Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV)
A swine flu virus and a pair of technology, applied in the field of animal health inspection, can solve the problems of exacerbation of disease and increase of fatality rate
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[0102] (1) Extraction of viral RNA: Take 200 mL of chicken embryo allantoic fluid of swine influenza virus and add it to a 1.5 mL centrifuge tube, add 600 μl Trizol solution, shake and mix, let stand at room temperature for 5 minutes, then add 200 μl chloroform, shake and mix, and let stand at room temperature 5min, centrifuge at 12000rpm at 4℃ for 10min; take the supernatant into a 1.5mL centrifuge tube, add 800μl of isopropanol containing 1‰DEPC, mix by inverting, centrifuge at 12000rpm for 10min, discard the supernatant, add 500μl of isopropanol containing 1‰DEPC 70% ethanol, centrifuge at 12000rpm at 4°C for 10min, discard the ethanol, dry at room temperature, add 20 μl DEPC water to dissolve, and store at -80°C for later use.
[0103] (2) NASBA amplification system:
[0104] 3 μl RNA of the sample to be tested
[0105] Buffer 4μl (containing 40mM / L PH8.5 Tris-HCL, 70mM / L KCl, 12mM / L MgCl 2 , 5mM / L DTT buffer)
[0106] 10mM / L dNTP 2μl
[0107] 20mM / L NTP 2μl
[0108] ...
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