A kind of universal pcr primer pair and its application
A primer pair and product technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of difficulty in distinguishing similar species or genera, weak sequence specificity, and weak versatility and other problems, to achieve the effect of strong versatility, strong specificity and low cost
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Embodiment 1
[0028] Example 1 Universal PCR sequencing identification of different microorganisms
[0029] After the sample was ground with liquid nitrogen, the genomic DNA of the 25 microorganisms to be detected in the sample was extracted with the Tiangen kit N96 Plant Genomic DNA Kit, and the extracted genomic DNA was used as the template DNA for the subsequent PCR reaction.
[0030] Use primers AP1 and AP2 to obtain the gene sequences of the 25 kinds of microorganisms by PCR amplification. The reaction system and reaction conditions are as follows: Reaction system: AP1 1 μmol / L, AP2 1 μmol / L, 0.5mmol / L dNTP, 1.5mmol / L magnesium Ions, 0.05U / μL DNA Taq polymerase and 10ng / μL template DNA; reaction conditions: (1) 95°C, denaturation for 5 minutes; (2) 95°C, denaturation for 30 seconds; (3) 50°C, annealing for 30 seconds; (4) Extend at 72°C for 2 minutes, wherein the steps (2)-(4) are repeated for 30 cycles; (5) Extend at 72°C for 5 minutes; (6) Store at 4°C;
[0031]Use primers EU27F and...
Embodiment 2
[0048] Example 2 Unknown sample sequencing identification
[0049] Separation and enrichment of 17 species of unknown microorganisms in the samples, among which the samples numbered UB1-UB8 are the bacterial cells separated from the streaked plate; the samples numbered UB9-UB15 are mold blocks in expired yogurt, and the bacterial cells were collected directly after washing; The samples numbered UB16-UB17 are bacteria powder, and the bacteria were collected directly after washing with water. Genomic DNA was extracted with Tiangen kit N96 Plant Genomic DNAKit, and the extracted genomic DNA was used as template DNA for subsequent PCR reactions.
[0050] Use primers AP1 and AP2 to amplify the gene sequence by PCR. Reaction system: AP1 1 μmol / L, AP2 1 μmol / L, 0.5 mmol / L dNTP, 1.5 mmol / L magnesium ion, 0.05 U / μL Taq DNA polymerase and 100 ng / μL Template DNA; reaction conditions: (1) 95°C, denaturation for 5 minutes; (2) 95°C, denaturation for 30 seconds; (3) 50°C, annealing for 30 ...
Embodiment 3
[0063] Example 3 PCR sequencing amplification of the sample to be detected under different reaction conditions
[0064] Get sample UA1-10 in embodiment 1 and PCR amplification according to the following conditions:
[0065] The PCR system includes: AP1 0.1μmol / L, AP20.1μmol / L, 0.2mmol / L dNTP, 1.0mmol / L magnesium ion, 0.01U / μL DNA polymerase and 1ng / μL template DNA.
[0066] Using the genomic DNA of UA1-10 in Example 1 as a template, the PCR reaction conditions include the following steps: (1) denaturation at 90°C for 3 minutes; (2) denaturation at 90°C for 60s; (3) annealing at 45°C for 10s; (4) 65°C Extending at °C for 1 min, wherein the steps (2)-(4) were repeated for 25 cycles; (5) extending at 65 °C for 3 min. The amplified PCR products were subjected to agarose gel electrophoresis.
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