Fluorescent quantitative PCR kit for detecting enterococcus faecium in microbial feed additive
A microbial feed, Enterococcus faecium technology, applied in the field of bioengineering, can solve the problems of uneven quality of commercially available products
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Embodiment 1
[0019] 1. Fluorescence quantitative PCR (qPCR) detection
[0020] (1) Mix the qPCR reaction solution, hot-start Taq enzyme and probes in a centrifuge tube according to the number of tested samples n (n=the number of samples to be tested + 2), shake on a vortex shaker, and divide each tube into Installed, cover the tube cover and set aside.
[0021] (2) Add the negative control solution to a dispensing tube, take the DNA of each sample and add it to the corresponding reaction tube; finally, take out the positive control and add it to another reaction tube.
[0022] (3) Fluorescence quantitative PCR reaction system: 10 μL Premix Ex Taq TM (2×), 2μL template, 1μL each of upstream and downstream primers and probes (10μmol / L), 5μL ddH 2 O, 20 μL total.
[0023] (4) Fluorescence quantitative PCR reaction conditions: pre-denaturation at 95°C for 30s, cycle 40 times according to the following parameters: denaturation at 95°C for 10s, fluorescence collection at 62°C for 20s, fluores...
Embodiment 2
[0040] Example 2 Parameter optimization
[0041]Determine the probe concentration: 10 μL Premix Ex TaqTM (2×), 2 μL template DNA, 1 μL (10 μmol / L) each of the upstream and downstream primers, respectively add 0.1, 0.5, 1, 2 μL (10 μmol / L) of double distilled water to make up for the probe 20 μL. The results showed that the amplification of the reaction system was good when the final concentration of the probe was 0.5 μM.
[0042] Determine primer concentration: 10 μL Premix Ex TaqTM (2×), 2 μL template DNA, 1 μL (10 μmol / L) probe, add 1 μL (10 μmol / L) of upstream and downstream primers respectively, 0.5, 1, 2, 4 μL (10 μmol / L) L), make up 20 μL with double distilled water. The results showed that when the final primer concentration was 0.5 μM, the reaction system amplified well.
[0043] Annealing temperature: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s, extension at 60°C, 62°C, and 65°C for 20s, 40 cycles. The results showed that when the annealing temp...
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