Primers for detecting influenza virus by RT-PCR and method for detecting influenza virus
An RT-PCR, influenza virus technology, applied in the field of RT-PCR detection technology, can solve the problems of time-consuming operation and high laboratory requirements, and achieve the effect of convenient application and simple operation
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Embodiment 1
[0043] Embodiment 1: Design and synthesis of influenza A virus H1N1RT-PCR oligonucleotide primers:
[0044] Download swine-origin influenza virus H1N1 and influenza A viruses H1N1, H3N2, H5N1 and other subtypes M gene and HA gene complete sequence, the software compares and analyzes the consistency of the M gene sequence of all influenza A viruses, and selects a relatively conserved region to design primers; swine H1N1, new type A H1N1 and seasonal influenza A All HA gene sequences of the virus H1N1 were compared together, and primers were selected for the conserved region and the variable region respectively. In primer design, 2 or less degenerate bases are allowed at the same variable site. Screen the extracted candidate primers that meet the following requirements: ①The probe length L is between 19~28bp; ②Tm value is between 42~59℃; ③GC% is between 25~75%; ④polyN≤4bp; ⑤Hairpin ≤4bp; ⑥ coverage >90%; ⑦ BLAST screening, specificity score > L×0.4. The optimal Tm value is se...
Embodiment 2
[0045] Embodiment two: the present invention detects the application example of unknown virus:
[0046] 1. Extraction of viral RNA:
[0047] Take 200 μL of virus sampling solution, add 500 μL of lysis solution, and extract 50 μL of viral RNA according to the instructions of RNeasy Mini Kit (Qiagen, catalog #74104).
[0048] 2. RT-PCR reaction:
[0049] 1) System configuration: use QIAGEN One step RT-PCR Kit (catalog#210212) reaction solution.
[0050]
[0051] 2) RT-PCR: put the reaction tube with the above reaction system in the PCR machine for RT-PCR, the reaction procedure is as follows
[0052] 60°C for 1 minute;
[0053] 42°C for 10 minutes;
[0054] 50°C for 30 minutes;
[0055] 95°C for 15 minutes;
[0056] Denaturation at 94°C for 30 seconds;
[0057] Anneal at 50°C for 30 seconds;
[0058]Extend at 72°C for 1 minute;
[0059] Go back to step 5, 34 cycles;
[0060] 72°C for 10 minutes;
[0061] Store at 4°C.
[0062] 3. Detection of RT-PCR products: Take...
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