Primer for amplifying molecules of microbes in pit mud of Luzhou-flavor liquor and detection method
A technology for microbial flora and Luzhou-flavor liquor, which is applied in the field of winemaking, can solve the problems that the information of microbial population is small and cannot reflect the structure and richness of microbial flora in pit mud well, and achieves the effect of quick and timely monitoring.
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Embodiment 1
[0048] The establishment of embodiment one inventive method
[0049] 1. Selection of primers
[0050] During the establishment of this method, primers for the amplification of the V6-V8 hypervariable region of 16S rDNA were used, and the product was about 470 bp, which was close to the optimal fragment length of DGGE. In addition, the primers used to amplify the V3 region which are widely used at present are also used, and the PCR products amplified by the two pairs of primers are about 240bp and 470bp respectively. Different primer sequences were amplified separately, the amplification of the V3 region referred to the existing literature, and the amplification of the V6-V8 region was optimized by gradient PCR.
[0051] 2. Optimization of amplification conditions
[0052] The amplification conditions of the V3 hypervariable region refer to the existing literature (Wang Qizan, Xu Qiufang, Jiang Peikun, etc. DGGE analysis of the 16S rDNA V3 region fragment PCR of the soil bact...
Embodiment 2
[0086] Embodiment 2 uses the method of the present invention to compare and detect the pit mud in different parts of the same cellar
[0087] (1) Extraction of microbial total DNA from pit mud, using E.Z.N.A.TM soil DNA kit to extract total microbial DNA.
[0088] (2) Primer sequences for amplifying 16S rDNA:
[0089] Primer I: 5'-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAA CGC GAA GAACCT TAC-3',
[0090] Primer II: 5'-ACG GGC GGT GTG TAC-3'.
[0091] (3) Amplification of prokaryotic 16S rDNA V6-V8 region: PCR reaction system: 50 μL: 1.0 μL Taq enzyme (5U / μL), 5.0 μL 10× buffer, 3.0 μL MgCl2 (25 mmol / L), 4.0 μL dNTPs Mixture (2.5 mmol / L each), 1.0 μL each of primers (10 μmol / L), 1.0 μL template DNA (100 ng / L), 34.0 μL sterilized double distilled water.
[0092] PCR amplification program: pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 45s, application of falling PCR, annealing at 65°C, each round of 1°C drop, finally down to 49°C, annealing for ...
Embodiment 3
[0097] Embodiment 3 Using the method of the present invention to compare and detect different cellar muds with different cellar ages with large differences in wine production quality
[0098] (1) Extraction of total microbial DNA from pit mud, using E.Z.N.A.TM soil DNA ktt to extract total microbial DNA.
[0099] (2) Primer sequences for amplifying 16S rDNA:
[0100] Primer I: 5'-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAA CGC GAA GAACCT TAC-3',
[0101] Primer II: 5'-ACG GGC GGT GTG TAC-3'.
[0102] (3) Amplification of prokaryotic 16S rDNA V6-V8 region: PCR reaction system: 50 μL: 1.0 μL Taq enzyme (5U / μL), 5.0 μL 10× buffer, 3.0 μL MgCl2 (25 mmol / L), 4.0 μL dNTPs Mixture ( 2.5 mmol / L each), 1.0 μL each primer (10 μmol / L), 1.0 μL template DNA (100 ng / L), 34.0 μL sterilized double distilled water.
[0103] PCR amplification program: pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 45s, application of falling PCR, annealing at 65°C, each round of...
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