Fluorescent quantitative PCR detection material and kit for detecting D/660 branch of influenza D virus
A fluorescence quantitative, influenza virus technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microorganism determination/inspection, etc., to achieve the effect of high sensitivity, loss reduction, and economic loss reduction
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Embodiment 1
[0039] Example 1 Table 1 Fluorescent quantitative PCR primers and probe sequences
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[0042] The known type D influenza virus of sheep is used as the detection material, and the negative control is pure water.
[0043] The fluorescent group of the specific fluorescent probe is a FAM fluorescent group; the quenching group is a BHQ1 quenching group. 1. Sheep influenza D virus RNA extraction
[0044] RNA was extracted using the TRIZOL method. After adding Trizol to the cells or tissues, place them at room temperature for 5 minutes to fully lyse; centrifuge at 12,000 rpm for 5 minutes to discard the precipitate, add Trizol to chloroform, shake and mix, and place at room temperature for 15 minutes; centrifuge at 12,000 rpm at 4°C for 15 minutes, and transfer the upper layer to another centrifuge tube; Add isopropanol (precipitate RNA) and mix well, place at room temperature for 5-10min; centrifuge at 12000rpm at 4°C for 10min, discard the supernatant, RNA si...
Embodiment 2
[0050] Example 2 Fluorescence quantitative PCR standard curve drawing
[0051] The reaction system is 20 μL, including 10 μL of fluorescent quantitative PCR enzyme mixture Luna universaprobeQpcr Mix, 2 μL of template cDNA, H 2 O (Nuclease-free) was supplemented to 20 μL, of which LunauniversaprobeQpcr Mix 10 μL contained 0.8 μL of pre-qPCR primers (10 μM), 0.8 μL of post-qPCR primers (10 μM), and 0.4 μL of probes (10 μM).
[0052] The reaction program was: 95°C pre-denaturation for 60 seconds, then 95°C for 15 seconds, 60°C for 30s and then 40-45 cycles.
[0053] The above-mentioned positive standard sample was calculated as the copy number by measuring the concentration of the positive plasmid, and then used ddH 2 O-fold dilution is 6 concentration gradients 10 -7 -10 -2 copies / μL, 20 μL systems were prepared with these 6 concentration gradients, and the Bio-rad fluorescent quantitative PCR instrument was used for amplification, and the established standard curve was as fo...
Embodiment 3
[0054] The sensitivity experiment of embodiment 3 fluorescence quantitative PCR probe method
[0055] With the positive standard sample described in embodiment 2, then calculate the copy number by measuring the concentration of positive plasmid, then use ddH 2 O-fold dilution is 6 concentration gradients 10 -7 -10 -2 copies / μL, prepare 20 μL systems with these 6 concentration gradients, and perform amplification with Bio-rad fluorescent quantitative PCR instrument. The reaction conditions are as in Example 2, and the results are shown in the appendix. image 3 shown. The extracted positive plasmid was diluted 10 times with ultrapure water, and the content of the plasmid was measured at 260nm and 280nm wavelengths, and the copy number of the plasmid DNA was obtained by the formula: the lowest detection concentration of real-time fluorescent quantitative PCR was 10.09 copies / μL.
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