplification; preferably, the method for extracting AIV nucleic acid includes mixing the sample to be tested with the direct-amplification nucleic acid release agent and heating at 95°C for 3 minutes, followed by centrifugation to collect the supernatant; more preferably, the direct-amplification nucleic acid release agent includes Tris-HCl buffer, EDTA, NaCl, and / or the centrifugation conditions include centrifugation at 3000×g for 1 minute.

[0023] In some embodiments of the present invention, the method for extracting AIV nucleic acid further includes vortexing the mixture before heating; preferably, the treatment time is 10 seconds.

[0024] In some embodiments of the present invention, the rapid detection method further includes extracting AIV from the sample to be tested using a magnetic bead method before extracting AIV nucleic acid; preferably, AIV is extracted using a magnetic bead method viral RNA / DNA nucleic acid extraction kit.

[0025] The beneficial effects of this invention are: This invention provides a rapid detection method for avian influenza virus. The method utilizes specific primer pairs and probes designed based on the conserved region of the AIV M gene, exhibiting high specificity and accurately detecting the presence and concentration of avian influenza virus in test samples. Based on ultra-rapid qPCR technology, this rapid detection method can complete detection within 30 minutes, with intra- and inter-batch coefficients of variation (CV) as low as 0.5%, and the detection results are virtually identical to those of traditional qPCR, demonstrating extremely high accuracy. When using an optimized amplification reaction system and procedure, this rapid detection method enables extraction-free direct amplification of AIV nucleic acid, achieving a detection sensitivity of 3.15 copies / μL, and exhibiting no cross-reactivity with other common avian pathogens. Furthermore, this invention proposes a method for detection by reconstituted lyophilized PCR detection premix with ultrapure water and then mixing it with the test sample to form the amplification reagent. This eliminates the dependence of PCR detection reagents on a cold chain, and the lyophilized PCR detection premix can be stably stored at room temperature for 12 months. This rapid detection method is expected to be applicable to on-site detection of avian influenza virus in various scenarios, including farms, ports, and primary healthcare institutions. Attached Figure Description

[0026] Figure 1 This represents the Ct values ​​obtained using primers and probes F1-R1-P1 and F2-R2-P2; Figure 2 This indicates the Ct values ​​obtained from the RT-qPCR reaction system and screening procedure; Figure 3 This indicates the results obtained from a specificity test; Figure 4 This indicates the results obtained from the sensitivity test; Figure 5 This indicates the results obtained from the precision test; Figure 6 This section compares the single-test time and single-test cost of the POCT method established in this invention with those of the traditional qPCR detection method. Figure 7 This indicates the results of the performance evaluation of the lyophilized PCR detection premix; Figure 8 This indicates the stability assessment results of the lyophilized PCR detection premix; Figure 9 This section provides a brief overview of the application implementation and the test results. Detailed Implementation

[0027] To further illustrate the present invention, the rapid detection method for avian influenza virus provided by the present invention will be described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] 1. Materials, reagents, and instruments The viral magnetic bead DNA / RNA extraction kit was purchased from Sairuisi Biotechnology (Jilin) ​​Co., Ltd. (Changchun, Jilin).

[0029] NaOH, Tris-HCl (pH = 8.0), 1 mmol EDTA, and SYBR Green dye mixture were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0030] Hifair ® The V Multiplex One Step RT-qPCR Probe Kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0031] The portable real-time quantitative PCR instrument Cs7 was purchased from Chengdu Peak Biotechnology Co., Ltd.

[0032] The ABI 7500 fluorescence PCR instrument was purchased from Thermo Fisher Scientific, Waltham, MA, USA.

[0033] Commercial avian influenza virus vaccine (H9 subtype JY strain, virulence titer ≥2×10⁻⁶) 7.3 EID 50The H5N6 subtype H5-Re13 strain, the H5N8 subtype H5-Re14 strain, and the H7N9 subtype H7-Re4 strain were purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.

[0034] 2. Preparation of viral nucleic acid Nucleic acid was extracted from avian influenza virus (AIV), porcine reproductive and respiratory syndrome virus (PRRSV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Marek's disease virus (MDV), infectious bursal disease virus (IBDV), chicken anemia virus (CAV), infectious laryngotracheitis virus (ILTV), egg drop syndrome virus (EDSV), and pullorum disease virus (PDV) using a magnetic bead method. Store at 80℃.

[0035] Viral nucleic acid was rapidly extracted using a direct-amplification nucleic acid release agent: 100 μL of avian influenza virus vaccine was mixed with 60 μL of nucleic acid release agent (10 mmol Tris-HCl buffer, 2 mmol EDTA, 0.2 mmol NaCl), vortexed for 10 seconds, heated at 95°C for 3 minutes, and centrifuged at 3000×g for 1 minute to remove impurities. The supernatant was used directly as a qPCR template. The extracted nucleic acid was then... Store at 80℃.

[0036] 3. Primer pair and probe design Download the conserved region sequence of the AIV M gene: Download the conserved region sequence of the avian influenza virus M gene from the GenBank database (GenBank accession numbers: MK463937, MG366926, MG366918, LC053495, MG366921, MG366924, MG366910, MG366929, AB916686, LC656336, MG366935, LC316734, MG366915, MG366927, DQ525415, EF070737, KF435052, MG366940, MG366941, AB937724, MW265186, MW265182, KF383481, MH375353, MH375877, MW314797). Two primer pairs and probes were designed based on this conserved region for screening. In addition, using the published whole genome sequences of representative PRRSV-1 and PRRSV-2 strains, a primer-probe pair was designed targeting the conserved region of the porcine reproductive and respiratory syndrome virus (PRRSV) M gene as an internal control gene. The primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd. The sequences of the primers and probes are shown in Table 1.

[0037] Table 1. Primer pairs and probe sequences

[0038] 4. Primer pair and probe screening Using AIV nucleic acid (5 mg / L) as a template, and with a blank control ddH2O, qPCR amplification was performed using the TaqGMan probe method with two sets of primers and probes designed in Table 1, F1-R1-P1 and F2-R2-P2, to screen for the primers and probes with the best amplification efficiency and sensitivity. By comparing the amplification curves, primers and probes with a higher amplification curve plateau, a smaller Ct value, and an "S"-shaped amplification curve were selected. The PCR amplification reagent was 25 μL, and the specific components and their amounts are shown in Table 2. The reaction program was set as follows: 50℃ for 2 min; 95℃ for 30 s, 95℃ for 10 s, 60℃ for 30 s; for a total of 40 cycles. The Ct values ​​obtained using these two sets of primers and probes, F1-R1-P1 and F2-R2-P2, are shown below. Figure 1 As shown, from Figure 1 As can be seen, compared with primer F2-P2-R2, primer F1-P1-R1 exhibits a lower Ct value and a higher plateau phase during amplification. This indicates that F1-P1-R1 has significant advantages in amplification efficiency and detection sensitivity. Therefore, F1-P1-R1 was selected for subsequent experimental studies.

[0039] Table 2 RT-qPCR reaction system

[0040] 5. RT-qPCR reaction system and screening procedure The RT-qPCR reaction system in Table 2 was used in conjunction with a portable real-time PCR system to screen primer concentrations, probe concentrations, and annealing temperatures (reaction program: 50℃ 2min; 95℃ 30s, 95℃ 10s, 60℃ 30s; 40 cycles in total).

[0041] With 3.15×10 7 3.15×10 6 3.15×10 5 A standard plasmid of avian influenza virus M gene (AIV-M) of 3.12 × 10⁶ copies / μL, and 3.12 × 10⁶ copies / μL. 6The standard plasmid containing the porcine reproductive and respiratory syndrome virus M gene (PRRSV-M) at a concentration of copies / μL was used as the DNA template. First, primer concentration gradients were set to 0.2, 0.4, 0.6, and 0.8 μmol / L (probe concentration fixed at 0.2 μmol / L, PRRSV primer concentration maintained at 0.4 μmol / L, and PRRSV probe concentration maintained at 0.1 μmol / L). After determining the optimal primer concentrations, the probe concentration gradient was adjusted to 0.2, 0.3, 0.4, and 0.5 μmol / L for further screening. Finally, annealing temperatures were selected at three gradients: 58°C, 60°C, and 62°C. Each experiment was repeated in triplicate. The screening results are shown below. Figure 2 As shown, Figure 2 The results showed that for different concentrations of amplification template, 0.8 mol / L primers and 0.5 μmol / L probes exhibited the lowest Ct values ​​and the highest amplification efficiencies, with 58℃ being the optimal annealing temperature for the best amplification effect. Based on these experimental results, the optimal reaction system and program for AIV RT-qPCR are: primer concentration 0.8 µmol / L, probe concentration 0.5 µmol / L, and annealing temperature 58℃.

[0042] 6. Performance evaluation of RT-qPCR for avian influenza virus Using primers and probes F1-R1-P1, and based on the selected RT-qPCR reaction system and procedure, the analytical performance of a portable real-time PCR device in the quantitative detection of avian influenza virus (AIV) via reverse transcription was systematically evaluated. The experimental design included three key validation modules: specificity analysis using a multiplex pathogen detection platform, sensitivity determination using serially diluted templates, and statistical validation of precision based on repeatability experiments. To objectively evaluate the detection efficiency of the portable device, parallel detections were performed simultaneously using a traditional benchtop real-time PCR system throughout the experiment.

[0043] Specificity assays: To verify the specificity of this detection method, we used primers and probes F1-R1-P1 to amplify the nucleic acids of other common pathogens affecting chickens using the selected RT-qPCR reaction system and program. These included Infectious Bronchitis Virus (IBV), Newcastle Disease Virus (NDV), Marek's Disease Virus (MDV), Infectious Bursal Disease Virus (IBDV), Chicken Anemia Virus (CAV), Egg Drop Syndrome Virus (EDSV), Phocine Distemper Virus (PDV), and Infectious Laryngotracheitis Virus (ILTV). Furthermore, genomic DNA from chickens and humans was analyzed to further validate the specificity of the detection method. Among these, a positive plasmid (3.15 × 10⁻⁶) was used. 7 (copies / μL) was used as a positive control, and dd H2O was used as a negative control.

[0044] Specific test results such as Figure 3 As shown, by Figure 3 It was observed that only the amplification template containing AIV RNA exhibited a typical "S" amplification curve during the amplification process. Other amplification templates containing nucleic acids of common chicken susceptible viruses (IBV, NDV, MDV, IBDV, CAV, ILTV, EDSV, PDV) and the negative control did not produce non-specific amplification signals (Ct value > 35). Simultaneous detection using a conventional benchtop quantitative PCR system yielded results consistent with the POCT results established in this invention. Therefore, the established AIV RT-qPCR exhibits extremely high specificity.

[0045] Sensitivity test: To determine the limit of detection (LOD) of the detection method, a concentration range of 3.15 × 10⁻⁶ was used. 8 Up to 3.15×10 0 Using AIV-M gene standard plasmids of copies / μL as templates, 10-fold serial dilutions were employed. Standard curves were established to evaluate the amplification efficiency of RT-qPCR, with each experiment performed in triplicate.

[0046] Sensitivity test results as follows Figure 4 As shown, from Figure 4 It can be seen that when the plasmid concentration is 3.15 × 10⁻⁶, 0The portable device could still stably detect a positive signal even at a concentration of copies / μL, thus determining the limit of detection (LOD) to be 3.15 × 10⁻⁶. 0 copies / μL. Simultaneous detection using a traditional benchtop quantitative PCR system yielded results consistent with the POCT results established in this invention. The standard curve showed good linearity (0.998), and the amplification efficiency reached 90.969%, meeting the international nucleic acid quantification standard (MIQE guidelines).

[0047] Precision test: To assess the reproducibility of the assay, three different concentrations (3.15 × 10⁻⁶) were used. 7 3.15×10 6 and 3.15×10 5 RT-qPCR amplification was performed using AIV-M gene standard plasmid (copies / μL) as template. In intra-assay reproducibility experiments, each template concentration was amplified three times using the same batch of RT-qPCR reaction mixture. In inter-assay reproducibility experiments, five batches of RT-qPCR reaction mixture were prepared weekly for five consecutive weeks, and the three template concentrations were tested. Furthermore, the experiments involved three different operators, three batches of reagents, and three brands of qPCR instruments (Xi'an Tianlong Gentier 96R, ABI 7500, and ABI StepOne Plus). The coefficient of variation (CV) was defined as follows: CV = (standard deviation ÷ mean) × 100%.

[0048] Precision test results as follows Figure 5 As shown, from Figure 5 It can be seen that under three different amplification concentration conditions (3.15×10), 7 3.15×10 6 and 3.15×10 5 The CVs for intra- and inter-batch repeatability tests were both less than 0.5% (copies / µL). Simultaneous detection using a conventional benchtop quantitative PCR system yielded results consistent with those established in this invention for point-of-care testing (POCT).

[0049] The above results demonstrate that the AIV RT-qPCR detection method established in this invention has good specificity, sensitivity, and reproducibility.

[0050] 7. Cost assessment of RT-qPCR for avian influenza virus A comparative study was conducted on the single-test time and cost of the POCT method established in this invention and the traditional qPCR detection method. The results are as follows: Figure 6As shown, the POCT method established by this invention can complete the detection within 30 minutes, and the cost of a single detection is only 32.04 yuan, which greatly saves the time and cost of on-site detection.

[0051] 8. Verification of the lyophilization effect of PCR detection premix solution PCR detection premix lyophilization method: According to Table 2, prepare 25 μL of PCR detection premix according to the required amounts of all components except nucleic acid template RNA (primer concentration 0.8 µmol / L, probe concentration 0.5 µmol / L, no nucleic acid template RNA added, ribonuclease-free H2O added directly after adding lyophilization protectant to 25 μL), and add 8 wt% trehalose, 3 wt% PEG20000 and 1.5 wt% mannitol as lyophilization protectants; after pre-freezing the PCR detection premix containing lyophilization protectants at -80℃ for 2 hours, perform vacuum freeze-drying: first stage -40℃ / 0.1 mbar drying for 12 hours, second stage 25℃ / 0.01 mbar drying for 3 hours.

[0052] Prepare a sample of RNA containing nucleic acid template (3.15 × 10⁻⁶) according to the description in Table 2. 6 The reaction system containing copies / μL of AIV-M gene standard plasmid was used as a control group for evaluating the detection performance of the subsequent lyophilized PCR detection premix.

[0053] Performance evaluation of lyophilized PCR detection premix: Add 20 μL of DEPC water to the lyophilized PCR detection premix, then transfer the solution to a PCR reaction tube and add 5 μL of nucleic acid template RNA (3.15 × 10⁻⁶). 6 (Copies / μL of AIV-M gene standard plasmid). Simultaneously, tests were performed on samples at three concentrations: high (Ct < 25), medium (25 ≤ Ct ≤ 30), and low (Ct > 30). In the same PCR assay, if the difference in Ct values ​​between the experimental group (using lyophilized PCR detection premix) and the control group for the same sample is ΔCt < 1, it indicates that the activity of the lyophilized PCR detection premix is ​​not significantly affected. Conversely, if ΔCt ≥ 1, it indicates that the activity of the lyophilized PCR detection premix has been affected; the larger the ΔCt value, the more severe the effect; ΔCt ≥ 10 indicates that the lyophilized PCR detection premix has been inactivated.

[0054] The Ct values ​​of the experimental group's reaction system (composed of lyophilized PCR detection premix and nucleic acid template RNA) and the control group's reaction system when detecting the same sample are as follows: Figure 7 As shown, from Figure 7 It can be seen that the Ct values ​​of the two are similar under high, medium and low template concentrations, indicating that the lyophilized PCR detection premix used in the experimental group has good detectability.

[0055] 9. Stability analysis of lyophilized PCR detection premix solution To evaluate the stability of the lyophilized PCR detection premix, the lyophilized PCR detection premix was prepared according to the lyophilization method described above. The lyophilized PCR detection premix was then stored at room temperature (18-25℃), 37℃, 56℃, and 80℃, respectively. Samples were periodically taken and reconstituted with 25 μL of DEPC water, and the activity was monitored. The results are as follows: Figure 8 As shown.

[0056] from Figure 8 As shown in the upper left figure, after 360 days of storage at room temperature, the ΔCt of the lyophilized PCR detection premix for high, medium, and low concentration samples was less than 1, indicating that the activity was essentially unaffected. Figure 8 As shown in the upper right figure, when the lyophilized PCR detection premix was stored at 37°C for 100 days, the activity of the premix significantly decreased, with ΔCt values ​​of 4.32, 4.80, and 5.59 for high, medium, and low concentration samples, respectively. Figure 8 As shown in the lower left figure, when the lyophilized PCR detection premix was stored at 56℃ for 70 days, the ΔCt values ​​for high, medium, and low concentration samples were 4.82, 5.61, and 6.19, respectively, and the low concentration samples were essentially undetectable. From... Figure 8 As shown in the lower right figure, when the lyophilized PCR detection premix was stored at 80℃ for one day, the ΔCt values ​​for high, medium, and low concentration samples all exceeded 1, indicating that the activity of the lyophilized PCR detection premix was significantly affected. These experimental results demonstrate that the lyophilized PCR detection premix of this invention can be stored for at least 360 days at room temperature and approximately 100 days at 37℃, effectively meeting the stability requirements of conventional reagents.

[0057] Application Examples To evaluate the detection capability of the method of the present invention, the inventors used lyophilized PCR detection premix to test 26 known positive samples, 10 known negative samples, 40 unknown blood samples, and 43 swab samples based on the screened RT-qPCR reaction system and procedure. A brief flowchart and results are as follows: Figure 9 As shown, Figure 9-B results showed that all known positive samples were positive, all known negative samples were negative, and of the 83 unknown samples, 11 were positive and 72 were negative, with a positive detection rate of 13.3%. To verify the accuracy of the detection results of the method of the present invention, the inventors simultaneously used the conventional qPCR method to test 26 known positive samples, 10 known negative samples, and 83 unknown samples. The detection results were completely consistent with the detection results of the method of the present invention. Figure 9 As shown in -B. The results indicate that the POCT technology for AIV established in this invention is expected to be applicable to on-site detection of avian influenza virus in various scenarios, including farms, ports, and primary healthcare institutions.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rapid detection method for avian influenza virus, characterized in that, The method includes using primer pairs and probes designed for the conserved region of the M gene of AIV, and amplifying the amplification reagent containing the sample to be tested based on ultra-fast qPCR technology. The sequence of the forward primer in the primer pair is: 5'-GGCCCCCTCAAAGCCGA-3'; the sequence of the reverse primer is: 5'-CGTCTACGCTGCAGTCC-3'; and the sequence of the probe is ROX-CACTGGGCACGGTGAGCGT-BHQ2.

2. The rapid detection method according to claim 1, characterized in that, The concentration of the primer pair in the amplification reagent is 0.2–0.8 μmol / L; preferably 0.6–0.8 μmol / L; more preferably 0.8 μmol / L. And / or, the concentration of the probe in the amplification reagent is 0.3–0.5 μmol / L; preferably 0.4–0.5 μmol / L; more preferably 0.5 μmol / L.

3. The rapid detection method according to claim 1 or 2, characterized in that, The amplification reagent further includes a PCR buffer; preferably, the PCR buffer includes Tris, KCl, MgCl2, dNTPs, and dUTPs; more preferably, the PCR buffer includes 100 mmol / L Tris, 50 mmol / L KCl, 5 mmol / L MgCl2, 25 mmol / L dNTPs, and 100 mmol / L dUTPs; even more preferably, the PCR buffer accounts for 48% to 50% of the volume of the amplification reagent, preferably 49%. And / or, the amplification reagent further includes DNA polymerase; preferably, the DNA polymerase is Taq DNA polymerase, and / or, the concentration of the DNA polymerase in the amplification reagent is 0.18–0.22 U / µL, preferably 0.2006 U / µL; And / or, the amplification reagent further includes reverse transcriptase; preferably, the concentration of the reverse transcriptase in the amplification reagent is 8-8.5 U / µL, more preferably 8.264 U / µL; And / or, the amplification reagent further includes UDG enzyme; preferably, the concentration of UDG enzyme in the amplification reagent is 0.4-0.45 U / µL, more preferably 0.4132 U / µL; And / or, the amplification reagent further includes an RNase inhibitor; preferably, the concentration of the RNase inhibitor in the amplification reagent is 16-17 U / µL, more preferably 16.528 U / µL.

4. The rapid detection method according to claim 3, characterized in that, The amplification reagent consists of the sample to be tested and a PCR detection premix; preferably, the PCR detection premix includes the primer pairs, probes, DNA polymerase, reverse transcriptase, PCR buffer, UDG enzyme, and RNase inhibitors described above.

5. The rapid detection method according to claim 4, characterized in that, The rapid detection method includes reconstituted a lyophilized PCR detection premix with ultrapure water and then mixing it with the sample to be tested to form an amplification reagent; preferably, the ultrapure water is DEPC water.

6. The rapid detection method according to claim 5, characterized in that, The lyophilized PCR detection premix was prepared by a method including the following steps: adding a lyophilization protectant to the PCR detection premix; pre-freezing the PCR detection premix containing the lyophilization protectant at -80℃ for 2 hours, and then performing vacuum freeze-drying: first stage drying at -40℃ / 0.1 mbar for 12 hours, and second stage drying at 25℃ / 0.01 mbar for 3 hours.

7. The rapid detection method according to claim 6, characterized in that, The lyophilization protectant includes trehalose, PEG20000, and mannitol; preferably, the content of trehalose in the PCR detection premix containing the lyophilization protectant is 8 wt%, and / or the content of PEG20000 in the PCR detection premix containing the lyophilization protectant is 3 wt%, and / or the content of mannitol in the PCR detection premix containing the lyophilization protectant is 1.5 wt%.

8. The rapid detection method according to any one of claims 1 to 6, characterized in that, The annealing temperature for the amplification is 58℃~62℃; preferably 58℃~60℃; more preferably 58℃. Alternatively, the amplification program is: 50℃ for 2 min; 95℃ for 30 s, 95℃ for 10 s, 58~62℃ for 30 s; for a total of 40 cycles; preferably, the amplification program is: 50℃ for 2 min; 95℃ for 30 s, 95℃ for 10 s, 58℃ for 30 s; for a total of 40 cycles.

9. The rapid detection method according to any one of claims 1 to 6, characterized in that, The rapid detection method further includes extracting AIV nucleic acid from the sample to be tested using a direct-amplification nucleic acid release agent before amplification; preferably, the method for extracting AIV nucleic acid includes mixing the sample to be tested with the direct-amplification nucleic acid release agent and heating at 95°C for 3 minutes, followed by centrifugation to collect the supernatant; more preferably, the direct-amplification nucleic acid release agent includes Tris-HCl buffer, EDTA, NaCl, and / or the centrifugation conditions include centrifugation at 3000×g for 1 minute.

10. The rapid detection method according to claim 9, characterized in that, The method for extracting AIV nucleic acid further includes vortexing the mixture before heating; preferably, the treatment time is 10 seconds. And / or, the rapid detection method further includes extracting AIV from the sample to be tested using a magnetic bead method before extracting AIV nucleic acid; preferably, AIV is extracted using a magnetic bead method viral RNA / DNA nucleic acid extraction kit.

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