A detection method for h5 and h7n9 subtype highly pathogenic avian influenza virus
An avian influenza virus, highly pathogenic technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of indistinguishability, economic losses in the poultry industry, etc., and achieve short detection time, The effect of high sensitivity and high hardware requirements
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Embodiment 1
[0033] Example 1: Determination of conserved sequence regions and screening of primer probes
[0034] The applicant studied the HA nucleotide sequences of different NA subtypes and different branches of the H5 subtype influenza virus by molecular biology methods (Table 1), and compared them with the HA sequences of H5 strains isolated and preserved in the applicant's laboratory , to determine its conserved region ( figure 1 ). Sequence comparison found that the HA sequence of the H5 subtype influenza virus is in the nt106–nt174 region, and the sequence of this region is as follows:
[0035] ATGGAAAAARAACGTYACTGTWACRCATGCYMAAGACATACTGGARAAGACACAYAAYGGGARGCTYTG;
[0036] In the nt210-nt242 region, the sequence is ATTGYAGTGTRGCWGGATGGCTHCTYGGRAAYCC, which is highly conserved; where, R=A or G, Y=C or T, M=or C, W=A or T.
[0037] Table 1: H5 virus strain information table used in the design of primers and probes in the present invention
[0038]
[0039]
[0040] Accordi...
Embodiment 2
[0056] Embodiment 2: Establish detection method
[0057] After establishing the detection reaction system and reaction conditions, 25 μl of 2×RT-PCR reaction buffer (containing dNTPs, Mg 2+ ), 1.5 μl of water, 2.0 μl of the upstream primer synthesized in the first step (the concentration is 10 μmol / L), 2.0 μl of the downstream primer synthesized in the first step (the concentration is 10 μmol / L), the probe synthesized in the first step 1.5 μl (concentration: 10 μmol / L), enzyme mixture (reverse transcriptase, RNase inhibitor, Taq enzyme with 5'→3' exo-cutting activity) 2.5 μl, viral nucleic acid to be detected 10.0 μl (from clinical samples or other samples extracted with a nucleic acid extraction kit); then the reaction system is sealed and placed on a fluorescent quantitative PCR instrument for reaction. Reaction conditions: first stage, reverse transcription 50°C / 10min; second stage, pre-denaturation 95°C / 2min; third stage, 95°C / 10s, 60°C / 30s, 40 cycles; Fluorescence was c...
Embodiment 3
[0063] Embodiment 3: the effect detection of primer and probe
[0064] 1. Sensitivity testing of H5 and H7N9 subtype highly pathogenic avian influenza viruses was carried out using the primer probes and methods established by the above screening, including the following steps:
[0065] The first step: extract viral RNA, and measure the viral RNA content with a micro-nucleic acid analyzer. Dilute the RNA by 10 times, take 10.0 μl of the diluted RNA template, and add it to 40.0 μl of qRT-PCR master mix;
[0066] The second step: use the established real-time fluorescent quantitative RT-PCR method to detect and determine its sensitivity;
[0067] The result shows that the real-time fluorescent quantitative RT-PCR method that the present invention establishes can detect the H5 virus RNA template of 0.1fg, can detect the virulent RNA template of 0.004fg H7N9 ( image 3 ).
[0068] 2. The specificity test of the real-time fluorescent quantitative RT-PCR detection method for H5 an...
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