Nucleic acid molecule composition, reagent and kit for detecting H9 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD and application

Abstract

The invention relates to the technical field of molecular biology, in particular to a nucleic acid molecule composition for detecting H9 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD, a reagent, a kit and application. The nucleic acid molecule composition comprises an upstream primer RAA-H9-F, a downstream primer RAA-H9-R and a specific crRNA-H9, wherein the upstream primer RAA-H9-F is used for detecting the nucleic acid molecule; the sequence of the upstream primer RAA-H9-F is as shown in SEQ ID No: 1, the sequence of the downstream primer RAA-H9-R is as shown in SEQ ID No: 2, and the sequence of the specific crRNA-H9 is as shown in SEQ ID No: 3. The nucleic acid molecule composition provided by the invention can realize visual detection of the H9 subtype avian influenza virus, is high in detection accuracy, short in time consumption, good in specificity and high in sensitivity, provides a basis for rapid, simple and accurate diagnosis of the H9 subtype avian influenza virus, and meets the requirements 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 H9 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 huge economic losses to the chicken industry. More importantly, AIV can be transmitted across species between mammals, posing a potential threat to human health and safety.

[0003] Avian influenza virus (AIV) belongs to influenza A virus. According to the difference in antigenicity of HA and neuraminidase (NA) proteins, it can be divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11). Theoretically, they can be combined into 198 different subtypes. These subtypes may differ in terms of pathogenicity and transmission ability of the virus. For example, subtypes such as H5N1 and H7N9 are highly pathogenic avian influenza subtypes that pose a greater threat to human health and poultry farming.

[0004] At present, nucleic acid detection technology is a 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 HA or NA gene) are amplified using specific primers. If there is avian influenza virus nucleic acid in the sample, after PCR amplification, the corresponding nucleic acid fragments can be detected by 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 infection and transmission risk of the virus. This method has high sensitivity and strong specificity, and can detect low concentrations of viruses, but it requires professional equipment and technicians, and the detection cost is relatively high.

[0005] For the H9 subtype avian influenza virus, there is currently no low-cost detection method that has high sensitivity, good specificity, and is suitable for clinical use. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a nucleic acid molecule composition for detecting H9 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD. The nucleic acid molecule composition provided by the present invention can realize the visual detection of H9 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 the rapid, simple and accurate diagnosis of H9 subtype avian influenza virus and meets the needs of clinical detection.

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

[0008] A nucleic acid molecule composition for detecting H9 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD, comprising an upstream primer RAA-H9-F, a downstream primer RAA-H9-R and a specific crRNA-H9;

[0009] The sequence of the upstream primer RAA-H9-F is: 5'-TATGGCAGCAGATAGAGAATCAACTCAAAA-3' (SEQ ID NO: 1),

[0010] The sequence of the downstream primer RAA-H9-R is: 5'-ATGCTCATCAAGTGTTTTCTGGTTTTCAAG-3' (SEQ ID NO: 2),

[0011] The sequence of the specific crRNA-H9 is: 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUAUUCACUUUGGAUGUUAUUUUUAUCAA-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 in the conserved region of the gene sequence for detection of H9 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 the lateral flow test strip, 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 a kit for detecting H9 subtype avian influenza virus, and the reagent or kit containing the nucleic acid molecule composition belongs to the protection scope of the present invention.

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

[0015] The present invention also provides a method for detecting H9 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-H9-F and a downstream primer RAA-H9-R;

[0017] S2: adding the RAA amplification product to the CRISPR-Cas13a system for incubation, wherein the CRISPR-Cas13a system includes specific crRNA-H9;

[0018] S3: Use LFD to detect the incubation products of CRISPR-Cas13a.

[0019] Preferably, the RAA amplification system in step S1 includes: A Buffer 29.4 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, nucleic acid template 2-14.1 μL, B Buffer 2.5 μL, and DNase / RNase-Free Wate is added to 50 μL.

[0020] Preferably, the RAA amplification system in step S1 is reacted at 42° C. for 30 min.

[0021] Preferably, the CRISPR-Cas13a system in step S2 includes: LwaCas13a protein (15 μg / mL) 0.5 μL, crRNA (100 μmol / L) 0.2 μL, probe (50 μmol / L) 1.28 μL, T7 RNA Polymerase 0.1 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.

[0022] Furthermore, the incubation temperature in step S2 is 37° C., and the incubation time is 20 min.

[0023] Furthermore, when the CRISPR-Cas13a incubation product was detected using LFD, two red bands appeared on LFD for positive results, one in the quality control area and one in the detection area. The 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 ​​LFD and there was no red band in the detection area, indicating a negative result. The negative result indicated that the amplified product did not contain the detection fragment.

[0024] The above-mentioned preferred reaction conditions can further improve the detection efficiency of H9 subtype avian influenza virus.

[0025] The present invention also provides the use of the nucleic acid molecule composition, reagent and kit in non-diagnostic detection of avian H9 subtype avian influenza.

[0026] Preferably, in the application of poultry farming environment monitoring, the presence of avian influenza virus nucleic acid in environmental samples is regularly detected to determine whether the virus is present in the environment, which can be used as an indicator of whether the cleaning and disinfection measures of the farm are effective. 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 prevent the outbreak of avian influenza.

[0027] 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 detection is mainly for public health and poultry trade safety.

[0028] Preferably, in the application of ecological monitoring of migratory bird migration routes, water samples, soil samples and migratory bird feces samples are collected in wetlands and other migratory bird gathering areas to detect avian influenza virus. This is because migratory birds are one of the natural hosts of avian influenza virus, and they may spread the virus during migration. The purpose of this monitoring is to understand the distribution and transmission dynamics of avian influenza virus in the natural ecological environment, so as to take preventive and control measures in advance to prevent the virus from spreading to poultry or humans.

[0029] The present invention has the following beneficial effects:

[0030] The present invention designs specific crRNA and RT-RAA primers according to the relatively conservative sequence of H9 subtype avian influenza virus. When crRNA recognizes the target sequence, LwaCas13a protein is activated to cut the reporter probe. As the test results are read by lateral flow test strips (LFD), the purpose of simple, efficient and visual detection of H9 subtype avian influenza virus is achieved. The method for detecting H9 subtype avian influenza virus provided by the invention can realize effective amplification of the target gene by reacting for 30 minutes under the condition of 42°C, and incubate the product for 20 minutes under the CRISPR detection system at 37°C after amplification, and then determine the product by LFD after incubation; the test results show that the nucleic acid molecule composition provided by the invention has good specificity for H9 subtype avian influenza virus, and has no cross reaction with H1, H3, H5, H6, H7, H10 subtype avian influenza virus and Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), 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 92.0%, and the method can be used for clinical rapid diagnosis and epidemic purification of H9 subtype avian influenza. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the electrophoresis diagram of the amplification product of the optimal primer in Example 1; wherein, M. DL1000 DNA Marker; 1. H9AIV; 2. negative control.

[0032] Figure 2 This is a graph showing the relationship between the amount of LwaCas13a protein used in the CRISPR-H9 detection system and the detection effect;

[0033] Figure 3 This is a graph showing the relationship between the amount of crRNA used and the detection effect in the CRISPR-H9 detection system;

[0034] Figure 4 This is a graph showing the relationship between the amount of TaqMan probe protein used and the detection effect in the CRISPR-H9 detection system;

[0035] Figure 5 This is a graph showing the relationship between the amount of T7 RNA Polymerase used and the detection effect in the CRISPR-H9 detection system;

[0036] Figure 6 This is a graph showing the relationship between the amount of NTP Buffer Mix used in the CRISPR-H9 detection system and the detection effect;

[0037] Figure 7 is a graph of the specific screening results in Example 2;

[0038] Figure 8 This is a diagram of the sensitivity screening results in Example 2. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0040] Example 1

[0041] 1. Plasmids and viruses

[0042] 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 Professional Laboratory (Guangzhou) of South China Agricultural University, and the experiments involving live viruses are all completed in the Animal Biosafety Level 3 (BSL3) Laboratory of South China Agricultural University.

[0043] The pUC57-H9 plasmid standard containing the target gene was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0044] 2. Reagents and instruments used

[0045] Table 1 Reagent Catalog

[0046]

[0047] Table 2 Instrument Catalog

[0048]

[0049]

[0050] 3. Experimental steps

[0051] 3.1 RNA extraction

[0052] According to the instructions of the RNA extraction kit provided by Shanghai Feijie Biotechnology Co., Ltd., the extraction process of total viral RNA was performed. The extraction steps are as follows: First, 100 μL of the virus sample was placed in a 2 mL EP tube, followed by the addition of 500 μL of lysis buffer (RA2), and the lysis buffer and the virus sample were mixed by inversion, and gently inverted 5 to 10 times to ensure sufficient mixing. Then, all the lysate was aspirated into the inner cannula with a filter membrane (for tissue samples, centrifugation was required to obtain the supernatant to avoid clogging caused by the inhalation of suspended impurities), and centrifuged at 12000 rpm for 60 s at 4 ° C. Subsequently, the liquid in the outer cannula was removed, and 500 μL of wash buffer (Wash Buffer, 45 mL of anhydrous ethanol was added to the inner cannula for the first use) was added. Centrifuge at 13000 rpm for 60 s, discard the liquid in the outer cannula, and repeat this washing step once. Then, the inner cannula was put back into the outer cannula, and no washing solution was added. The tube was centrifuged at 13000 rpm for 2 minutes at 4°C to remove the residual liquid. Finally, the inner cannula was transferred to a 1.5 mL EP tube that had been treated with high pressure. 25 to 50 μL of eluent (or DNase / RNase-Free Water with a pH value greater than 7.0) was added to the central area of ​​the filter membrane of the inner cannula, and the total viral RNA was obtained by centrifugation at 13000 rpm for 60 seconds after standing at room temperature for 1 minute. The quality of the extracted RNA product was 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 for the conserved region of the HA gene of the H9 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 better bind to the template and avoid generating more dimers and hairpin structures;

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

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

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

[0061] According to this principle, the use of Primer Premier 5.0 software for manual design can automatically generate primer sequences based on the above design principles using its built-in algorithms and databases. After the design is completed, the primers need to be biologically evaluated, such as checking the specificity, sensitivity, and nonspecific amplification risk of the primers. In addition, the performance of the primers, such as amplification efficiency and specificity, needs to be verified through agarose gel electrophoresis experiments to ensure the effectiveness of the primers in practical applications.

[0062] The present application designed the optimal primers as shown in Table 3 according to the above principles. The electrophoresis diagram of the amplified product (180 bp single band) is shown in Figure 1 .

[0063] Table 3 Optimal primers for H9 subtype avian influenza virus

[0064]

[0065] 3.3 Design of crRNA

[0066] In order to ensure the effectiveness of the CRISPR system in detecting avian influenza virus, the HA gene sequence of the H9 subtype avian influenza virus published by the Global Initiative on Sharing Avian Influenza Data (GISAID) was analyzed for conserved regions, and the crRNA sequence was specifically compared with other avian influenza virus subtypes and other avian disease viruses using BLAST to design crRNA, and the optimal specific crRNA shown in Table 4 was obtained.

[0067] Table 4 Optimal specific crRNA for H9 subtype avian influenza virus

[0068]

[0069] 3.4 RT-RAA amplification and DNA purification

[0070] The reference sequences used for genetic evolution analysis, in addition to the sequences identified by this laboratory, were extracted from the H9 virus strain sample nucleic acid using an RNA extraction kit, and then RT-RAA amplified the template according to the instructions of the RNA Constant Temperature Rapid Amplification Kit (Basic Type). The specific amplification steps are as follows:

[0071] (1) Take out the required components of the kit 30 minutes in advance, thaw 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 in sequence (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 thermostatic device and incubated at 42°C 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 by EZ-10 column 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 above mixed solution to the adsorption column, let stand at room temperature for 2 min, and centrifuge at 5000 rpm for 1 min.

[0081] (3) Pour out the liquid in the collection tube. Add 500 μL Wash Solution to the adsorption column and centrifuge at 8000 rpm for 1 min. Pour out the liquid in the collection tube and put the adsorption column back into the original collection tube.

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

[0083] (5) Place the adsorption column in a clean 1.5 mL centrifuge tube. Add 30-50 μL of Eliution Buffer or DNase / RNase-Free Water to the center of the adsorption membrane 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 Loading Buffer (10×) was added to each well, and the mixture was spotted into the wells of the 2% agarose gel containing nucleic acid dye prepared in advance, and electrophoresis was performed under a constant voltage of 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-H9 detection system

[0086] First, prepare the CRISPR-Cas13a-H9 reaction system: including 1.6μL crRNA (100μmol / L), 0.64μL TaqMan probe (25μmol / L), 0.25μL T7 RNA Polymerase, 1.6μL NTP Buffer Mix, 1μL Murine RNase Inhibitor (40U / μL), and 2.0μL 10×T7 RNA Polymerase Buffer. Add 0.25, 0.5, 1, 2, 4, and 8μL LwaCas13a protein (60μg / mL) to the reaction mixture, respectively, add 0.6μL cDNA (100ng), make up to 20μL with DNase / RNase-Free Water, and react at 37℃ for 20min. Determine the optimal amount of LwaCas13a gene editing protein based on the criterion of the strongest fluorescence signal in the shortest time of CRISPR-Cas13a detection. The reaction system was prepared with the optimal LwaCas13a protein dosage, and 0.1, 0.2, 0.4, 0.8, and 1.6 μL crRNA (100 μmol / L) were added to the reaction system to determine the optimal crRNA concentration. The CRISPR-Cas13a detection system was optimized using the sequential control variable method, and the concentrations of other substances were set as follows: TaqMan probe (25 μmol / L) 0.16, 0.32, 0.64, 1.28, 2.56 μL; T7 RNA Polymerase 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 μL; NTP Buffer Mix 0.2, 0.4, 0.8, 1.2, 1.6, 2.0 μL, and finally the optimal reaction system of CRISPR-Cas13a-H9 was determined.

[0087] In order to optimize the detection effect of the CRISPR detection system on the H9 subtype avian influenza virus, the optimal dosage of the five key components in the system, namely LwaCas13a protein, crRNA, TaqMan probe, T7 RNA Polymerase, and NTP Buffer Mix, was explored and obtained as follows: Figure 2 to Figure 7 The data shown in the figure are used to sort out the optimal detection system of CRISPR-Cas13a-H9 as shown in Table 5.

[0088] Table 5 CRISPR-Cas13a-H9 optimal detection system

[0089]

[0090]

[0091] 3.6 Usage of CRISPR-LFD nucleic acid test strips

[0092] This study used a chromatography double antibody sandwich method to detect the labeled probe of CRISPR-Cas13a. When designing the probe, one end is labeled with biotin (Biotin) and the other end is labeled with 6-carboxyfluorescein (6-FAM). This test strip can be used to detect the 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 absorption pad. When the volume of the amplified product is 50-100 μL, the nucleic acid product can be directly detected in the EP tube. When the product volume is less than 50 μL, DNase / RNase-Free Water should be added to 50 μL, and the test should be performed after pipetting and mixing.

[0094] (2) After the CRISPR system reaction is completed, open the EP tube and insert the binding pad end (arrow end) of the test strip into the EP tube. The liquid level must not exceed the upper end of the binding pad. After the judgment area is completely soaked (about 1 to 2 minutes), the test strip can be taken out after the quality control line (C line) is colored. 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. If the results are read after 10 minutes, they are considered invalid.

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

[0097] Example 2

[0098] 1. Specificity detection

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

[0100] The results are as follows Figure 7 As shown in the figure, specifically: H9 virus strain showed positive results; H1, H3, H5, H6, H7, H10 subtype avian influenza viruses and NDV, IBV, IBDV, DTMUV showed negative results; negative control results were normal. This shows that the CRISPR-LFD detection method for H9 subtype avian influenza virus established in this study has good specificity and has no cross-reaction with other subtypes of avian influenza virus and other poultry disease viruses.

[0101] 2. Sensitivity testing

[0102] The pUC57-H9 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 for RT-RAA amplification and CRISPR-Cas13a reaction, and DNase / RNase-Free Water was used to set up a negative control 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 8 detection gradients of 10 copies / μL were positive, and the 1 copy / μL detection concentration was consistent with the negative control result and was negative. Therefore, the sensitivity of the H9 subtype avian influenza virus CRISPR-LFD detection method established in this study is 10 copies / μL.

[0104] 3. Compliance and clinical sample testing

[0105] In order to further evaluate the clinical application ability of the CRISPR-LFD detection method for H9 subtype avian influenza virus, this study used the "gold standard" for avian influenza virus detection - virus isolation and identification method and the established CRISPR-LFD-H9 method for compliance evaluation. According to the results of virus isolation and identification, 52 clinical samples were selected, including 25 H9 subtype avian influenza viruses and 27 non-H9 subtype avian influenza viruses (2 H1 subtypes, 4 H3 subtypes, 6 H5 subtypes, 4 H6 subtypes, 5 H9 subtypes, 1 H10 subtype avian influenza virus, and 5 other poultry disease viruses). The clinical data are shown in Table 6. Among the 52 clinical samples with clear background, the CRISPR-LFD-H9 method detected 23 positive samples and 29 negative samples, with a compliance rate of 92.0% (23 / 25*100%).

[0106] At the same time, the national standard GB / T 18936-2020 fluorescence quantitative PCR method was used to detect the 52 clinical samples. The results are shown in Table 6. Among the 52 known clinical samples, the fluorescence quantitative PCR method detected 24 positive samples and 28 negative samples. The conformity rate of the CRISPR-LFD-H9 method established in this study with the national standard fluorescence quantitative PCR method was 95.8% (23 / 24*100%).

[0107] Table 6 Clinical data statistics of CRISPR-LFD-H9 method

[0108]

[0109] In summary, the present invention designs a nucleic acid molecule composition for detecting H9 subtype avian influenza virus by combining designed primers and specific crRNA, and establishes a detection method for H9 subtype avian influenza virus (CRISPR-LFD-H9 method) based on the nucleic acid molecule composition. The 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 dripped onto 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, and good accuracy. Compared with conventional PCR, the method has a detection instrument without large equipment, shorter time consumption, and rapid detection, and realizes rapid nucleic acid amplification detection under non-laboratory conditions, which provides reliable guarantee for early clinical detection and epidemiological investigation of avian influenza disease, and can specifically detect the presence of H9 subtype avian influenza virus, which can greatly reduce the occurrence of misdiagnosis of avian influenza virus infection, and is conducive to promotion and application in clinical practice.

[0110] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A nucleic acid molecule composition for detecting H9 subtype avian influenza virus based on RT-RAA / CRISPR-Cas13a / LFD, characterized in that: It includes an upstream primer RAA-H9-F, a downstream primer RAA-H9-R and a specific crRNA-H9; the sequence of the upstream primer RAA-H9-F is shown in SEQ ID No: 1, the sequence of the downstream primer RAA-H9-R is shown in SEQ ID No: 2, and the sequence of the specific crRNA-H9 is shown in SEQ ID No:

3.

2. A reagent for detecting H9 subtype avian influenza, characterized in that: The invention comprises the nucleic acid molecule composition according to claim 1.

3. A kit for detecting H9 subtype avian influenza, characterized in that: The invention comprises the nucleic acid molecule composition according to claim 1.

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

5. A method for detecting H9 subtype avian influenza virus using the nucleic acid molecule composition according to claim 1, 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-H9-F and a downstream primer RAA-H9-R; S2: adding the RAA amplification product to the CRISPR-Cas13a system for incubation, wherein the CRISPR-Cas13a system includes specific crRNA-H9; S3: Use LFD to detect the incubation products of CRISPR-Cas13a.

6. Use of the nucleic acid molecule composition according to claim 1 in non-diagnostic detection of avian H9 subtype avian influenza.

7. Use of the reagent according to claim 2 in non-diagnostic detection of avian H9 subtype avian influenza.

8. Use of the kit according to claim 3 or 4 in non-diagnostic detection of avian influenza H9 subtype.

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