A nucleic acid releasing agent for H5N1 virus and a preparation method and application thereof

Abstract

The present application relates to the technical field of rapid detection of H5N1 avian influenza virus, and particularly relates to a nucleic acid releasing agent for H5N1 virus, and a preparation method and application thereof.The nucleic acid releasing agent is composed of the following components with a mass ratio of (50-80):1:(3.9-12.5):(0.02-0.25):(15-63):(12-20):(10-87): guanidine hydrochloride solution, TritonX-100, isopropyl alcohol, Tween-20, ethanol, EDTA and DEPC water.The nucleic acid releasing agent provided by the present application can efficiently release viral nucleic acid and remove interfering substances, and lays a foundation for detecting samples with low viral load;then, in combination with primer screening, reaction system and freeze-drying condition optimization, a visual freeze-drying RT-RAA detection system is constructed, and finally, rapid, sensitive, specific and visual detection of H5N1 virus is realized.

Description

Technical Field

[0001] This invention relates to the technical field of rapid detection of H5N1 avian influenza virus, specifically to a nucleic acid release agent for H5N1 virus, its preparation method, and its application. Background Technology

[0002] The H5N1 avian influenza virus belongs to the Orthomyxoviridae family and is an influenza A virus. It is a highly pathogenic strain that can be transmitted between birds and humans. Human infection has a high mortality rate, posing a serious threat to public health, livestock farming, and wildlife protection. The subtype classification of the H5N1 virus is determined by two key surface glycoproteins—hemagglutinin (HA) and neuraminidase (NA). Therefore, detection of the H5N1 virus in samples requires monitoring the HA and NA genes. Furthermore, detecting only a single gene cannot accurately distinguish H5N1 from other influenza A virus subtypes (such as H5N2, H5N6, and H1N1). Only by simultaneously detecting both the HA and NA genes, confirming that they are H5 and N1 types respectively, can the presence of the H5N1 virus in the sample be accurately diagnosed. This is a core prerequisite for accurate virus typing and ensuring detection specificity.

[0003] Currently, detection methods for the H5N1 virus have been developed to a certain extent, and can be mainly divided into the following categories based on their detection principles: virus isolation and culture methods, serological detection methods, nucleic acid detection methods, and antigen detection methods. While virus isolation and culture methods offer high accuracy, they suffer from complex operation, long detection cycles, or reliance on sophisticated instruments. Serological and antigen detection methods, while convenient, lack sufficient sensitivity. In contrast, isothermal amplification techniques in nucleic acid detection (such as RT-RAA and RT-LAMP) do not require a thermal cycler and can rapidly amplify nucleic acids under isothermal conditions, shortening the detection cycle to within 30 minutes. Furthermore, they can be combined with lateral flow test strips (LFD) for visual interpretation, making them suitable for rapid on-site testing scenarios. Lyophilized isothermal amplification reagents can be stored and transported at room temperature, further improving the convenience and applicability of the detection. However, isothermal amplification techniques sometimes struggle to reliably detect early-infection samples with low viral loads and are susceptible to non-specific amplification due to interfering substances, affecting detection sensitivity. Therefore, developing a simple, rapid, sensitive, and highly specific visual detection method for H5N1 virus based on HA and NA gene targeting has significant practical application value. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a nucleic acid release agent for H5N1 virus, its preparation method and application, so as to solve the problem of difficulty in detecting early infection samples with low viral load in the prior art.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] A nucleic acid release agent for H5N1 virus comprises the following components: guanidine hydrochloride solution, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water; wherein the mass ratio of the guanidine hydrochloride solution, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water is (50~80): 1: (3.9~12.5): (0.02~0.25): (15~63): (12~20): (10~87), wherein the concentration of guanidine hydrochloride in the guanidine hydrochloride solution is 50~53 mmol / L.

[0007] This invention provides a novel nucleic acid release agent formulation for H5N1 virus. This nucleic acid release agent can rapidly disrupt viral particles and host cell structures, efficiently releasing viral nucleic acids (such as the HA and NA target genes of H5N1), while simultaneously removing interfering substances during the nucleic acid release process. This provides a pure and complete nucleic acid template for subsequent RT-RAA amplification, further laying the foundation for detecting samples with low viral loads. Furthermore, a comparison of the amplification effect of this formulation with commercially available nucleic acid release agents shows that its UV fluorescence is superior to that of the commercially available products.

[0008] The second technical solution provided by this invention is:

[0009] A method for preparing a nucleic acid release agent for H5N1 virus includes the following steps: Guanidine hydrochloride solution, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water are mixed in a mass ratio of (50-80): 1: (3.9-12.5): (0.02-0.25): (15-63): (12-20): (10-87) to obtain the nucleic acid release agent, wherein the concentration of guanidine hydrochloride in the guanidine hydrochloride solution is 50-53 mmol / L. Further, the 50-53 mmol / L guanidine hydrochloride solution is prepared by diluting 0.48-0.51 g of guanidine hydrochloride to 100 mL with water.

[0010] The preparation method of the nucleic acid release agent for H5N1 virus of the present invention is simple and inexpensive, and is suitable for large-scale promotion and use.

[0011] The third technical solution provided by this invention is:

[0012] A visual detection method for H5N1 virus based on freeze-dried RT-RAA technology, wherein the H5N1 virus sample is subjected to nucleic acid release using the above-mentioned nucleic acid release agent for H5N1 virus or a nucleic acid release agent prepared by the above-mentioned method for preparing nucleic acid release agent for H5N1 virus.

[0013] To release viral nucleic acid from the sample more efficiently, preferably, the volume ratio of the nucleic acid release agent to the H5N1 virus sample is (1~1.3):1.

[0014] To further improve the amplification effect and efficiency, preferably, the following steps are included: mixing the H5N1 virus sample after nucleic acid release with lyophilization reagent in water, amplifying it in a constant temperature metal bath at 39~41℃ for 20 min, and then observing it under ultraviolet fluorescence irradiation.

[0015] Preferably, the lyophilization reagent comprises lyophilized enzyme mixture powder, upstream primer, downstream primer, fluorescent dye, lyophilization protectant, and magnesium acetate. Before lyophilization, the volume ratio of the enzyme mixture, upstream primer, downstream primer, fluorescent dye, lyophilization protectant, magnesium acetate solution, and nucleic acid release agent is (12.5~13): 1: 1: (1.5~2.5): (3~3.5): (2.5~3): (2.5~3). This amplification system results in higher amplification efficiency and avoids reagent waste.

[0016] Furthermore, the main components of the enzyme mixture are recombinant enzyme, single-stranded binding protein, DNA polymerase, dNTPs, ATP, and buffer solution.

[0017] Preferably, the volume ratio of the enzyme mixture, upstream primer, downstream primer, fluorescent dye, lyophilization protectant, magnesium acetate solution and nucleic acid release agent is 12.5: 1: 1: 1.5: 3: 2.5: 2.5.

[0018] Preferably, the sequences of the upstream and downstream primers used to detect the HA gene in the H5N1 virus are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the sequences of the upstream and downstream primers used to detect the NA gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0019] To better amplify the target fragment and increase the intensity of ultraviolet fluorescence, preferably, the final concentration of both the upstream and downstream primers in the RT-RAA amplification reaction system is 0.40 μM; and the concentration factor of the fluorescent dye used is 12.5.

[0020] Preferably, the H5N1 virus visualization detection method includes the following steps:

[0021] (1) Mix the H5N1 virus sample with the nucleic acid release agent at a volume ratio of 1:1, lyse it, let it stand at room temperature for 2 min, and then take 5 μL of the supernatant as a template for RT-RAA amplification;

[0022] (2) Add water to the lyophilized reagent and the amplification template obtained in step (1) and place them in a reaction tube. After mixing the reaction tube thoroughly by hand, place it in a handheld centrifuge and centrifuge at 2000 rpm for 10 s. Then place it in a constant temperature metal bath at 39℃ for 20 min and observe the ultraviolet fluorescence effect after the reaction.

[0023] The technical solution of the present invention achieves the following beneficial technical effects:

[0024] 1. The minimum gene copy number of the visual detection method for H5N1 virus of the present invention is 10. 3 The number of copies / mL indicates that the detection method of the present invention has high sensitivity and can be used to detect early-stage infected samples with low viral load.

[0025] 2. The detection method of the present invention has high specificity and can effectively identify the H5N1 virus. It does not cross-react with influenza A virus, influenza B virus, or respiratory syncytial virus.

[0026] 3. In the simulated on-site testing scenario, the gene copy number is 10. 3 copies / mL, 10 5 The test samples at copies / mL were effectively amplified, and the fluorescence intensity of samples of the same dilution concentration from different batches showed good uniformity, indicating that the H5N1 virus visualization detection method of the present invention has good repeatability and stability. Attached Figure Description

[0027] Figure 1 This is a diagram showing the screening results of the HA gene primer pairs of the present invention;

[0028] Figure 2 This is a diagram showing the screening results of the NA gene primer pairs of this invention;

[0029] Figure 3 This is a UV fluorescence intensity diagram of the fluorescent dye at 12.5x in the RT-RAA amplification system of this invention;

[0030] Figure 4 The images show the RT-RAA amplification effects of the nucleic acid releasing agents in Examples 1-3 of this invention.

[0031] Figure 5 The image shows the RT-RAA amplification effect of the nucleic acid releasing agent in Example 1 of this invention and a conventional nucleic acid releasing agent.

[0032] Figure 6 This is a graph showing the ultraviolet fluorescence intensity of RT-RAA amplification after reacting at different temperatures for 20 min in Experiment Example 2 of this invention.

[0033] Figure 7This is a graph showing the ultraviolet fluorescence intensity of RT-RAA amplification corresponding to different concentrations of lyophilization protectant in Experimental Example 3 of this invention.

[0034] Figure 8 This is a graph showing the sensitivity test results of the H5N1-RT-RAA system (HA gene) in Experiment Example 4 of this invention;

[0035] Figure 9 This is a graph showing the sensitivity test results of the H5N1-RT-RAA system (NA gene) in Experiment Example 4 of this invention;

[0036] Figure 10 This is a diagram showing the specific results of the H5N1-RT-RAA system (HA gene) in Experimental Example 5 of this invention;

[0037] Figure 11 This is a diagram showing the specific results of the H5N1-RT-RAA system (NA gene) in Experiment Example 5 of this invention;

[0038] Figure 12 This is a graph showing the stability results of the H5N1-RT-RAA system (HA gene) in Experiment Example 6 of this invention;

[0039] Figure 13 This is a graph showing the stability results of the H5N1-RT-RAA system (NA gene) in Experiment Example 6 of this invention;

[0040] Figure 14 This is a graph showing the results of the on-site detection experiment (HA gene) of the lyophilized reagent in Experiment Example 6 of the present invention;

[0041] Figure 15 This is a diagram showing the results of the on-site detection experiment (NA gene) using the lyophilized reagent in Experiment Example 6 of this invention. Detailed Implementation

[0042] The virus sample used in the embodiments of the present invention is the H5 subtype avian influenza virus HI antigen (AIV-H5-Re13), purchased from the Harbin Veterinary Research Institute; the avian influenza virus H5N1 gene plasmid was synthesized by Shanghai Sangon Biotech; and the influenza A virus, influenza B virus, and respiratory syncytial virus were provided by the Health Center headquarters.

[0043] The chemical abbreviations involved in this invention are: Tween-20, disodium ethylenediaminetetraacetate (EDTA), and nuclease-free water (DEPC water).

[0044] The following are the reagents and instruments used in the embodiments of this invention:

[0045] The RT-RAA basic nucleic acid amplification reagent was purchased from Jiangsu Qitian Biotechnology Co., Ltd., model S00R00A (this kit contains RT-RAA enzyme mixture, magnesium acetate solution, nuclease-free purified water, and Buffer V basic buffer, etc.); the one-step reverse transcription fluorescent quantitative probe kit was purchased from Shanghai Sangon Biotech Co., Ltd., model L526KA9342 (this kit contains 2×OneStep RT-qPCR Probe Mix, reverse transcriptase mixture, and enzyme-free water); SYBR Green I nucleic acid dye was purchased from Solarbio, model SY1020; and the lyophilization protectant was purchased from Zhuhai Baorui Biotechnology Co., Ltd., model 2024052903.

[0046] Nucleic acid release reagents: guanidine hydrochloride (purchased from Beijing Solarbio), Triton X-100 (purchased from Beijing Solarbio), ethanol (purchased from Tianjin Zhiyuan), isopropanol (purchased from Tianjin Zhiyuan), Tween-20 (purchased from Tianjin Huasheng), EDTA (ethylenediaminetetraacetic acid) (purchased from Beijing Solarbio), and conventional nucleic acid release products were purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.

[0047] The enzyme mixture is a matching reagent for the RT-RAA basic nucleic acid amplification reagent (purchased from Jiangsu Qitian Biotechnology Co., Ltd., model number S00R00A). Its main components are recombinase, single-stranded binding protein, DNA polymerase, dNTPs, ATP and buffer.

[0048] The instruments used in the experiment were a constant temperature metal bath (PMC), a constant temperature water bath (China Beili, SYG-4), a fully automated nucleic acid analysis system (Switzerland, Qiagen), a portable digital display constant temperature heater, and a handheld ultraviolet meter.

[0049] The preparation methods for the test samples involved in the following embodiments and experimental examples adopt the following steps:

[0050] (1) Construction of standard curve for HA gene plasmid: Prepare HA standard solutions with four concentration gradients, covering a concentration range of 10. 7 ~10 4 Copies / mL, each concentration was serially diluted, each concentration point was measured 3 times, and the average value was taken. A curve was plotted with the concentration log value on the x-axis and the average ct value on the y-axis.

[0051] (2) The H5 subtype avian influenza virus antigen (AIV-H5-Re13) was dissolved in 2 mL of double-distilled water. The initial concentration of the diluted antigen was calculated to be 1.950*10 using a standard curve plotted with the HA gene plasmid. 10 The antigen was serially diluted 10-fold (copies / mL) and used as a test sample.

[0052] The following describes the preliminary preparations for the examples and experimental cases, specifically involving primer design, construction of recombinant plasmids, and screening of optimal primer pairs:

[0053] (1) Primer design: The HA and NA gene sequences of H5N1 subtype avian influenza virus were retrieved from the GenBank database of NCBI and imported into AlignX software for comparison and selection of conserved regions. Then, DNASTAR software was used to perform BLAST sequence homology analysis to select highly conserved nucleic acid sequences (see Table 1). RT-RAA primers for HA and NA genes were designed, and 4 pairs of primers for each gene were synthesized (see Table 2).

[0054] (2) Construction of recombinant plasmid: The target gene HA and NA segments of the H5N1 subtype avian influenza virus were cloned into the pUC57 vector to synthesize a recombinant plasmid (synthesized by Shanghai Sangon Biotech).

[0055] Table 1. Amplified target gene sequences

[0056]

[0057] Table 2. RT-RAA primer pairs for amplifying target genes

[0058]

[0059] (3) Screening of RT-RAA primers: The four pairs of upstream and downstream primers for HA and NA in step (2) were combined in pairs to perform basic RT-RAA amplification on the recombinant plasmids. After capillary electrophoresis, it was found that H5-HA RT-RAA F2 / R3 (160bp fragment size) and H5-NA RT-RAA F3 / R2 (108bp fragment size) showed no specific bands, no primer dimers, and no dragging (e.g.) Figure 1 , 2 As shown in the figure, these two sets of primers have good amplification effects on the target band, so they were used in subsequent experiments. The basic RT-RAA amplification system consisted of 25 μL of RT-RAA enzyme mixture, 2 μL each of forward and reverse primers, 5 μL of recombinant plasmid, 5 μL of magnesium acetate solution, and 5 μL of nuclease-free water to a final volume of 50 μL; the amplification method was the basic RT-RAA amplification method.

[0060] (4) Screening of optimal primer and fluorescent dye concentrations: The H5-HA RT-RAAF2 / R3 and H5-NA RT-RAA F3 / R2 selected in step (3) were used to perform basic RT-RAA amplification on the recombinant plasmid (the method is the same as in step 3), and the fluorescence effect after amplification was observed. The amount of each primer added in the amplification system was set to 1 μL (final concentration of 0.2 μM), 1.2 μL (final concentration of 0.24 μM), 1.6 μL (final concentration of 0.32 μM), and 2.0 μL (final concentration of 0.40 μM), respectively. The fluorescent dye used was a 10000x fluorescent dye diluted with dilution factors of 100×, 50×, 25×, 12.5×, and 6.25×, respectively. Then, primers of different concentrations and fluorescent dyes of different concentrations were combined in pairs to screen out the combination with the highest fluorescence intensity after amplification. This invention has shown through observation that using a combination of 12.5× fluorescent dye and primers of different concentrations yields better amplification results (e.g., Figure 3 As shown, Figure 3 (NC is the negative control, the same below). The amount of primer added was 2.0 μL, that is, the final concentration in the system was 0.40 μM. After being combined with 12.5× fluorescent dye, the amplification fluorescence intensity was the highest.

[0061] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0062] I. Specific Examples of the H5N1 Virus Nucleic Acid Release Agent and its Preparation Method of the Present Invention

[0063] Example 1

[0064] The H5N1 virus nucleic acid release agent of this embodiment is composed of 50 mmol / L guanidine hydrochloride, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water. The mass ratio of 50 mmol / L guanidine hydrochloride, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water is 50: 1: 3.98: 0.02: 15: 20: 10.

[0065] The nucleic acid release agent for the H5N1 virus in this embodiment is prepared by directly mixing the above components.

[0066] Example 2

[0067] The H5N1 virus nucleic acid release agent of this embodiment is composed of 50 mmol / L guanidine hydrochloride, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water. The mass ratio of 50 mmol / L guanidine hydrochloride, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water is 40:0.5:4:0.05:10:10:35.45. The H5N1 virus nucleic acid release agent of this embodiment is prepared by directly mixing the above components.

[0068] Example 3

[0069] The H5N1 virus nucleic acid release agent of this embodiment is composed of 50 mmol / L guanidine hydrochloride, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water, wherein the mass ratio of 50 mmol / L guanidine hydrochloride, Triton X-100, isopropanol, Tween-20, ethanol, EDTA, and DEPC water is 30:0.4:5:0.1:25:5:34.5. The H5N1 virus nucleic acid release agent of this embodiment is prepared by directly mixing the above components.

[0070] Experimental Example 1: Verification of the amplification effect of the nucleic acid releasing agents in Examples 1-3

[0071] In this experimental example, the H5N1 virus nucleic acid release agent from Examples 1-3 and a commercially available nucleic acid release agent (purchased from Anpu Future Biotechnology Co., Ltd.) were used to lyse the same sample to be tested, and then RT-RAA amplification was performed to compare the amplification effects. The results are as follows: Figure 4 , 5 As shown. By observation Figure 4 The results of UV fluorescence irradiation showed that the nucleic acid releasing agents of Examples 1-3 could all be effectively amplified, and the amplification fluorescence effect of Example 1 was better than that of Examples 2 and 3. This experimental example further compared the amplification results of Example 1 with those of conventional commercial nucleic acid releasing agents and found that... Figure 5 The nucleic acid release agent of Example 1 has a similar amplification effect to, or even better than, conventional commercial nucleic acid release agents.

[0072] The specific experimental method was as follows: the nucleic acid release agents from Examples 1-3 and the purchased commercial nucleic acid release agents were mixed with the same sample at a volume ratio of 1:1, lysed, and incubated at room temperature for 2 minutes. Then, 5 μL of the supernatant was used as a template for RT-RAA amplification; the sample used was 10 3 copies / mL of AIV-H5N1-Re13 antigen.

[0073] The steps for RT-RAA amplification are as follows: Add 5 μL of the above template, 25 μL of RT-RAA enzyme mixture, 2 μL each of H5-HA RT-RAA F2 / R3 and H5-NA RT-RAA F3 / R2 upstream and downstream primers, 5 μL of 12.5× fluorescent dye, and 5 μL of magnesium acetate solution to the reaction tube. Make up the volume to 50 μL with nuclease-free water. After thoroughly mixing the reaction tube by hand, centrifuge at 2000 rpm for 10 s in a handheld centrifuge, and then react at 39℃ for 20 min before observing the results under ultraviolet fluorescence irradiation.

[0074] Experimental Example 2: Screening of different reaction temperatures and times in the RT-RAA amplification system

[0075] This experimental example screened the optimal reaction temperature and reaction time for the amplification system. The screening process is as follows:

[0076] (1) Mix the nucleic acid release agent of Example 1 with 10 6 AIV-H5N1-Re13 antigen of copies / mL was mixed and lysed at a volume ratio of 1:1, incubated at room temperature for 2 min, and then 5 μL of the supernatant was used as a template for RT-RAA amplification.

[0077] (2) Add 5 μL of the template from step (1), 25 μL of RT-RAA enzyme mixture, 2 μL each of the upstream and downstream primers of H5-HA RT-RAA F2 / R3 and H5-NA RT-RAA F3 / R2, 3 μL of 12.5× fluorescent dye, and 5 μL of magnesium acetate solution to the reaction tube. Make up the reaction system to 50 μL with nuclease-free water. After thoroughly mixing the reaction tube by hand, centrifuge briefly at 2000 rpm for 10 s in a handheld centrifuge. Amplification was performed at different reaction temperatures and times. The reaction temperatures were set to 37℃, 39℃, and 41℃, and the reaction times were set to 20 min, 30 min, and 40 min, respectively. The UV fluorescence irradiation results were observed after combining the two. According to the observation results, the UV fluorescence intensity was stronger after combining 39℃ and 20 min (e.g., Figure 6 As shown in the figure, 39℃ and 20min were therefore used as the reaction conditions for subsequent experiments.

[0078] Experiment Example 3: Screening of the Amount of Lyophilization Protectant Added to the RT-RAA Amplification System

[0079] This experimental example screens the amount of lyophilization protectant added to the amplification system. The specific steps are basically the same as those in Experiment 2, except that: in step (2), an additional lyophilization protectant is added to the RT-RAA reaction system, and the results are tested according to different proportions in Table 4. Figure 7 As shown. According to Figure 7The combined UV fluorescence intensity results showed that when the proportion of lyophilization protectant reached 12% (i.e., 6 μL of lyophilization protectant was added to every 50 μL of reaction system), the fluorescence intensity was the brightest, the lyophilized sample was dense and had a good morphology, did not disperse when shaken, and could be quickly dissolved by adding deionized water.

[0080] Table 4. Amounts of each component added to the lyophilized RT-RAA reaction system

[0081]

[0082] II. Specific Examples of the Application of the Nucleic Acid Release Agent for H5N1 Virus of the Present Invention

[0083] Example 4

[0084] The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology of the present invention comprises the following steps:

[0085] (1) Dispense each component of the RT-RAA reaction system in Table 4 according to the amount used, and then freeze it immediately at -80℃. After it is completely frozen, use the SCIENTZ-10YG freeze dryer of Xinzhi Biotechnology to freeze-dry it to obtain the freeze-dried reagent.

[0086] The freeze-drying process is as follows: Pre-freeze at -50℃ for 24 hours under extreme vacuum conditions, then raise the temperature from -50℃ to -30℃ within 20 minutes, maintain the temperature at -30℃ for 4 hours for sublimation drying, raise the temperature from -30℃ to 26℃ within 60 minutes, maintain the temperature at 26℃ for 4 hours for desorption drying, and the freeze-drying process is now complete. Quickly complete the packaging, vacuum the bag using a flat bag intelligent packaging machine, and then seal it.

[0087] Table 4 RT-RAA Reaction System

[0088]

[0089] (2) Preparation of RT-RAA amplification template: The nucleic acid release agent of Example 1 was mixed with 10 6 AIV-H5N1-Re13 antigen of copies / mL was mixed and lysed at a volume ratio of 1:1, incubated at room temperature for 2 min, and then 5 μL of the supernatant was used as a template for RT-RAA amplification.

[0090] (3) RT-RAA amplification: Add 5 μL of amplification template and 2 μL of nuclease-free water to the lyophilized reagent. After thoroughly mixing the reaction tube by hand, place it in a handheld centrifuge for a short time, and then place it in a constant temperature metal bath at 39℃ for 20 min. Observe the ultraviolet fluorescence effect after the reaction.

[0091] Experiment Example 4: Sensitivity of the Visual Detection Method for H5N1 Virus

[0092] This experimental example is to determine the sensitivity of the detection method in Example 4. Specifically, the AIV-H5N1-Re13 antigen was set to 10. 7 -10 1 Seven different concentrations (copies / mL) were measured, and a negative control (NC) was set up. The results are as follows: Figure 8 , 9 As shown. The detection method is the same as in Example 4. According to... Figure 8 , 9 The ultraviolet fluorescence results show that the minimum gene copy number of the visual detection method for H5N1 virus of this invention is not less than 10. 3 copies / mL.

[0093] Experimental Example 5: Specificity of the Visual Detection Method for H5N1 Virus

[0094] This experimental example was designed to determine the specificity of the detection method in Example 4. Specifically, the AIV-H5N1-Re13 antigen was replaced with a positive nucleic acid sample of influenza A, influenza B, or respiratory syncytial virus, and RT-RAA amplification was performed. The ultraviolet fluorescence effects were compared, and the results are as follows: Figure 10 , 11 As shown. The detection method is the same as in Example 4. According to... Figure 10 , 11 The ultraviolet fluorescence results show that the H5N1-RT-RAA system of the present invention does not cross-react with influenza A virus, influenza B virus, or respiratory syncytial virus, and the method can effectively identify H5N1 virus.

[0095] Experiment Example 6: Stability of the Visual Detection Method for H5N1 Virus

[0096] In this experimental example, the lyophilized reagent obtained in step (1) of Example 4 was placed in a 37°C constant temperature incubator for 30 days for acceleration. Then, different diluted samples were visually tested using the lyophilized reagent before and after acceleration to compare the stability of the unaccelerated (0d) and accelerated (30d) samples. The test results are as follows: Figure 12 , 13 As shown. The different dilutions were 10... 7 copies / mL, 10 5 copies / mL, 10 3 copies / mL of AIV-H5N1-Re13 antigen, the detection method is the same as in Example 4. According to Figure 12 , 13The test results show that both direct use of the lyophilized reagent and use after storage at 37°C for 30 days can detect H5N1 virus in samples of different dilution concentrations. This also indicates that the sensitivity of the lyophilized reagent in Example 4 did not decrease after storage at 37°C for 30 days, and it has strong temperature tolerance.

[0097] To further verify the practical application effect of the lyophilization system in Example 4, this experimental example used a digital display portable rechargeable heating container and a portable ultraviolet fluorescent lamp to simulate an on-site detection scenario. Samples treated with the nucleic acid release agent from Example 1 were randomly selected for lyophilized RT-RAA amplification detection. Three replicates and a negative control were set up. The results are as follows: Figure 14 , 15 As shown. The test method is the same as in Example 4. Figure 14 , 15 10 of them 5 copies / mL, 10 3 Copies / mL refers to the concentration of the sample itself. According to... Figure 14 , 15 The ultraviolet fluorescence results show that all samples can be effectively amplified, and the fluorescence intensity of samples of the same dilution concentration from different batches is relatively uniform, indicating that the H5N1 virus visualization detection method of Example 4 of this invention has good repeatability and stability.

[0098] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A nucleic acid release agent for H5N1 virus, characterized in that, It is composed of the following components: guanidine hydrochloride solution, Triton X-100, isopropanol, Tween-20, ethanol, EDTA and DEPC water; the mass ratio of guanidine hydrochloride solution, Triton X-100, isopropanol, Tween-20, ethanol, EDTA and DEPC water is (50~80): 1: (3.9~12.5): (0.02~0.25): (15~63): (12~20): (10~87), wherein the concentration of guanidine hydrochloride in the guanidine hydrochloride solution is 50~53 mmol / L.

2. A method for preparing a nucleic acid release agent for H5N1 virus, characterized in that, Includes the following steps: The nucleic acid release agent was prepared by mixing guanidine hydrochloride solution, Triton X-100, isopropanol, Tween-20, ethanol, EDTA and DEPC water in a mass ratio of (50~80): 1: (3.9~12.5): (0.02~0.25): (15~63): (12~20): (10~87), wherein the concentration of guanidine hydrochloride in the guanidine hydrochloride solution was 50~53 mmol / L.

3. A visual detection method for H5N1 virus based on freeze-dried RT-RAA technology, characterized in that, In the visualization detection method, the H5N1 virus sample is subjected to nucleic acid release using the nucleic acid release agent for H5N1 virus as described in claim 1 or the nucleic acid release agent prepared by the preparation method of the nucleic acid release agent for H5N1 virus as described in claim 2.

4. The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology according to claim 3, characterized in that, The volume ratio of the nucleic acid release agent to the H5N1 virus sample is (1~1.3):

1.

5. The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology according to claim 3, characterized in that, The steps include: mixing the H5N1 virus sample after nucleic acid release with lyophilization reagent in water, amplifying it in a constant temperature metal bath at 39~41℃ for 20 min, and then observing it under ultraviolet fluorescence irradiation.

6. The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology according to claim 5, characterized in that, The lyophilized reagents include lyophilized powder of enzyme mixture, upstream primer, downstream primer, fluorescent dye, lyophilization protectant and magnesium acetate. Before lyophilization, the volume ratio of enzyme mixture, upstream primer, downstream primer, fluorescent dye, lyophilization protectant, magnesium acetate solution and nucleic acid release agent is (12.5~13): 1: 1: (1.5~2.5): (3~3.5): (2.5~3): (2.5~3).

7. The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology according to claim 6, characterized in that, The volume ratio of the enzyme mixture, upstream primer, downstream primer, fluorescent dye, lyophilization protectant, magnesium acetate solution, and nucleic acid release agent is 12.5: 1: 1: 1.5: 3: 2.5: 2.

5.

8. The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology according to claim 6, characterized in that, The sequences of the upstream and downstream primers used to detect the HA gene in H5N1 virus are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the sequences of the upstream and downstream primers used to detect the NA gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

9. The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology according to any one of claims 6-8, characterized in that, The final concentration of both the upstream and downstream primers in the RT-RAA amplification reaction system was 0.40 μM; the concentration factor of the fluorescent dye used was 12.

5.

10. The H5N1 virus visualization detection method based on freeze-dried RT-RAA technology according to any one of claims 5-8, characterized in that, Includes the following steps: (1) Mix the H5N1 virus sample with the nucleic acid release agent at a volume ratio of 1:1, lyse it, let it stand at room temperature for 2 min, and then take 5 μL of the supernatant as the RT-RAA amplification template. (2) Add water to the lyophilized reagent and the amplification template obtained in step (1) and place them in a reaction tube. After mixing the reaction tube thoroughly by hand, place it in a handheld centrifuge and centrifuge at 2000 rpm for 10 s. Then place it in a constant temperature metal bath at 39℃ for 20 min and observe the ultraviolet fluorescence effect after the reaction.

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