H7 subtype avian influenza virus detection method

Abstract

The invention provides a detection method of an H7 subtype avian influenza virus to realize safe, specific, rapid, sensitive, simple and rapid detection of the H7 subtype avian influenza virus so as to make up for the defects of the existing traditional detection technology. Compared with RT-PCR (Reverse Transcription-Polymerase Chain Reaction), fluorescent RT-PCR and fluorescent RT-RAA (Reverse Transcription-Reverse Amplification Amplification) methods, a nucleic acid constant-temperature test strip detection method does not need a complicated temperature change process, an amplification reaction can be rapidly completed under a constant-temperature condition, a reaction product does not need electrophoresis or fluorescent signal analysis, test strip detection can be performed only through simple dilution treatment, and a detection result can be observed in real time. The method is simple to operate, gets rid of the constraint of complex instruments, greatly shortens the reaction time, and is very suitable for on-site emergency rapid detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus microorganism detection, and particularly relates to a detection method of H7 subtype avian influenza virus. BACKGROUND

[0002] Avian influenza virus (AIV) is a single-stranded negative-strand RNA virus, which is composed of 8 independent RNA segments. One of the significant biological characteristics is that there are many subtypes and frequent variations. So far, thousands of strains with different virulence have been isolated from birds. According to the antigenic differences of hemagglutinin (HA) and neuraminidase (NA) on the surface of the virus particles, the AIV is divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11). Currently, the highly pathogenic avian influenza (HPAI) caused by H5 and H7 subtypes of avian influenza virus not only threatens the health of birds but also causes public health problems.

[0003] At present, the detection of H7 subtype avian influenza virus is mainly carried out by real-time fluorescent quantitative RT-PCR method and RT-PCR method. Both of the two methods need precise experimental instruments, and the time consumption is relatively long. The detection is suitable for laboratory detection. From sample collection and transportation to laboratory to obtain the detection result, at least one day is needed, which delays the confirmation and prevention and control of the epidemic. SUMMARY

[0004] The purpose of the present application is to provide a detection method of H7 subtype avian influenza virus, so as to realize the safe, specific, sensitive, rapid and convenient detection of H7 subtype avian influenza virus.

[0005] The present application first provides a primer pair and a probe set for detecting H7 subtype avian influenza virus, which are primer pair H7 forward and H7 reverse and probe H7 probe for identifying the HA gene of H7 subtype avian influenza virus. The primer pair and probe sequence information are as follows: Forward primer H7 forward: 5'-Biotin-CCTATGATCACAGYAAATACAGGGAAGAGGCAAT-3'(SEQ ID NO:1) Reverse primer H7 reverse: 5'-CTAGAAGTATGAAACATGATGCCCCGAAGCTAA-3'(SEQ ID NO:2) Probe H7 probe: 5'-FAM-CCCGAAGCTAAACCAAAGTATCACATCTT / THF / GTAGCCGCTGCTTAGTT-[C3-spacer]-3'; Wherein, the upstream primer 5' end is added with Biotin (BIO) modification, the probe 5' end is added with FAM fluorescence modification, the 3' end is added with C3 spacer modification, and the 30th base from the 5' end of the probe is substituted with Tetrahydrofuran (THF). The application also provides a product for detecting H7 subtype avian influenza virus, which uses the primer pair and the probe described above; and the product further comprises reagents for reverse transcription recombinase-mediated isothermal amplification and a nucleic acid detection test strip.

[0006] In another aspect, the application provides a nucleic acid isothermal amplification test strip detection method for detecting H7 subtype avian influenza virus in a clinical sample, which comprises the following steps: 1) RT-RAA reaction An RT-RAA reaction system 50 μl is prepared, containing reaction buffer 25 μl, 10 pmol / μl H7 forward and H7 reverse each 2.1 μl, 10 pmol / μl H7 probe 0.6 μl, RNase Free dH2O 14.2 μl, magnesium acetate 5.0 μl, RT test strip basic reaction unit (containing freeze-dried powder of recombinase, reverse transcriptase and polymerase), and sample RNA template 1 μl to be detected. The detection reaction condition is set as: constant temperature amplification at 39℃ for 20 min.

[0007] 2) Test strip detection Take 15 μl of the reaction product into a 1.5 ml centrifuge tube, dilute 10 times by adding 135 μl of pure water, mix thoroughly, and then place the nucleic acid test strip into the centrifuge tube rack, with the sample end inserted below the liquid level. After standing for 7-10 min, the result is read.

[0008] 3) Result determination Quality control standard: the test strip control band appears a color band, and the result is valid; the test strip control band does not appear a color band, and the result is invalid. The negative control appears no amplification curve, and the positive control appears an amplification curve, and the experimental data is valid, otherwise the experimental result is considered invalid.

[0009] Result description and determination: the test strip control band and the detection band both appear color bands, and the sample is judged as positive; the test strip control band appears a color band and the detection band has no color band, and the sample is judged as negative.

[0010] The application provides a method for rapidly detecting H7 subtype avian influenza virus based on nucleic acid isothermal amplification and test strip detection technology, so as to realize safe, specific, rapid, sensitive and simple rapid detection of H7 subtype avian influenza virus, and make up for the deficiency of the existing traditional detection technology. Compared with RT-PCR, fluorescent RT-PCR and fluorescent RT-RAA method, the nucleic acid isothermal test strip detection method does not need complex temperature change process, can rapidly complete the amplification reaction under constant temperature condition, and the reaction product does not need electrophoresis or fluorescence signal analysis, but only needs simple dilution treatment to carry out test strip detection and observe the detection result in real time. The method is simple in operation, free from the shackles of complex instruments, greatly shortens the reaction time, and is very suitable for on-site emergency rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 : the sensitivity detection result graph of the detection method in the application; Figure 2 : the specificity detection result graph of the detection method in the application. DETAILED DESCRIPTION

[0012] The application combines RAA technology and nucleic acid test strip technology to establish a nucleic acid isothermal amplification test strip detection method, which has the advantages of simple operation, short time, and no need for precise instruments, and can be used for on-site rapid detection of the disease in farms, live poultry trading markets and quarantine stations, so as to gain valuable time for early prevention and control of the disease.

[0013] The application will be described in detail below in combination with examples.

[0014] Example 1: Design and screening of H7 subtype avian influenza virus RT-RAA detection primer and probe The primer is designed according to the hemagglutinin coding gene HA gene of H7 subtype avian influenza virus, and the segment where the primer probe is located needs to be able to comprehensively cover the H7 subtype avian influenza virus. Therefore, 918 published HA gene sequences of H7 subtype avian influenza virus are found through NCBI, a conservative region is analyzed according to gene comparison, and after homology analysis, a specific conservative fragment is selected for fluorescence quantitative detection primer and probe design. Meanwhile, the RAA test strip detection method has specific requirements for the length of the primer and the probe, the primer length is 30-35 bp, and the probe length is 46-52 bp (at least 30 bases from the 5' end to the THF site, and at least 15 bases from the THF site to the 3' end). Because the primer and the probe are relatively long in length, the GC content of the primer and the probe, and the secondary structure and primer dimer which may affect the experimental effect also need to be considered. Therefore, in order to ensure the amplification efficiency, the design and screening of the primer and the probe have high requirements.

[0015] Two upstream primers and two downstream primers were designed and combined to screen the best primers. The 5' end of the upstream primers was modified with Biotin. The primer sequences were as follows: Upstream primer H7-F1: 5'-Biotin-CCTATGATCACAGYAAATACAGGGAAGAGGCAAT-3', Downstream primer H7-F2: 5'-Biotin-CACAGCAAATACAGGGAAGAGGCAATGCAAAATAG-3'(35bp); Downstream primer H7-R1: 5'-GAAGTATGAAACATGATGCCCCGAAGCTAA-3'(30bp) Downstream primer H7-R2: 5'-CTAGAAGTATGAAACATGATGCCCCGAAGCTAA-3'(33bp) Two probes were designed to screen the best probe. The 5' end of the probes was modified with FAM fluorescence, and the 3' end was modified with C3 spacer. The 30th base of the 5' end of H7-P1 probe was replaced with tetrahydrofuran (THF), and the 33rd base of the 5' end of H7-P2 probe was replaced with tetrahydrofuran (THF). The modified probe sequences were as follows: H7-P1: 5'-FAM-CCCGAAGCTAAACCAAAGTATCACATCTT / THF / GTAGCCGCTGCTTAGTT -[C3-spacer]-3' H7-P2: 5'-FAM-CAAAGTATCACATCTTTGTAGCCGCTGCTTAG / THF / GACTGGGTCAATCTG[C3-spacer] -3' The primers and probes were combined to form six combinations to screen the best primer and probe combination.

[0016] Combination 1: H7-F1, H7-R1, and H7-P1 Combination 2: H7-F1, H7-R2, and H7-P1 Combination 3: H7-F1, H7-R1, and H7-P2 Combination 4: H7-F2, H7-R1, and H7-P1 Combination 5: H7-F2, H7-R2, and H7-P1 Combination 6: H7-F2, H7-R2 and H7-P2 The screening of primer pairs and probes includes the following steps 1) Preparation of RT-RAA reaction system The reaction buffer 25 μl, 10 pmol / μl H7 forward and H7 reverse 2.1 μl each, 10 pmol / μl H7 probe 0.6 μl, RNase Free dH2O 14.2 μl, magnesium acetate 5.0 μl, RT test strip basic reaction unit (containing freeze-dried powder of recombinase, reverse transcriptase and polymerase), sample RNA template to be detected 1 μl. The detection reaction condition is set as: constant temperature amplification at 39°C for 20 min.

[0017] 2) Test strip detection Take 15 μl of the reaction product into a 1.5 ml centrifuge tube, dilute 10 times by adding 135 μl of pure water, mix thoroughly, and then place it on the centrifuge tube rack. Take out the nucleic acid test strip, insert the sample end below the liquid level in the centrifuge tube, and stand for 7-10 min to read the results.

[0018] 3) Result determination Quality control standard: the test strip control band appears color band, the result is valid, the test strip control band does not appear color band, the result is invalid. The negative control appears no amplification curve, and the positive control appears amplification curve, then the experimental data is valid, otherwise the experimental result is considered invalid.

[0019] Result description and determination: the test strip control band and detection band appear color band, the sample is judged as positive; the test strip control band appears color band and the detection band has no color band, the sample is judged as negative.

[0020] The experimental results show that under the same reaction conditions, the amplification efficiency of H7 combination 2 is better than that of other combinations, therefore the primer pair and probe of H7 combination 2 are selected as the optimum, and the sequence information of the finally determined primer and probe is as follows: Forward primer H7 forward: 5'-Biotin-CACAGCAAATACAGGGAAGAGGCAATGCAAAATAG-3'(SEQ ID NO: 1) Reverse primer H7 reverse: 5'-CTAGAAGTATGAAACATGATGCCCCGAAGCTAA-3'(SEQ ID NO: 2) Probe H7 probe: 5′-FAM-CCCGAAGCTAAACCAAAGTATCACATCTT / THF / GTAGCCGCTGCTTAGTT -[C3-spacer]-3′ Example 2: Detection sensitivity and specificity 1. Detection sensitivity Six groups of H7 subtype avian influenza virus RNA templates with different concentrations were set up for nucleic acid isothermal test strip detection.

[0021] H7 subtype influenza virus RNA was extracted according to the instructions of the RNA extraction kit, and digital PCR quantification was performed using the universal fluorescence detection method for avian influenza virus recommended in the national standard "Avian Influenza Diagnostic Technology" (GB / / T18936-2020). The concentration of the extracted viral RNA was determined and diluted in a 10-fold gradient to 10 5 copies / μl, 10 4 copies / μl, 10 3 copies / μl, 10 2 copies / μl, 10 1 1 μl of each of the 100 copies / μl, 1 copy / μl, and 0.1 copy / μl samples was used as the reaction template. RT-RAA nucleic acid amplification was performed according to the aforementioned loading method. 15 μl of the reaction product was transferred to a 1.5 ml centrifuge tube and diluted 10-fold with 135 μl of purified water. Mix thoroughly and place in a centrifuge tube rack. Remove the nucleic acid test strip and insert the sample tip below the liquid level in the centrifuge tube. Allow to stand for 7-10 minutes and read the results.

[0022] The results showed that the screened primer and probe combination could ensure the sensitivity of detection, and the detection sensitivity was 1 copy / reaction at the final RNA concentration.

[0023] 2. Detection specificity Add the components to the reaction system described above, using RNA from H1N2, H3N2, H4N2, H5N1, H5N2, H5N6, H6N2, H7N3, H7N9, H9N2, H10N7, H11N2, Newcastle disease virus, and infectious bronchitis virus as templates. The RT-RAA assay reaction conditions are: amplification at 39°C for 20 minutes. Transfer 15 μl of the reaction product to a 1.5 ml centrifuge tube, dilute 10-fold with 135 μl of pure water, mix thoroughly, and place in a centrifuge tube rack. Remove the nucleic acid test strip and insert the sample tip below the liquid level in the centrifuge tube. Let stand for 7-10 minutes before reading the result.

[0024] The results show that color bands appear on all test strip control lines, indicating that the test results are valid. Among them, the detection lines of H7N3 subtype avian influenza virus and H7N9 subtype avian influenza virus test strips appear color bands, which are determined to be positive, and the detection lines of other samples do not appear color lines, which are determined to be negative. The results show that the H7 subtype avian influenza virus nucleic acid isothermal test strip detection method established in the application can realize specific detection of H7 subtype avian influenza virus, and does not have cross reaction with other related viruses.

[0025] Example 3: Detection application of actual samples 1. Sample collection: Pharyngeal swab samples of various poultry in 3 live poultry wholesale markets were collected, with a total of 90 samples, including 60 chicken samples, 24 duck samples and 6 goose samples. PBS solution (pH 7.0-7.4, 0.01 mol / L) was used as a preservative solution (containing penicillin 2000 IU / ml, streptomycin 2000 IU / ml, nystatin 1000 IU / ml, BSA 5 mg / ml). After sample collection, the samples were sealed in an ice-cooled incubator and sent to the laboratory for processing within 24 h or stored at -70℃.

[0026] 2. Sample preparation The cotton swab was placed in a centrifuge tube containing 1 ml of sample preservative solution, vortexed, centrifuged at 10000 r / min for 5 min at 4℃, and the supernatant was used for nucleic acid extraction.

[0027] 3. Nucleic acid extraction The extracted RNA was stored at -70℃ for standby use.

[0028] 4. Identification 4.1 Comparative method: referring to the H7 subtype avian influenza virus fluorescence RT-PCR detection method in the national standard “Highly Pathogenic Avian Influenza Diagnosis Technology” (GB / T 18936-2020), as a comparative method.

[0029] 4.2 Sample detection The collected clinical samples were detected by the national standard method and the method of the application.

[0030] 4.3 Detection results The results show that the coincidence rate of the detection results by the national standard method and the method in the application is 100%.

[0031] Table 1: Data table of detection results

[0032] In summary, the method in the application can quickly and accurately identify the H7 subtype avian influenza virus, and meets the current demand for detection of the H7 subtype avian influenza virus. Meanwhile, the method in the application adopts the constant temperature detection technology and the nucleic acid test strip technology, is simple to operate, short in time consumption, and can realize on-site rapid detection of the H7 subtype avian influenza virus.

Claims

1. A primer pair and probe set for detecting H7 subtype avian influenza virus, characterized in that: The primer pair and probe set, wherein the sequence of the forward primer is as follows: 5′- CCTATGATCACAGYAAATACAGGGAAGAGGCAAT-3′; The sequence of the reverse primer is as follows: 5′-CTAGAAGTATGAAACATGATGCCCCGAAGCTAA -3′; The sequences of the probes are as follows: 5′-CCCGAAGCTAAACCAAAGTATCACATCTT / THF / GTAGCCGCTGCTTAGTT-[C3-spacer]-3′.

2. The primer pair and probe set according to claim 1, wherein The 5′ end of the upstream primer is modified by adding biotin.

3. The primer pair and probe set according to claim 1, wherein The 5' end of the probe is modified with FAM fluorescence, the 3' end is modified with a C3 spacer, and the 30th base from the 5' end of the probe is substituted with tetrahydrofuran.

4. Use of the primer pair and probe set according to claim 1 in preparing a product for detecting H7 subtype avian influenza virus.

5. A product for detecting H7 subtype avian influenza virus, characterized in that: The product comprises the primer pair and probe set according to claim 1.

6. The product according to claim 5, wherein The product also contains a reagent for reverse transcription recombinase-mediated isothermal amplification and a nucleic acid detection test strip.

7. A method for detecting H7 subtype avian influenza virus in clinical samples, characterized in that: The method is to use the preparation according to claim 6 for detection.

8. The method according to claim 7, wherein The method comprises the following steps: 1) RT-RAA reaction Prepare a 50 μl RT-RAA reaction system containing 25 μl of reaction buffer, 2.1 μl each of 10 pmol / μl H7 forward and H7 reverse, 0.6 μl of 10 pmol / μl H7 probe, 14.2 μl of RNase-free dH2O, 5.0 μl of magnesium acetate, a basic reaction unit of an RT test strip, and 1 μl of the sample RNA template to be tested. The reaction conditions were set as follows: isothermal amplification at 39°C for 20 min. 2) Test strip detection Take 15μl of the reaction product into a 1.5ml centrifuge tube, add 135μl of pure water to dilute 10 times, mix thoroughly and place on a centrifuge tube rack, take out the nucleic acid test strip, insert the sample end into the centrifuge tube below the liquid level, let it stand for 7-10 minutes and read the results; 3) Result determination Quality control standard: If the control strip of the test strip shows a colored band, the result is valid; if the control strip does not show a colored band, the result is invalid; if both the negative control and the test strip show a colored band and there is no amplification curve, and the positive control shows an amplification curve, the experimental data is valid; otherwise, the experimental result is considered invalid; Result description and judgment: If both the control zone and the test zone of the test strip have color bands, the sample is judged to be positive; if the control zone of the test strip has color bands and the test zone has no color bands, the sample is judged to be negative.

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