A nucleic acid molecule composition, reagent, kit and application for detecting H7 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD

Abstract

The present invention relates to the field of molecular biology technology, and in particular to a nucleic acid molecule composition, reagent, kit and application for detecting H7 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD. The nucleic acid molecule composition includes an upstream primer RAA-H7-F, a downstream primer RAA-H7-R and a specific crRNA-H7; the sequence of the upstream primer RAA-H7-F is shown in SEQ ID No: 1, the sequence of the downstream primer RAA-H7-R is shown in SEQ ID No: 2, and the sequence of the specific crRNA-H7 is shown in SEQ ID No: 3. The nucleic acid molecule composition provided by the present invention can realize the visual detection of H7 subtype avian influenza virus, and the detection accuracy is high, the time is short, the specificity is good, and the sensitivity is high, which provides a basis for quickly, simply and accurately diagnosing H7 subtype avian influenza virus and meets the needs of clinical detection.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and more specifically, to a nucleic acid molecule composition, reagent, kit and application for detecting H7 subtype avian influenza based on RT-RAA / CRISPR-Cas13a / LFD. Background Art

[0002] Avian influenza virus (AIV) has been prevalent worldwide in recent years, causing significant economic losses to the poultry industry. More importantly, AIV can cross species between mammals, posing a potential threat to human health and safety.

[0003] Avian influenza virus (AIV) belongs to the influenza A virus family. Based on the antigenicity of the virus's hemagglutinin (HA) and neuraminidase (NA) proteins, it can be divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11). Theoretically, these can combine to form 198 different subtypes. These subtypes may differ in pathogenicity and transmissibility. For example, subtypes such as H5N1 and H7N9 are highly pathogenic avian influenza subtypes that pose a significant threat to human health and the poultry industry.

[0004] Currently, nucleic acid detection technology is the most widely used method for avian influenza virus detection, mainly through polymerase chain reaction (PCR) technology. First, the viral nucleic acid in the sample is extracted, and then the gene fragments of the avian influenza virus (such as the HA or NA gene) are amplified using specific primers. If avian influenza virus nucleic acid is present in the sample, after PCR amplification, the corresponding nucleic acid fragments can be detected through gel electrophoresis, fluorescence quantitative PCR, and other methods. Fluorescence quantitative PCR technology can not only detect the presence of viral nucleic acid, but also quantitatively analyze the content of viral nucleic acid, which is of great significance for assessing the degree of viral infection and the risk of transmission. This method has high sensitivity and strong specificity and can detect low concentrations of virus, but it requires professional equipment and technicians, and the detection cost is relatively high.

[0005] For the H7 subtype avian influenza virus, there is currently no low-cost detection method that is highly sensitive, specific, and suitable for clinical use. Summary of the Invention

[0006] In order to overcome the deficiencies in the prior art, the present invention aims to provide a nucleic acid molecule composition for detecting H7 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD. The nucleic acid molecule composition provided by the present invention can realize visual detection of H7 subtype avian influenza virus, and has high detection accuracy, short detection time, good specificity and high sensitivity, providing a basis for rapid, simple and accurate diagnosis of H7 subtype avian influenza virus, and meeting the needs of clinical detection.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A nucleic acid molecule composition for detecting H7 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD, comprising an upstream primer RAA-H7-F, a downstream primer RAA-H7-R, and a specific crRNA-H7;

[0009] The sequence of the upstream primer RAA-H7-F is: 5'-TATTTGGTGCTATAGCGGGGTTCATTGAAA-3' (SEQ ID NO: 1),

[0010] The sequence of the downstream primer RAA-H7-R is: 5'-TTGATCTATTGCCGATTGAGTGCTTTTGTA-3' (SEQ ID NO: 2),

[0011] The sequence of the specific crRNA-H7 is: 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAAUCUGCAGCAGUUCCCUCUCCCUGUG-3' (SEQ ID NO: 3).

[0012] The present invention integrates RT-RAA technology, CRISPR-Cas13a system and lateral flow technology to establish a CRISPR-LFD nucleic acid detection method. Specific CRISPR-crRNA and RT-RAA primer sets are designed within the conserved region of the gene sequence for detection of H7 subtype avian influenza virus. The fluorescent signal in the CRISPR detection is converted into an intuitive and easy-to-read signal, and the results are quickly read through lateral flow test strips, eliminating the dependence on special equipment. The detection sensitivity of this method is 10 copies / μL, and it does not cross-react with other avian disease pathogens. In the detection of clinical samples, this technology can also complete the detection within 1 hour, and the detection results have a high consistency with the virus isolation and identification and fluorescent quantitative PCR methods.

[0013] Preferably, the nucleic acid molecule composition is prepared into a reagent or kit for detecting H7 subtype avian influenza virus, and the reagent or kit containing the nucleic acid molecule composition falls within the scope of protection of the present invention.

[0014] Preferably, the above kit further comprises LwaCas13a protein and reagents for RAA amplification.

[0015] The present invention also provides a method for detecting H7 subtype avian influenza virus using the aforementioned nucleic acid molecule composition, which comprises the following steps:

[0016] S1: Extract the viral nucleic acid template and perform RAA amplification on the viral nucleic acid template using a primer set consisting of an upstream primer RAA-H7-F and a downstream primer RAA-H7-R;

[0017] S2: adding the RAA amplified product to the CRISPR-Cas13a system for incubation, wherein the CRISPR-Cas13a system includes specific crRNA-H7;

[0018] S3: Detect the incubation products of CRISPR-Cas13a using LFD.

[0019] The specific steps of the above method are:

[0020] Preferably, the RAA amplification system in step S1 includes: 29.4 μL of A Buffer, 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 2-14.1 μL of nucleic acid template, 2.5 μL of B Buffer, and DNase / RNase-Free Water is added to 50 μL.

[0021] Preferably, the RAA amplification system in step S1 is reacted at 42° C. for 30 min.

[0022] Preferably, the CRISPR-Cas13a system in step S2 includes: LwaCas13a protein (15 μg / mL) 2.0 μL, crRNA (100 μmol / L) 0.5 μL, probe (50 μmol / L) 3.2 μL, T7 RNA Polymerase 0.4 μL, NTP Buffer Mix 2.0 μL, Murine RNase Inhibitor (40 U / μL) 1.0 μL, 10×Cas13a Reaction Buffer 2.0 μL, 10×T7 RNA Polymerase Buffer 2.0 μL, cDNA (100 ng) 0.6 μL, and DNase / RNase-Free Water is supplemented to 20.0 μL.

[0023] Furthermore, the incubation temperature in step S2 is 37° C., and the incubation time is 20 min.

[0024] Furthermore, when the CRISPR-Cas13a incubation product was detected using LFD, two red bands appeared on the LFD, indicating a positive result, one in the quality control area and one in the detection area. A positive result indicated that the amplified product contained the nucleic acid fragment to be detected; a red band appeared in the quality control area of ​​the LFD and there was no red band in the detection area, indicating a negative result. A negative result indicated that the amplified product did not contain the detection fragment.

[0025] The above-mentioned preferred reaction conditions can further improve the detection efficiency of H7 subtype avian influenza virus.

[0026] The present invention also provides the use of the nucleic acid molecule composition, reagent, and kit in non-diagnostic detection of avian H7 subtype avian influenza.

[0027] Ideally, in poultry farming environmental monitoring applications, the presence of avian influenza virus nucleic acid in environmental samples could be regularly tested to determine if the virus is present in the environment, serving as an indicator of the effectiveness of cleaning and disinfection measures at the farm. If the virus is detected, the farm can promptly strengthen disinfection and isolation measures to prevent the virus from spreading among the poultry population and thus prevent an avian influenza outbreak.

[0028] Preferably, in the monitoring application of poultry trading markets, samples are collected from different stalls in the market for avian influenza virus testing. Once the virus is found, the relevant area can be closed and disinfected. This testing is mainly for public health and poultry trade safety.

[0029] For ecological monitoring of migratory bird routes, preferably, water, soil, and bird feces samples are collected from wetlands and other areas where migratory birds gather to test for avian influenza viruses. This is because migratory birds are a natural reservoir for avian influenza viruses and may spread the virus during migration. The goal of this monitoring is to understand the distribution and transmission dynamics of avian influenza viruses in natural ecological environments, so that preventative measures can be implemented in advance to prevent the virus from spreading to poultry or humans.

[0030] The present invention has the following beneficial effects:

[0031] The present invention designs specific crRNA and RT-RAA primers based on the relatively conserved sequence of the H7 subtype avian influenza virus. When the crRNA recognizes the target sequence, it activates the LwaCas13a protein to cleave the reporter probe. The test results are read using a lateral flow test strip (LFD), achieving the purpose of simple, efficient and visual detection of the H7 subtype avian influenza virus. The method for detecting H7 subtype avian influenza virus provided by the present invention can achieve effective amplification of the target gene by reacting at 42°C for 30 minutes. After amplification, the product is incubated in a CRISPR detection system at 37°C for 20 minutes, and the incubated product is then determined by LFD. Test results show that the nucleic acid molecule composition provided by the present invention has good specificity for H7 subtype avian influenza virus and has no cross-reaction with H1, H3, H5, H6, H9, and H10 subtype avian influenza viruses, as well as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), and duck Tembusu virus (DTMUV); the minimum detection limit can reach 10 copies / μL; the method has good repeatability and stability; the clinical detection compliance rate reaches 94.4%, and the method can be used for clinical rapid diagnosis and epidemic control of H7 subtype avian influenza. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the electrophoresis diagram of the amplification product of the optimal primer in Example 1; wherein, M. DL1000 DNA Marker; 1. H7AIV; 2. negative control.

[0033] Figure 2 This is a graph showing the relationship between the amount of LwaCas13a protein used in the CRISPR-H7 detection system and the detection effect;

[0034] Figure 3 This is a graph showing the relationship between the amount of crRNA used and the detection effect in the CRISPR-H7 detection system;

[0035] Figure 4 This is a graph showing the relationship between the amount of TaqMan probe protein used and the detection effect in the CRISPR-H7 detection system;

[0036] Figure 5 This is a graph showing the relationship between the amount of T7 RNA Polymerase used in the CRISPR-H7 detection system and the detection effect;

[0037] Figure 6 This is a graph showing the relationship between the amount of NTP Buffer Mix used in the CRISPR-H7 detection system and the detection effect;

[0038] Figure 7 This is a diagram of the specificity screening results in Example 2;

[0039] Figure 8 This is a diagram of the sensitivity screening results in Example 2. DETAILED DESCRIPTION

[0040] The present invention is further described below with reference to the embodiments, but the scope of protection claimed in the present invention is not limited to the scope described in the embodiments.

[0041] Example 1

[0042] 1. Plasmids and viruses

[0043] The H1, H3, H5, H6, H7, H9, and H10 subtype avian influenza viruses, as well as Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, and duck Tembusu virus used in the present invention are all preserved by the National Avian Influenza Laboratory (Guangzhou) of South China Agricultural University. Experiments involving live viruses are all completed in the animal biosafety level 3 (BSL3) laboratory of South China Agricultural University.

[0044] The pUC57-H7 plasmid standard containing the target gene was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0045] 2. Reagents and instruments used

[0046] Table 1 Reagent Catalog

[0047]

[0048] Table 2 Instrument catalog

[0049]

[0050] 3. Experimental steps

[0051] 3.1 RNA extraction

[0052] Total viral RNA was extracted according to the instructions for the RNA extraction kit provided by Shanghai Feijie Biotechnology Co., Ltd. The extraction steps are as follows: First, 100 μL of viral sample was placed in a 2 mL EP tube. 500 μL of lysis buffer (RA2) was then added. The lysis buffer and viral sample were thoroughly mixed by inverting the tube, gently inverting the tube 5-10 times to ensure thorough mixing. Next, the lysate was pipetted into an inner cannula with a filter membrane (for tissue samples, the supernatant should be obtained by centrifugation to avoid clogging caused by the absorption of suspended impurities). The tube was centrifuged at 12,000 rpm for 60 seconds at 4°C. The liquid in the outer cannula was then removed, and 500 μL of wash buffer (45 mL of anhydrous ethanol should be added for the first use) was added to the inner cannula. The tube was centrifuged at 13,000 rpm for 60 seconds, the liquid in the outer cannula was discarded, and the washing step was repeated once. Then, place the inner cannula back into the outer cannula and centrifuge at 13,000 rpm for 2 minutes at 4°C without adding wash buffer to remove any residual liquid. Finally, transfer the inner cannula to a 1.5 mL EP tube that has been autoclaved. Add 25 to 50 μL of eluent (or DNase / RNase-free water with a pH greater than 7.0) to the center of the filter membrane of the inner cannula. After standing at room temperature for 1 minute, centrifuge at 13,000 rpm for 60 seconds to obtain total viral RNA. The quality of the extracted RNA product is then assessed by measuring the OD260 / 280 ratio (1.8 to 2.1).

[0053] 3.2 Design of RT-RAA Primers

[0054] RT-RAA primers were designed targeting the conserved region of the HA gene of the H7 subtype avian influenza virus. The design principles are as follows:

[0055] (1) Contains the crRNA target region;

[0056] (2) Primer size is 30-35 bp;

[0057] (3) The GC content of the primers should be 40% to 60% to ensure better binding to the template and avoid the generation of more dimers and hairpin structures;

[0058] (4) The Tm value should be kept between 50 and 100°C to avoid melting difficulties;

[0059] (5) To ensure amplification efficiency, primers with product lengths of 100 to 200 bp are preferred;

[0060] (6) Avoid more than 5 consecutive C or G at the 5' end of the primer. The maximum allowed length of the mononucleotide repeat sequence is 5 nucleotides.

[0061] Based on this principle, manual design using Primer Premier 5.0 software automatically generates primer sequences based on the aforementioned design principles, leveraging its built-in algorithms and database. After design, the primers must undergo biological evaluation, such as checking their specificity, sensitivity, and risk of nonspecific amplification. Furthermore, agarose gel electrophoresis is required to verify primer performance, such as amplification efficiency and specificity, to ensure their effectiveness in practical applications.

[0062] The present application designed the optimal primers as shown in Table 3 according to the above principles, and the electrophoresis diagram of the amplified product (187 bp single band) is shown in Figure 1 .

[0063] Table 3 Optimal primers for H7 subtype avian influenza virus

[0064]

[0065] 3.3 Design of crRNA

[0066] To ensure the effectiveness of the CRISPR system in detecting avian influenza viruses, the HA gene sequence of the H7 subtype avian influenza virus published by the Global Initiative on Sharing Avian Influenza Data (GISAID) was used to analyze the conserved regions. BLAST was used to compare the crRNA sequence with other avian influenza virus subtypes and other poultry disease viruses to design crRNAs. The optimal specific crRNAs shown in Table 4 were obtained.

[0067] Table 4 Optimal specific crRNA for H7 subtype avian influenza virus

[0068]

[0069] 3.4 RT-RAA amplification and DNA purification

[0070] Reference sequences used for genetic evolution analysis, in addition to sequences identified by our laboratory, were extracted from H7 virus strain samples using an RNA extraction kit and then amplified using RT-RAA according to the instructions for the RNA Constant Temperature Rapid Amplification Kit (Basic). The specific amplification steps are as follows:

[0071] (1) Take out the required components of the kit 30 minutes in advance, melt them at room temperature, and shake to mix.

[0072] (2) Add 29.4 μL of A Buffer to each dry powder reaction tube (Note: A Buffer must be completely melted and mixed, otherwise it will affect the experimental results);

[0073] (3) Add 2 μL of upstream primer (10 μM) and 2 μL of downstream primer (10 μM) to each reaction tube;

[0074] (4) Add 2 to 14.1 μL of nucleic acid template to the reaction tube (the volume of template added can be adjusted according to actual needs, and the volume of DNase / RNase-Free Water added can be adjusted accordingly, so that the total volume of template and DNase / RNase-Free Water is 14.1 μL);

[0075] (5) Finally, add 2.5 μL of B Buffer to the reaction tube and mix thoroughly (be sure to shake the reaction tube upside down 8 to 10 times to mix thoroughly);

[0076] (6) After mixing, the reaction solution was spun (or rapidly centrifuged) to the bottom of the tube, and then the reaction tube was immediately placed in a thermostat at 42°C and incubated for 30 min.

[0077] (7) The reaction is completed and the RT-RAA amplification product is obtained.

[0078] The obtained RT-RAA amplification product was purified using an EZ-10 column-based DNA purification kit. The specific purification steps are as follows:

[0079] (1) Transfer the DNA mixture to a 1.5 mL centrifuge tube and add 3 volumes of Cleanup Solution.

[0080] (2) Place the adsorption column in a 2 mL collection tube. Transfer the mixture to the adsorption column, let it stand at room temperature for 2 minutes, and centrifuge it at 5000 rpm for 1 minute.

[0081] (3) Discard the liquid in the collection tube. Add 500 μL of Wash Solution to the adsorption column and centrifuge at 8000 rpm for 1 min. Discard the liquid in the collection tube and return the adsorption column to the original collection tube.

[0082] (4) Add 500 μL of Wash Solution to the adsorption column and centrifuge at 8000 rpm for 1 min. Discard the liquid in the collection tube and centrifuge again to remove the remaining Wash Solution.

[0083] (5) Place the adsorption column in a clean 1.5 mL centrifuge tube. Add 30–50 μL of Solution Buffer or DNase / RNase-Free Water to the center of the adsorption column and incubate at 50°C for 2 min. Centrifuge at 10,000 rpm for 1 min. The purified DNA, including PCR products, can be used directly in subsequent experiments or stored at -20°C.

[0084] After DNA purification, 5 μL of 10× Loading Buffer was added to each well. The mixture was then spotted onto a pre-prepared 2% agarose gel containing a nucleic acid dye and electrophoresed at 120 V. After electrophoresis, the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification.

[0085] 3.5 Establishment and optimization of the avian influenza virus CRISPR-H7 detection system

[0086] First, prepare the CRISPR-Cas13a reaction system: including 1.6 μL of crRNA (100 μmol / L), 0.64 μL of TaqMan probe (50 μmol / L), 0.25 μL of T7 RNA Polymerase, 1.6 μL of NTP Buffer Mix, 1 μL of Murine RNase Inhibitor (40 U / μL), and 2.0 μL of 10×T7 RNA Polymerase Buffer. Add 0.125, 0.25, 0.5, 1, 2, 4, and 8 μL of LwaCas13a protein (15 μg / mL) to the reaction mixture, respectively, add 0.6 μL of cDNA (100 ng), make up to 20 μL with DNase / RNase-Free Water, and react at 37°C for 20 min. Determine the optimal amount of LwaCas13a gene editing protein based on the criterion of the strongest fluorescence signal appearing in the shortest time of CRISPR-Cas13a detection. The optimal crRNA concentration was determined by adding 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 μL of crRNA (100 μmol / L) to the reaction system using the optimal LwaCas13a protein dosage. The CRISPR-Cas13a detection system was optimized using the sequential control variable method. The concentrations of other substances were set as follows: TaqMan probe (50 μmol / L) at 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 μL; T7 RNA polymerase at 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μL; and NTP buffer mix at 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 μL. The optimal CRISPR-Cas13a-H7 reaction system was determined.

[0087] In order to optimize the detection effect of the CRISPR detection system for H7 subtype avian influenza virus, the optimal dosage of the five key components in the system, LwaCas13a protein, crRNA, TaqMan probe, T7 RNA Polymerase, and NTP Buffer Mix, was explored and obtained as follows: Figures 2 to 7 The data shown in Table 5 were used to formulate the optimal detection system for CRISPR-Cas13a-H7.

[0088] Table 5 CRISPR-Cas13a-H7 optimal detection system

[0089]

[0090]

[0091] 3.6 How to use CRISPR-LFD nucleic acid test strips

[0092] This study used a chromatography-based double-antibody sandwich assay to detect CRISPR-Cas13a labeled probes. The probes were designed with biotin labeled on one end and 6-carboxyfluorescein (6-FAM) labeled on the other. This test strip can then be used to detect Cas enzyme cleavage products in the CRISPR system. The specific steps are as follows:

[0093] (1) Take out the corresponding number of test strips according to the number of test samples and mark them on the absorbent pad. When the volume of the amplified product is 50-100 μL, the nucleic acid product can be directly detected in the EP tube. If the product volume is less than 50 μL, it needs to be supplemented to 50 μL with DNase / RNase-Free Water, pipetted and mixed before testing.

[0094] (2) After the CRISPR system reaction is completed, open the EP tube and insert the conjugated pad end (arrow end) of the test strip into the EP tube. The liquid level must not exceed the upper end of the conjugated pad. Wait until the reading area is completely soaked (about 1-2 minutes). After the quality control line (C line) is colored, the test strip can be taken out. The test results can be directly read according to the color of the test strip.

[0095] (3) Observe the results within 10 minutes after the quality control line (T line) develops color. If the result is read after 10 minutes, it will be considered invalid.

[0096] (4) Record the test results, seal the test strips and discard them in a designated safe place.

[0097] Example 2

[0098] 1. Specificity detection

[0099] Nucleic acids were extracted from avian influenza viruses of subtypes H1, H3, H5, H6, H7, H9, and H10, as well as NDV, IBV, IBDV, and DTMUV. The extracted nucleic acids were amplified by RT-RAA, and the products were purified. The optimized CRISPR-Cas13a detection reaction system was used, with DNase / RNase-Free Water as a negative control. The reaction was incubated at 37°C for 20 minutes, and the fluorescence signal was monitored in real time to verify its specificity.

[0100] The results are as follows Figure 7 Specifically, the H7 virus strain showed a positive result; the H1, H3, H5, H6, H9, and H10 subtypes of avian influenza viruses, as well as NDV, IBV, IBDV, and DTMUV, showed negative results; and the negative control had a normal result. This indicates that the CRISPR-LFD detection method for the H7 subtype of avian influenza virus established in this study has high specificity and does not cross-react with other subtypes of avian influenza viruses or other poultry disease viruses.

[0101] 2. Sensitivity testing

[0102] The pUC57-H7 plasmid was used as a standard and diluted 10-fold with DNase / RNase-Free Water to set different copy numbers (1 to 10 8 The plasmid containing 100 copies / μL was used as a template (the amplification template was the pUC57-H7 standard plasmid) for RT-RAA amplification and CRISPR-Cas13a reaction, and a negative control was set with DNase / RNase-Free Water to test the detection limit of the reaction.

[0103] The results are as follows Figure 8 As shown, specifically: 10 copies / μL to 10 8 The results of the eight detection gradients of 10 copies / μL were positive, and the 1 copy / μL detection concentration was consistent with the negative control result and showed negative. Therefore, the sensitivity of the CRISPR-LFD detection method for H7 subtype avian influenza virus established in this study is 10 copies / μL.

[0104] 3. Compliance and clinical sample testing

[0105] To further evaluate the clinical applicability of the CRISPR-LFD detection method for H7 subtype avian influenza virus, this study used the "gold standard" for avian influenza virus detection—virus isolation and identification methods—to evaluate the concordance rate with the established CRISPR-LFD-H7 method. Based on the virus isolation and identification results, 44 clinical samples were selected, including 18 H7 subtype avian influenza viruses and 26 non-H7 subtype avian influenza viruses (2 H1 subtype, 4 H3 subtype, 6 H5 subtype, 4 H6 subtype, 5 H9 subtype, 1 H10 subtype avian influenza virus, and 4 other avian disease viruses). The clinical data are shown in Table 6. Among the 44 clinical samples with clear background, the CRISPR-LFD-H7 method detected 17 positive samples and 27 negative samples, with a concordance rate of 94.4% (17 / 18 * 100%).

[0106] The 44 clinical samples were also tested using the national standard GB / T 18936-2020 fluorescence quantitative PCR method. The results are shown in Table 6. Among the 44 known clinical samples, the fluorescence quantitative PCR method detected 17 positive samples and 27 negative samples. The concordance rate between the CRISPR-LFD-H7 method established in this study and the national standard fluorescence quantitative PCR method was 100% (17 / 17 * 100%).

[0107] Table 6 Clinical data statistics of CRISPR-LFD-H7 method

[0108]

[0109] In summary, the present invention designs a nucleic acid molecule composition for detecting H7 subtype avian influenza virus by combining designed primers and specific crRNA, and establishes a detection method for H7 subtype avian influenza virus (CRISPR-LFD-H7 method) based on this nucleic acid molecule composition. This method can be rapidly amplified under 42°C, and the detection of the target gene can be completed within 1 hour of reaction. The reaction product is added dropwise to the test strip and the test result can be observed within 3 to 5 minutes. The method has strong specificity, high sensitivity, and a minimum detection limit of up to 10 copies / μL, with good accuracy. Compared with conventional PCR, it has a detection instrument without large equipment, takes less time, and has rapid detection, realizing rapid nucleic acid amplification detection under non-laboratory conditions, providing reliable guarantee for early clinical detection and epidemiological surveys of avian influenza disease, and can specifically detect the presence of H7 subtype avian influenza virus, which can greatly reduce the occurrence of misdiagnosis of avian influenza virus infection and is conducive to its promotion and application in clinical practice.

[0110] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid molecule composition for detecting H7 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD, characterized in that: It includes an upstream primer RAA-H7-F, a downstream primer RAA-H7-R and a specific crRNA-H7; the sequence of the upstream primer RAA-H7-F is shown in SEQ ID No: 1, the sequence of the downstream primer RAA-H7-R is shown in SEQ ID No: 2, and the sequence of the specific crRNA-H7 is shown in SEQ ID No:

3.

2. A reagent for detecting H7 subtype avian influenza virus, characterized in that: The invention also comprises the nucleic acid molecule composition according to claim 1.

3. A kit for detecting H7 subtype avian influenza virus, characterized in that: The invention also comprises the nucleic acid molecule composition according to claim 1.

4. The kit according to claim 3, wherein The kit also includes LwaCas13a protein and reagents for RAA amplification.

5. A method for detecting H7 subtype avian influenza virus using the nucleic acid molecule composition according to claim 1 for non-diagnostic purposes, characterized in that: The following steps are involved: S1: Extract the viral nucleic acid template and perform RAA amplification on the viral nucleic acid template using a primer set consisting of an upstream primer RAA-H7-F and a downstream primer RAA-H7-R; S2: adding the RAA amplified product to the CRISPR-Cas13a system for incubation, wherein the CRISPR-Cas13a system includes specific crRNA-H7; S3: Detect the incubation products of CRISPR-Cas13a using LFD.

6. Use of the nucleic acid molecule composition according to claim 1 in detecting H7 subtype avian influenza virus for non-diagnostic purposes.

7. Use of the reagent according to claim 2 in detecting H7 subtype avian influenza virus for non-diagnostic purposes.

8. Use of the kit according to claim 3 or 4 in detecting H7 subtype avian influenza virus for non-diagnostic purposes.

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