Detection kit used for detecting pig Kobu virus
A detection kit and the technology of the kit, applied in the detection field, can solve the problems of insufficient positive rate and low detection positive rate, and achieve the effects of simple operation, good repeatability and high accuracy
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Embodiment 1
[0053] Example 1 The total RNA of porcine Kobu virus is extracted
[0054] Take 400 μl of serum from positively infected pigs, add 400 μl of Trizol, shake vigorously for 1-2 minutes, and mix thoroughly; add 100 μl of chloroform / isoamyl alcohol (24:1), shake vigorously for 30 seconds, and centrifuge at 12,000 rpm for 5 minutes; Carefully transfer to a sterile EP tube, add 200μl of absolute ethanol, and mix well; transfer all the above solution to a GenClean column in a 2ml collection tube, place at room temperature for 2min, and centrifuge at 8000rpm for 1min; carefully take out the column and discard Collect the waste liquid in the tube, put the column back into the collection tube; add 450 μl RPEsolution (rinsing solution plus 5 times absolute ethanol) to the column, centrifuge at 10000 rpm for 30 s at room temperature; pour off the waste liquid, repeat washing once, carefully take out the column, Discard the waste liquid in the collection tube, put the column back into the c...
Embodiment 2
[0055] Example 2 Primer Design
[0056] According to the 3D gene sequence (AB624488) of porcine Kobu virus published in GenBank, a pair of primers were designed using Primer5.0 software, and the 3D gene partial fragment (Kobu-3D) 101bp could be amplified with the designed primers. The primer sequences are as follows:
[0057] Upstream primer P1: 5'-GGACCCAGACACTTACGAACA-3'
[0058] Downstream primer P2: 5'-TCGGACCAGAATGGAAAGCA-3'
[0059] Primers were synthesized by Dalian Baobio Biotechnology Co., Ltd.
Embodiment 3
[0060] 3D gene RT-PCR amplification of porcine Kobu virus of embodiment 3
[0061] Take 5 μl of RNA sample and add DEPC water to make up 11 μl, then add 1 μl of reverse transcription primer, bathe in 70°C water for 10 minutes, put it on ice for 5 minutes, and centrifuge slightly; take it out, add 4 μl of 5×Buffer, 2 μl of dNTP, 1 μl of RNasinhibiter, and centrifuge briefly; 42 ℃ water bath for 2 minutes, quickly add 1 μl reverse transcriptase, and seal it with parafilm; 42 ℃ water bath for 60 minutes, transfer to 70 ℃ water bath for 15 minutes, and the reverse transcription is completed; take the cDNA obtained by reverse transcription as a template for PCR amplification;
[0062] The PCR amplification system is: Premix Ex Tap 12 μl, the concentration is 0.5 μmol / L primers P1 and P2 0.5 μl each, cDNA 1 μl, and finally make up to 25 μl with sterilized double distilled water;
[0063] The PCR amplification reaction program is:
[0064] (1) Pre-denaturation at 95°C for 5 minutes;...
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